Accepted Manuscript

The antioxidant, antibacterial, antibiofilm activity of essential oil from Citrus medica L.
var. sarcodactylis and its nanoemulsion

Zaixiang Lou, Jie Chen, Fuhao Yu, Hongxin Wang, Xingran Kou, Chaoyang Ma, Song
Zhu

PII: S0023-6438(17)30138-X
DOI: 10.1016/j.lwt.2017.02.037
Reference: YFSTL 6066

To appear in: LWT - Food Science and Technology

Received Date: 8 September 2016

Revised Date: 15 February 2017
Accepted Date: 19 February 2017

Please cite this article as: Lou, Z., Chen, J., Yu, F., Wang, H., Kou, X., Ma, C., Zhu, S., The antioxidant,
antibacterial, antibiofilm activity of essential oil from Citrus medica L. var. sarcodactylis and its
nanoemulsion, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.02.037.

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.
 ACCEPTED MANUSCRIPT

1 The antioxidant, antibacterial, antibiofilm activity of essential oil from Citrus

2 medica L. var. sarcodactylis and its nanoemulsion

4 Zaixiang Lou*, Jie Chen, Fuhao Yu, Hongxin Wang*，Xingran Kou, Chaoyang Ma,

PT
5 Song Zhu

RI
6 State Key Laboratory of Food Science and Technology, School of Food Science and

SC
7 Technology, Jiangnan University, Wuxi 214122, P.R.China

8 * Corresponding author. Tel.: + 86 510 85917795; fax: + 86 510 85917795

9 E-mail address: louzaixiang@126.com

U
AN
10
M

11
D
TE
C EP
AC

1
 ACCEPTED MANUSCRIPT

12 Abstract

13 In this work, the nanoemulsion of essential oil from Citrus medica L. var.

14 sarcodactylis was prepared using a spontaneous emulsification method. Meanwhile,

15 the effect of nanoemulsification on the antioxidant, antibacterial and antibiofilm

PT
16 activity of essential oil was evaluated. GC–MS analysis indicated nineteen

RI
17 compounds in the essential oil, and the essential oil was dominated by D-Limonene

SC
18 (50.04%). The minimum and stable droplets (73nm) of essential oil nanoemulsion

19 were formulated when the concentration of essential oil in oil phase was 50% (w/w).

20
U
The hydroxyl radicals, DPPH radicals scavenging ability, iron reducing power,
AN
21 antibacterial and antibiofilm activity of nanoemulsified essential oil were significantly
M

22 higher than those of pure essential oil at same concentration. Moreover, the

nanoemulsified essential oil exhibited higher inhibitive effect on the bacteria in tofu
D

23
TE

24 than pure essential oil. Therefore, nanoemulsification significantly enhanced the

25 antioxidant, antibacterial and antibiofilm activity/efficiency of essential oils, which

EP

26 will promote the abroad utilization of essential oils.

C

27 Keywords: nanoemulsion, essential oil, antioxidant, antibacterial, antibiofilm

AC

28

29

2
 ACCEPTED MANUSCRIPT

30 1 Introduction

31 The Citrus medica L. var. sarcodactylis is a plant of Rutaceae family. In China, it

32 is popularly named “Buddha hand citron” in markets. Customarily, it is used as a

33 medicinal material or functional vegetables (Peng et al., 2009; Wu, Li, Yang, Zhan, &

PT
34 Tu, 2013), and it is rich of essential oil.

RI
35 It has been widely reported that essential oils from natural plants exhibit

SC
36 antioxidant, antiradical, and antimicrobial properties (Adrar, Oukil, & Bedjou, 2016;

37 Bey-Ould Si Said et al., 2016; Kacem et al., 2016; Omar et al., 2016). Now, they have

38
U
been widely used as functional ingredients in food and pharmaceutical industries
AN
39 (Alviano et al., 2005; Kerekes et al., 2013). The antibacterial ability of essential oils
M

40 leads to their wide application in many food and beverage products to prolong the

shelf life. Consumer’s growing demand for natural rather than synthetic additives
D

41
TE

42 makes the essential oils highly desirable due to their natural property.

