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A Microemulsificação Do Óleo Essencial de Cravo Melhora Seu Controle in Vitro e in Vivo DePenicillium Digitatum
A Microemulsificação Do Óleo Essencial de Cravo Melhora Seu Controle in Vitro e in Vivo DePenicillium Digitatum
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
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.
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1 Microemulsification of clove essential oil improves its in vitro and in vivo control
2 of Penicillium digitatum
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5 Shoukui He, Xiaoyun Ren, Yangfan Lu, Yunbin Zhang, Yifei Wang*, Linjun Sun
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8 Road 100, Shanghai 201418, People’s Republic of China
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10 *Correspondence
13 Email: wangyifei@sit.edu.cn
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17 Abstract:
20 vivo microbial growth inhibition limit its application. Hence, this study aimed to
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21 strengthen the antifungal activity of CEO by loading it in microemulsion system. Two
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22 CEO microemulsions (ME-1, CEO/ethanol/Tween 80 = 1:2:7; ME-2,
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24 Penicillium digitatum in vitro and in navel oranges. Microemulsification of CEO
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25 caused a reduction of MIC (minimum inhibitory concentration) from 0.50 µl/ml to
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26 0.25 µl/ml, while the MFC (minimum fungicidal concentration) remained unchanged
28 incidence of navel oranges treated with ME-1 and ME-2 was significantly (p < 0.05)
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30 treatment after 5 days’ storage at 25 oC. In the vapor phase, CEO microemulsions had
31 the best control of lesion diameter and decay development. Additionally, the
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33 ability to suppress spore germination and germ tube elongation of P. digitatum. The
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36 Key words: clove essential oil; microemulsion; antifungal activity; navel oranges;
37 Penicillium digitatum
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39 1. Introduction
40 Navel oranges (Citrus sinensis L., Osbeck) are popular due to their highly
41 nutritional value. Unluckily, they are readily affected by pathogens (i.e., Penicillium
42 digitatum and Penicillium italicum) (Zeng, Zhang, Chen, & Fu, 2012). For citrus fruit,
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43 green mold caused by P. digitatum accounts for 90% of postharvest losses (Kellerman,
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44 Erasmus, Cronjé, & Fourie, 2014). Traditionally, chemical fungicides are primarily
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46 environmental problems and human toxicity (Xu, Yan, Ni, Chen, Zhang, & Zheng,
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47 2014), natural antifungal products should be developed as alternatives.
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48 In recent years, environmentally friendly essential oils (EOs) have been
50 activities (Davidson, Critzer, & Taylor, 2013). Since EOs can be derived from organic
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53 be the production of EOs (Feng, & Zheng, 2007). Indeed, EOs of citronella (Chen et
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54 al., 2014), Laurus nobilis (Xu, Yan, Ni, Chen, Zhang, & Zheng, 2014), lemongrass
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55 and cinnamon (Maqbool, Ali, Alderson, Mohamed, Siddiqui, & Zahid, 2011) have
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56 exhibited inhibitory activity against postharvest fungal pathogens in vitro and in vivo.
57 Despite the high efficiency of EOs in in vitro tests, the same effect in food is achieved
58 only with higher concentrations (Burt, 2004; Hulin, Mathot, Mafart, & Dufosse, 1998).
59 This fact may imply a challenging issue of EOs utilization in food industry, namely,
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63 foodborne pathogens (Teixeira, Leite, Domingues, Silva, Gibbs, & Ferreira, 2007).
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65 microemulsion was effective in inhibiting Bacillus subtilis, Escherichia coli and
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66 Staphylococcus aureus (Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Shen,
67 Weng, Zhao, Feng, & Zheng, 2009). In this sense, microemulsion could be an
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68 approach to attain a balance between antimicrobial efficiency and odor acceptability
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69 of EOs. However, up to now very little research has been carried out to formulate EO
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70 microemulsions for antifungal purposes.
72 the antimicrobial activity of various EOs. From those, cinnamon and clove EOs were
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74 established in our group controlled postharvest gray mold of pears without adverse
75 influence on fruit qualities (Wang, Zhao, Yu, Zhang, He, & Yao, 2014), indicating that
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76 such technologies are very promising to satisfy consumer demand for commercial
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77 formulations of EOs. Consequently, the present work aimed to (i) develop clove oil
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79 (iii) evaluate their capacity to control green mold in navel oranges by direct contact
82 2.1 Materials
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85 Shanghai Shenyu Pharmaceutical & Chemical Co., Ltd. (Shanghai, China). These
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87 essential oil (CEO) from Syzygium aromaticum was purchased from Dongshi Essence
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88 Perfume Co., Ltd. (Shanghai, China) and was stored at 4 °C in dark bottles prior to
89 use. According to the information given by the supplier, this oil was produced by
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90 steam distillation method. Its major components were found to be eugenol (76.190%)
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91 and β-caryophyllene (15.088%) (He et al., 2014).