43 However, the use of essential oils is highly limited due to their poor water
EP

44 solubility.The oil-in-water (O/W) emulsions or nanoemulsions provide good solutions

C

45 to improve the solubility of essential oils (Chang, McLandsborough, & McClements,

AC

46 2012; Xu et al., 2012; Ziani, Chang, McLandsborough, & McClements, 2011).

47 Through these emulsion-based delivery systems, the lipophilic ingredients could be

48 quickly integrated into aqueous-based food systems owing to their improved water

49 dispersibility. The nanoemulsions could be used to incorporate lipophilic components

50 into various aqueous-based food products, including dairy products and clear

3
 ACCEPTED MANUSCRIPT

51 beverages. Furthermore, the activity of nanoemulsified compounds may be improved

52 due to the excellent dispersity, stability and penetration abilities (Hatanaka et al., 2010;

53 Huang, Yu, & Ru, 2010; Mason, Wilking, Meleson, Chang, & Graves, 2007).

54 The aim of this work was to analyze the chemical composition of essential oil of

PT
55 Citrus medica L. var. sarcodactylis, prepare its nanoemulsion, as well as compare the

RI
56 antioxidant, antibacterial, antibiofilm activity of pure and nanoemulsified essential oil.

SC
57 Furthermore, the application of the essential oils in tofu would be investigated.

58 2 Materials and Methods

59 2.1 Materials
U
AN
60 The fruits (Citrus medica L. var. sarcodactylis) were provided by Zhejiang
M

61 golden hand Biological Technology Co., Ltd. The strains S. aureus ATCC 25923 and

E. coli ATCC 25922 were provided by China General Microbiological Culture

D

62
TE

63 Collection Center (Beijing, China).

64 2.2 Preparation of essential oil

EP

65 Fresh Citrus medica L. var. sarcodactylis (500 g) was ground in a blender. The
C

66 essential oils were obtained by hydrodistillation at 100℃ with a Clevenger-type

AC

67 apparatus. The essential oils obtained were separated from water and dried over

68 anhydrous Na2SO4, and stored at 4 ℃.

69 2.3 Chemical composition analysis

70 The components of the essential oil were identified by GC–MS analyses (Li et al.,

71 2015; Wianowska & Dawidowicz, 2016). Gas chromatography-mass spectrometry

4
 ACCEPTED MANUSCRIPT

72 (Varian 1200L) was incorporated with a relatively nonpolar capillary column (DB-5,

73 30 m length, 0.25 mm film thickness, 0.25 mm internal diameter). The injection port

74 and the interface were held at 220 and 260 ℃, respectively. The temperature was

75 programmed from 50℃ to 220℃ at 10℃ per minute and a hold at 220℃for 15 min

PT
76 with helium as the carrier gas. Mass spectra: electronic impact, ionisation potential 70

RI
77 eV, ion source temperature 200 ℃ and mass range 30 Da–500 Da. Most of the

SC
78 compounds were identified according to Kováts Indexes in reference to n-alkanes

79 (NIST, 2010) and mass spectra (authentic chemicals and Wiley spectral library

80 collection).
U
AN
81 2.4 Formation of nanoemulsions
M

82 The preparation ofnanoemulsion was carried out based on the method presented by

Chang (Chang, McLandsborough, & McClements, 2013) with some modifications.

D

83
TE

84 Briefly, an organic phase (containing essential oils, surfactant and medium chain

85 triglycerides, MCT) was added to an aqueous phase which was citrate buffer (5 mM,
EP

86 pH 6.0). Meanwhile, the system was magnetically stirred (500 rpm). First, the oil
C

87 (essential oil and MCT, 10 g) and surfactant (TWEEN 80, 20 g) were mixed together.
AC

88 Then, the mixture of oil and surfactant was titrated into 70 g of aqueous phase at 2

89 mL/min. The mean particle diameters of nanoemulsions were measured using

90 dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK). The

91 nanoemulsions were stored at room temperature for its intrinsic stability and observed

92 for phase separation or creaming.