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92 2.2 Preparation of pathogen
93 The navel orange fruit pathogen (P. digitatum), obtained from Institute of
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95 potato dextrose agar (PDA) at 4 oC. P. digitatum was incubated for one week on PDA
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101 CEO microemulsions were prepared as previously described (Wang, Zhao, Yu,
102 Zhang, He, & Yao, 2014). Two kinds of microemulsions were prepared: (1) ME-1: 1.0
103 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
104 Tween 80. Briefly, CEO and ethanol were vigorously mixed at predetermined weight
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105 ratios in glass test tubes sealed with removable caps at 25 oC. Tween 80 was then
106 added to the mixture under constant stirring using a magnetic stirrer (78-1B, Shanghai,
107 China) for 30 min. CEO microemulsions were stored at 4 oC and allowed to
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109 condition. In the two formulated microemulsions, evaporation loss was negligible.
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110 After equilibration for a period of 24 h, the resulting microemulsions were diluted to
111 the instrument sensitivity range with deionized water to measure the mean particle
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112 diameter using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a
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113 He-Ne laser (633.0 nm). The measurements were performed at a 90o scattering angle.
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114 ME-1 and ME-2 showed a droplet size of 241.1 nm and 150.0 nm, respectively. The
115 stability of the developed microemulsions was then examined according to Zhang, Li,
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116 Zhu, Feng, and Zheng (2011) with modifications. ME-1 and ME-2 were subjected to
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117 centrifugation at 4000 g for 15 min and 1-month storage at 20 oC. It was observed that
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121 concentration) of CEO microemulsions were determined using 2-fold serial dilution
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122 method (Jordán, Lax, Rota, Lorán, & Sotomayor, 2013) in order to assess their in
123 vitro antifungal activity. Aliquots (50 µl) of P. digitatum suspensions (approximately
124 1.0×107 spores/ml) were inoculated into 5 ml of potato dextrose broth (PDB)
125 containing suitable amounts of CEO microemulsions (0.0625-2 µl/ml). Both negative
126 (sterile PDB) and positive (5 ml of PDB + 50 µl of inoculum) controls were included.
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127 The samples were then incubated at 28 oC with shaking (200 rpm) for 48 h. The MIC
128 was recorded as the lowest concentration of microemulsions that completely inhibited
130 In order to determine MFC, 100 µl of each case without visible microbial growth
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131 was plated on PDA. Plates were incubated at 28 oC for 48 h. The lowest concentration
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132 at which P. digitatum failed to grow on PDA plates was defined as MFC. The MIC
133 and MFC of pure oil was also determined in a similar way. The assay was performed
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134 in triplicate.
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135 The fungistatic and fungicidal effects of CEO microemulsions were considered
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136 to be significantly (p < 0.05) enhanced when their MIC and MFC values were equal
137 to or higher than a twofold decrease compared with those of pure oil, respectively
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139 2.5 The capacity of CEO microemulsions to control green mold in navel oranges
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141 Navel oranges (Citrus sinensis L., Osbeck), harvested at commercial maturity,
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142 were obtained from Gannan, China. Samples of uniform ripeness, size and free of
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143 injuries were selected. The test fruits were surface-sterilized in sodium hypochlorite
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144 (0.1%) for 60 s, scoured under running water and air dried at 25 oC. Fruits were gently
146 suspension of P. digitatum (approximately 1.0×105 spores/ml) was pipetted into each
147 wound. Samples were then treated with 30 µl CEO microemulsions or pure oil at the
148 level of 0.25 µl/ml. SDW served as the control. After air drying for 1.5 h, the oranges
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149 were stored in enclosed plastic boxes to maintain a high relative humidity
151 wound number) and corresponding lesion diameter of oranges were examined after 5
152 days. Each treatment consisted of three replicates of 30 wounds each, and the
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153 experiment was performed twice.