5
 ACCEPTED MANUSCRIPT

93 2.5 Iron reducing power

94 The capacity of essential oils to reduce Fe3+ was assessed according to a reported

95 method (Bolanos de la Torre, Henderson, Nigam, & Owusu-Apenten, 2015; Zhang,

96 Shen, Zhu, & Xu, 2015).The essential oils were mixed with 2.5 ml of PBS buffer (pH

PT
97 6.5, 0.2 M) and 2.5 ml of potassium ferricyanide (1%). This mixture was incubated at

RI
98 50℃for 20 min. After the addition of trichloroacetic acid (2.5 ml, 10%), the new

SC
99 mixture was centrifuged for 10 min at 650×g. Then, the upper layer (2.5 ml) was

100 mixed with deionized water (2.5 ml) and ferric chloride (0.5 ml). The absorbance was

101
U
measured at 700 nm. A higher absorbance indicates a higher reducing power.
AN
102 2.6 Hydroxyl radicals scavenging assay
M

103 The hydroxyl radicals scavenging activity was measured according to the method

of Tsai et al. (Tsai, Stern, Chiou, Chern, & Liu, 2001) and Sheih et al. (Sheih, Wu, &
D

104
TE

105 Fang, 2009). The generating system of hydroxyl radicals was based on the Fenton

106 reaction (Fe2+ + H2O2), so the reaction mixture included 1.0 ml of 3µM indoxyl-β
EP

107 -glucuronide (IBG), 0.1 ml of 1.0 mM FeSO4, 1.6 ml of 3% H2O2, 0.05 ml of 10 mM

C

108 EDTA. The photons were measured using chemiluminescence instrument (Xi’an
AC

109 ruimai Co., Ltd., China). A 10 µL of essential oil sample was added to the reaction

110 mixture, and the degree of reduction of the chemiluminescence (CL) counts

111 represented the scavenging capabilities on the hydroxyl radicals. The experiments

112 were carried out in triplicates.

113 2.7 The 1,1-Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay

6
 ACCEPTED MANUSCRIPT

114 The DPPH radical-scavenging activity of essential oils was evaluated by a

115 reported. method (Parvathy, Negi, & Srinivas, 2009). The samples were assayed at

116 different concentrations, making them up to 1 mL with 50 mM Tris-HCl buffer (pH

117 7.4), followed by addition of 4 mL of 2,2-diphenyl-1-picrylhydrazyl (0.1 mM solution

PT
118 in ethanol) and mixing of the contents by shaking. A control in these experiments was

RI
119 prepared by the same protocol without the addition of sample. The tubes were

SC
120 incubated for 20 min in the dark. The absorbance was then measured at 517 nm. The

121 radical-scavenging activity was calculated using the following formula:

122
U
Radical-scavenging activity (%) = (Control OD -Sample OD)/Control OD×100 %.
AN
123 The experiments were carried out in triplicates.
M

124 2.8 Antibacterial activity

The antibacterial assay of the essential oils was tested by the pour plate method
D

125
TE

126 according to the reports of Parvathy et al. (Parvathy et al., 2009). To flasks containing

127 18 ml of melted agar, different concentrations of test material were added. Equivalent
EP

128 amounts of nanoemulsion without the essential oil were used as controls. One

hundred microlitre (about 105 cfu/ml) of culture were inoculated into the flasks under
C

129
AC

130 aseptic conditions. The media were then poured into sterilised Petri plates and

131 incubated at 37℃ for 26 h. The inhibitory effect of the essential oils was calculated

132 using the following formula: Inhibition (%) =(1-T/C)×100%. Where C is cfu/ml of

133 control and T is cfu/ml of test sample. The minimum inhibitory concentration (MIC)

134 was reported as the lowest concentration of the essential oil capable of inhibiting the

7
 ACCEPTED MANUSCRIPT

135 complete growth of the bacterium tested.

136 2.9 Effect on biofilm formation

137 The anti-biofilm activity of essential oils against pathogens was studied using the

138 method of crystal violet staining (Carvalho et al., 2012; Yuhua Chang, Weimin Gu, &

PT
139 Lynne McLandsborough, 2012; Quave, Plano, Pantuso, & Bennett, 2008; Schito et al.,

RI
140 2011). Serial dilutions of essential oil were added to the wells of a 24-well culture

SC
141 plate (Sarstedt, Newton, NC) containing 800µl of fresh broth medium and 100µl

142 bacteria suspension (108 cfu/ml). After incubation for 24 h, growth medium was

143
U
removed. The contents of the wells were rinsed 3 times with PBS, fixed by drying for
AN
144 2 h at 37℃. Once the wells were fully dried, 1 ml of 0.1% crystal violet stain was
M

145 added into wells to stain for 15 min. The excess stain was rinsed off with water and

then 1.0 ml of 95% (v/v) ethanol was added to each well to release the stain from the
D

146
TE

147 biofilm. One hundred microlitres from each well was then transferred to a new plate

148 for spectrophotometric analysis (OD570nm). For all the assays, a positive control
EP

149 without essential oil and a negative control without inoculation were prepared. The
C

150 procedure was performed in triplicates.

AC

151 2.10 The application of essential oils in tofu

152 Fresh tofu was bought from a local market in Wuxi. The tofu was cut to 15 g

153 portions and sterilized. The portions were randomly divided into three groups, control

154 group and two groups with treatment of essential oils. Three replicates were

155 performed. The tofu of the control group was dipped in sterile distilled water for 2

8
 ACCEPTED MANUSCRIPT

156 min. The treated groups were immersed into 2.0 mg/ml of pure essential oil or

157 nanoemulsified essential oil for 2 min. Then the tofu samples were taken out and

158 dried for 5 min on the asepsis work table (Shanghai Sujing Co., Ltd., Shanghai).

159 Eighty microliter of bacterial suspension (105cfu/ml) was inoculated onto each portion

PT
160 of tofu. Each tofu portion was put in an individual culture dish, and cultured at 22 ℃.

RI
161 Then, the numbers of viable bacteria were determined according to the method of

SC
162 Makwana et al. (Makwana, Choudhary, Haddock, & Kohli, 2015) in 48 hours.

163 2.11 Statistics analysis

164
U
.All the experiments were carried out in triplicates. The data represents the mean
AN
165 of triplicate values. An analysis of variance was applied using SPSS. Tukey’s test was
M

166 applied to compare the mean values when ANOVA showed significant differences. A

p value of less than 0.05 was considered statistically significant.

D

167
TE

168 3 Results and Discussion

169 3.1 The extraction yield of essential oil from Citrus medica L. var. sarcodactylis
EP

170 The extraction yield of essential oil increased fast during the first 3 hours. The
C

171 yield of essential oil increased from 0.805% to 0.882% when the extraction time
AC

172 increased from 1h to 3h. However, the yield of the essential oil leveled off after 3h.

173 Thus, the optimum extraction time was 3h, and the yield of essential oil was 0.882%.

174 3.2 Chemical composition of the essential oil from Citrus medica L. var.

175 sarcodactylis

9
 ACCEPTED MANUSCRIPT

176 The composition of the essential oil of Citrus medica L. var. sarcodactylis was

177 analyzed by GC–MS. Nineteen compounds in the essential oil were identified.

178 Quantitative and qualitative analytical results were shown in Table 1.

179 Nineteen components have been identified from the essential oil, while the

PT
180 remaining part of the oil has not been identified. The major compounds of the

RI
181 essential oil were D-Limonene (50.04%), ç-Terpinene (32.11%), à-Pinene (2.75) and

SC
182 1,3,6-Octatriene, 3,7-dimethyl-, (Z)- (1.64%).