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154 2.5.2 Vapor contact assay
155 Vapor contact test was used to determine the antifungal effects of vapors of CEO
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156 microemulsions as previously stated (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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157 Oranges were placed in 8-L plastic boxes after wounded and inoculated with the
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158 pathogen as described above. For each box, a culture dish (3.5 cm diameter)
159 containing 30 µl SDW (control), pure oil (0.025 µl/ml) or CEO microemulsions
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160 (0.025 µl/ml) was placed in the center. These boxes were then sealed using
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161 polyethylene bags. Disease incidence (infected wound number/total wound number)
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162 and corresponding lesion diameter of oranges were examined after 5 days’ storage at
163 25 oC. Each treatment consisted of three replicates of 30 wounds each, and the
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166 Pure oil and CEO microemulsions were added to 15-ml tubes containing 5 ml
167 PDB to achieve a final level of 0.25 µl/ml, respectively. SDW was added as the
168 control. Then a spore suspension (approximately 1×107 spores/ml) of 100µl was
169 inoculated into each tube. Spore germination (%) and germ tube length (µm) were
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171 germinated when the germ tube length equaled or exceeded that of the spore. At least
172 150 spores per replicate were microscopically examined for the presence of germ
173 tubes. The percentage of germinated spores was then estimated from the evaluated
174 spores. Furthermore, germ tube length (µm) was also determined. Each treatment
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175 consisted of three replicates, and the experiment was performed twice.
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176 2.7 Data analysis
177 Statistical analyses were conducted with SAS version 8.2, via one-way ANOVA.
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178 Duncan's test was then used to detect statistical significance. Differences in mean
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179 values were considered significant when p < 0.05.
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180 3. Results
182 The MIC and MFC values of CEO microemulsions in comparison to pure oil,
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184 of CEO caused a reduction of MIC from 0.50 µl/ml to 0.25 µl/ml, while the MFC
185 remained unchanged at 0.50 µl/ml. The lower MIC of CEO microemulsions suggested
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187 3.2 The capacity of CEO microemulsions to control green mold in navel oranges
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189 Direct addition assay was used to evaluate inhibitory effect of CEO
190 microemulsions on navel orange green mold. As shown in Fig. 2, pure oil and
191 microemulsions greatly delayed decay development. Moreover, the disease incidence
192 of navel oranges treated with microemulsion ME-1 and ME-2 was significantly (p <
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193 0.05) reduced by 34.51% and 20.93%, respectively, in comparison to the treatment
196 Data shown in Fig. 3 indicated that pure oil and microemulsions in vapor phase
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197 could also significantly (p < 0.05) inhibit P. digitatum on navel oranges after 5 days’
storage at 25 oC. CEO microemulsions exhibited the best control of lesion diameter
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199 and decay development (57.34%-59.52%, 8.28-8.59 mm), followed by pure oil
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200 (72.84%, 9.54 mm).
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201 3.3 Spore germination assay
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202 Determination of spore germination and germ tube elongation could help to
203 explain the antifungal activity of CEO microemulsions. In the control group, all
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204 spores germed and the germ tubes were too long and enwind each other (Fig. 4).
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205 However, pure oil and microemulsions significantly (p < 0.05) inhibited fungal spore
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206 germination rate and reduced germ tube length. The average suppressive efficacy was
207 in the following order: ME-1 ≈ ME-2 > pure oil > SDW control.