183 3.3 Preparation of essential oil nanoemulsions

184
U
The nanoemulsion of essential oil of Citrus medica L. var. sarcodactylis was
AN
185 prepared according to the method described in Section 2.4.The influences of the
M

186 composition of oil phase on the initial size of the oil droplets were investigated. The

oil phase which comprised of essential oil and carrier oil (MCT) was mixed prior to
D

187
TE

188 emulsification. The results of Z-average diameter of the nanoemulsion in Figure 1

189 indicated that the oil phase composition had a significant influence on the formation
EP

190 and stability of the essential oil nanoemulsion. As the concentration of essential oil in
C

191 the lipid phase increased, the mean diameter of the droplet firstly decreased and then
AC

192 increased. The smallest droplets (d≈73nm) were obtained when there were 50%

193 (w/w) essential oil and 50% (w/w) MCT in the lipid phase. Thus, the optimum

194 nanoemulsion consisted of 10% (w/w) oil (containing 50% essential oil and 50%

195 MCT, w/w), 20%(w/w) surfactant and 70% (w/w) citrate buffer (5 mM). There are

196 many possible reasons for the effect of essential oil on the diameter of the droplets.

10
 ACCEPTED MANUSCRIPT

197 The properties of oil phase (such as viscosity, interfacial tension and polarity) and the

198 surfactant properties (such as solubility, partitioning and curvature) will be influenced

199 by the composition of oil phase. Thus, the composition of oil phase highly affected

200 the formation of small oil droplets during emulsification.

PT
201 The stability of the essential oil nanoemulsion has also been investigated. It has

RI
202 been found that the nanoemulsion consisted of 10% (w/w) oil, 20% (w/w) surfactant

SC
203 and 70% (w/w) aqueous phase, with an oil phase composition of 50% essential oil and

204 50% MCT, was stable over 30 days. Neither phase separation nor creaming was

205
U
observed. The mean particle size of the essential oil nanoemulsion was 75 nm after 30
AN
206 days, so there was little change in the particle size. And the nanoemulsion was also
M

207 colorless, transparent.

3.4 The antioxidant activity of pure and nanoemulsified essential oils

D

208
TE

209 It has been widely reported that there is close correlation between antioxidant

210 activity and iron reducing power (Bolanos de la Torre et al., 2015;
EP

211 Canabady-Rochelle et al., 2015; Zhang et al., 2015). Thus, the iron reducing power of
C

212 essential oil was measured to evaluate the antioxidant activity. The results were
AC

213 shown in Figure 2. It was found that the reducing power of essential oils was

214 increased with the increase of the concentration of essential oils. After emulsification,

215 the iron reducing power of essential oil was obviously enhanced. At a concentration

216 of 0.48 mg/ml, the iron reducing power of nanoemulsified essential oil was 0.218,

217 while the reducing power of pure essential oil was 0.106. At other concentrations, the

11
 ACCEPTED MANUSCRIPT

218 reducing power of nanoemusified essential oil was also higher than that of pure

219 essential oil.

220 The antioxidant activity of essential oils determined using hydroxyl radicals

221 scavenging assay was shown in Figure 3. The hydroxyl radicals scavenging activity of

PT
222 both kinds of essential oils increased with the increase of their concentrations. At 0.06

RI
223 mg/ml, the radicals scavenging activity of pure essential oil (9.6%) was significantly

SC
224 lower than that of nanoemulsified essential oil (35.7%). At the concentration of 0.48

225 mg/ml, the radicals scavenging activity of nanoemulsified essential oil was 58.7%,

226
U
while the radicals scavenging activity of pure essential oil was only 26.1%.
AN
227 The DPPH radical-scavenging activities of both kinds of essential oils increased
M

228 with the increase of concentration (Fig. 4). At 0.12 mg/mL, scavenging ability of

nanoemulsified essential oil was significantly higher (51.6%) than that of pure
D

229
TE

230 essential oil (30.5%). At 0.48 mg/mL, radical scavenging ability of nanoemulsified

231 and pure essential oil were 72.4% and 44.3%, respectively.
EP

232 The pure essential oil was not solute in aqueous system, which might reduce
C

233 their antioxidant activity. Especially when the essential oils were certainly diluted,
AC

234 they might lose antioxidant activity. However, the essential oil nanoemulsions had an

235 excellent solubility in aqueous system, which were suitable for efficient delivery of

236 active ingredients, and their large surface area enable rapid penetration of active

237 compounds. Therefore, nanoemulsified essential oil can scavenge the radicals more

238 efficiently, and exhibit higher reducing power, thereby enhancing the radicals

12
 ACCEPTED MANUSCRIPT

239 scavenging ability and reducing power. Thus, nanoemulsification significantly

240 improved the antioxidant activity of the essential oil from Citrus medica L. var.

241 sarcodactylis.

242 3.5 The antibacterial activity of essential oils

PT
243 The inhibitive effect of pure essential oil and nanoemulsified essential oil on

RI
244 bacteria was in Figure 5. The results indicated that the inhibition rate of

SC
245 nanoemulsified essential oil was significantly higher than that of pure essential oil. At

246 a concentration of 0.12 mg/ml, the inhibition rate of pure essential oil on S. aureus

247
U
was 40.8%, while the inhibition rate of nanoemulsified essential oil was 80.4%. When
AN
248 the concentration was 0.48 mg/ml, the inhibition rate of nanoemulsified essential oil
M

249 on S. aureus was 100%, so the growth of bacteria was completely inhibited, and the

MIC of nanoemulsified essential oil against S. aureus was 0.48mg/ml. At the same
D

250
TE

251 concentration, the inhibition rare of pure essential oil was only 67.2%. As shown in

252 Figure 5 b, the inhibition rate of nanoemulsified essential oil against E. coli was also
EP

253 significantly higher than that of pure essential oil.

C

254 3.6 The effect of essential oils on the formation of biofilm

AC

255 The effect of two kinds of essential oils on the formation of biofilm of S. aureus

256 was shown in Figure 6. The results showed that both kinds of essential oils exhibited

257 inhibitive effect on the formation of biofilm. The inhibitive effect of nanoemulsified

258 essential oil was significantly higher than that of pure essential oil. At a concentration

259 of 0.24mg/ml, the inhibition rate of pure essential oil on biofilm formation was 55.6%,

13
 ACCEPTED MANUSCRIPT

260 while the inhibition rate of nanoemulsified essential oil was 91.5%. At 0.48 mg/ml,

261 the inhibition rate of nanoemulsified essential oil was 100%, thus the formation of

262 biofilm was completely inhibited. Inhibition of adherence is the first important step

263 for the prevention of biofilm formation (Y. Chang, W. Gu, & L. McLandsborough,

PT
264 2012). The anti-biofilm ability of the essential oil nanoemulsion may be attributed to

RI
265 its inhibition capability on the attachment of bacteria on surfaces.

SC
266 For pathogens, biofilm plays a key role in nullifying the effect of antibacterial

267 and antibiofilm agents. Inhibition on the formation of biofilm was the key step to

268
U
reduce the pathogenic effect of bacteria. The treatment of essential oils, especially the
AN
269 nanoemulsified essential oil, showed significant inhibition on the formation of biofilm.
M

270 Their usage in food industries is highly expected. Hence, food based nanoemulsions

of essential oil may act as a good alternatives to control food-borne pathogens.

D

271
TE

272 3.7 The application of essential oils in tofu

273 The antibacterial activity of pure essential oil and nanoemulsified essential oil
EP

274 against S. aureus in tofu portions was evaluated. For the tofu treated with essential
C

275 oils, it was found that there was no effect on the flavor and color of tofu. Only little
AC

276 fruit aroma was added. The result was shown in Figure 7. It was found that S. aureus

277 was completely inhibited within 48 h by the nanoemulsified essential oil, and there

278 was no bacteria growing from 12 h to 48 h. As for the pure essential oil, the inhibitive

279 effect on S. aureus was much lower. After 48h of treatment, there was 8.4 Log cfu/g

280 in the samples treated with pure essential oil, which was significantly higher (8.4 Log

14
 ACCEPTED MANUSCRIPT

281 cfu/g) than those treated with nanoemulsified essential oil. Compared to the samples

282 treated with the pure essential oil, there was more than 99.99% reduction in

283 population of S. aureus in the samples treated with nanoemulsified essential oil at 48

284 h. The results indicated that nanoemulsified essential oil exhibited a much higher

PT
285 efficiency in the inhibition of S. aureus in tofu, and it could be used to extend the

RI
286 shelf life of tofu. The nanoemulsified essential oil might be a promising antibacterial

SC
287 for tofu preservation.