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208 4. Discussion
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210 compounds for food, cosmetic, pharmaceutical and oil recovery applications (Ma &
211 Zhong, 2015), only a limited number of reports relevant to their use for antimicrobial
213 new and promising application. In the current work, we explored the possibility of
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218 compared to pure oil. This phenomenon may result from the small droplet sizes of
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219 CEO microemulsions (ME-1, 241.1 nm; ME-2, 150.0 nm), which could improve the
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220 transport mechanisms through P. digitatum cell membrane (Donsìa, Annunziatab,
221 Sessaa, & Ferraria, 2011). Similarly, microemulsion was found to improve the
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222 antimicrobial activity of glycerol monolaurate (the glycerol monoester of lauric acid)
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223 (Fu, Feng, & Huang, 2006; Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Lu,
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224 Wang, Shen, Feng, & Zheng, 2008b). In the food additive industry, there are
226 sub-lethal levels instead of putting reliance on individual food preservatives at high
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227 concentrations (Roller, 1999). In this regard, enhanced fungistatic activity may imply
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230 Hence, the in vivo antifungal activity of CEO microemulsions in the liquid and vapor
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231 phases was determined subsequently. In agreement with a previous study, pure CEO
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232 had some control effects on citrus green mold caused by P. digitatum (Shao, Cao, Xu,
233 Xie, Yu, & Wang, 2015). Additionally, better antifungal activity was found in CEO
235 previously formulated in our laboratory exhibited higher in vivo antifungal activity
236 than pure ones (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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237 The use of EO in vapor phase is an appealing approach to reduce its sensory
238 effect (Tyagi, Malik, Gottardi, & Guerzoni, 2012). The antimicrobial activity
239 generated by CEO vapor has been reported in previous literature. Tullio et al. (2007)
240 found that CEO vapor had a wide antifungal spectrum. Vapors of EO combinations of
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241 clove and cinnamon exerted a synergism or antagonistic effect for the inhibition of
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242 bacteria, depending on EO concentration applied (Goni, Lopez, Sanchez, Gomez-Lus,
243 Becerril, & Nerin, 2009). In the present paper, we evaluated the effect of vapors of
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244 CEO microemulsions in controlling a major postharvest pathogen P. digitatum. Better
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245 control activity was observed in microemulsified CEO. Moreover, CEO
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246 microemulsions in the liquid and vapor phases exhibited quite similar antifungal
249 Exploring the mechanism of action could help to elucidate the remarkable
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250 performance of CEO microemulsions. In this study, spore germination and germ tube
251 elongation of P. digitatum was significantly (p < 0.05) suppressed by pure oil and
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252 microemulsions, which was indirect evidence of disruption and dysfunction of cell
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253 walls and membranes. Furthermore, the greatest inhibition was observed in P.
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254 digitatum treated with CEO microemulsions. Thus, it can be inferred that the
255 enhanced antifungal activity of CEO microemulsions might be due to the increase in
256 cell membrane permeabilization caused by the small droplet sizes of these
257 formulations.
258 In conclusion, improved control activity of CEO against P. digitatum in vitro and
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260 CEO microemulsions exhibited stronger inhibitory activity on spore germination and
261 germ tube elongation than pure oil. Due to the enhanced antifungal activity of
262 microemulsified CEO in the vapor and liquid phases, lower antimicrobial
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263 concentrations are required for utilization in food. This fact is of paramount
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264 importance from a safety and organoleptic point of view. The sensory effect of CEO
265 might thus be reduced. These results suggest that CEO microemulsions established in
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266 this work may be potential commercial formulations to control postharvest green
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267 mold of navel oranges. Further investigations should be conducted to explore the
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268 effects of these formulations on fruit quality traits, such as sensory acceptability,
269 weight loss, firmness, total soluble solids, ascorbic acid and color.
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270 Acknowledgment
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272 Commission Science and Technology Innovation Foundation (12YZ165) and Science
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275 References
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358 digitatum.
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359 Figure 2 The inhibitory effects of clove essential oil microemulsions on P.digitatum
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360 decay development in artificially wounded and inoculated navel oranges. The disease
361 incidence (A) and lesion diameter (B) were measured after 5 days’ storage at 25 oC.
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362 Vertical bars represent standard deviation. Different letters signify significant
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363 differences (p < 0.05).
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364 Figure 3 The inhibitory effects of vapors of clove essential oil microemulsions on
365 P.digitatum decay development in artificially wounded and inoculated navel oranges.
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366 The disease incidence (A) and lesion diameter (B) were measured after 5 days’
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367 storage at 25 oC. Vertical bars represent standard deviation. Different letters signify
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369 Figure 4 Effects of clove essential oil microemulsions on spore germination (A),
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370 germ tube elongation (B) and microscope images (C) of P. digitatum. Data represent
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371 mean ± standard deviation. Different lowercase letters within a column indicate
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372 significant differences (p < 0.05). ND (not determined), the germ tube length could
373 not be determined because the germ tubes were too long and enwind each other.
374 Germination and germ tube length were determined microscopically (C) after 18 h
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378 Figure 1
379
(A)
380 ME-2
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Formulations
382 ME-1
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384 Pure oi
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0.00 0.25 0.50
MIC (µl/ml)
(B)
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395 Figure 2
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Infected fruits (%)
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401 20
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411 Figure 3
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Lesion diameter (mm)
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427 Figure 4
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Highlights
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oranges.
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Microemulsions decreased spore germination and germ tube elongation of P.
digitatum.
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