288 4 Conclusions

289
U
Through GC-MS analysis, nineteen compounds were identified in the essential oil
AN
290 of Citrus medica L. var. sarcodactylis. After the optimization of oil phase
M

291 composition, a stable nanoemulsion of essential oil with minimum droplets was

obtained. The nanoemulsification significantly increased the antioxidant, antibacterial

D

292
TE

293 and antibiofilm activity of essential oil. Moreover, the nanoemulsified essential oil

294 exhibited much higher efficiency in inhibiting the bacteria in tofu than pure essential
EP

295 oil, indicating a better application prospect of essential oil nanoemulsions. Therefore,
C

296 nanoemulsification provides an excellent way to significantly improve the activity and
AC

297 efficiency of liposoluble active agents, and greatly expands their application in

298 various aqueous foods.

299 Acknowledgments

300 The authors gratefully acknowledge the financial support provided by Project

301 31201433 of the national natural Science Foundation of PR China.

15
 ACCEPTED MANUSCRIPT

302 References

303 Adrar, N., Oukil, N., & Bedjou, F. (2016). Antioxidant and antibacterial activities of

304 Thymus numidicus and Salvia officinalis essential oils alone or in combination.

305 Industrial Crops and Products, 88, 112-119.

PT
306 Alviano, W. S., Mendonça-Filho, R. R., Alviano, D. S., Bizzo, H. R., Souto-Padrón, T.,

RI
307 Rodrigues, M. L. Souza, M. M. G. (2005). Antimicrobial activity of Croton

SC
308 cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic

309 microorganisms. Oral Microbiology and Immunology, 20(2), 101-105.

310
U
Bey-Ould Si Said, Z., Haddadi-Guemghar, H., Boulekbache-Makhlouf, L., Rigou, P.,
AN
311 Remini, H., Adjaoud, A., Madani, K. (2016). Essential oils composition,
M

312 antibacterial and antioxidant activities of hydrodistillated extract of Eucalyptus

globulus fruits. Industrial Crops and Products, 89, 167-175.

D

313
TE

314 Bolanos de la Torre, A. A. S., Henderson, T., Nigam, P. S., & Owusu-Apenten, R. K.

315 (2015). A universally calibrated microplate ferric reducing antioxidant power

EP

316 (FRAP) assay for foods and applications to Manuka honey. Food Chemistry,
C

317 174, 119-123.

AC

318 Canabady-Rochelle, L. L. S., Harscoat-Schiavo, C., Kessler, V., Aymes, A., Fournier,

319 F., & Girardet, J.-M. (2015). Determination of reducing power and metal

320 chelating ability of antioxidant peptides: Revisited methods. Food Chemistry,

321 183, 129-135.

322 Carvalho, F. G., Puppin-Rontani, R. M., Fucio, S. B., Negrini Tde, C., Carlo, H. L., &

16
 ACCEPTED MANUSCRIPT

323 Garcia-Godoy, F. (2012). Analysis by confocal laser scanning microscopy of

324 the MDPB bactericidal effect on S. mutans biofilm CLSM analysis of MDPB

325 bactericidal effect on biofilm. J Appl Oral Sci, 20(5), 568-575.

326 Chang, Y., Gu, W., & McLandsborough, L. (2012). Low concentration of

PT
327 ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria

RI
328 monocytogenes by inhibiting its initial adherence. Food Microbiology, 29(1),

SC
329 10-17.

330 Chang, Y., Gu, W., & McLandsborough, L. (2012). Low concentration of

331
U
ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria
AN
332 monocytogenes by inhibiting its initial adherence. Food Microbiol, 29(1),
M

333 10-17.

Chang, Y., McLandsborough, L., & McClements, D. J. (2012). Physical properties

D

334
TE

335 and antimicrobial efficacy of thyme oil nanoemulsions: influence of ripening

336 inhibitors. J Agric Food Chem, 60(48), 12056-12063.

EP

337 Chang, Y., McLandsborough, L., & McClements, D. J. (2013). Physicochemical

C

338 properties and antimicrobial efficacy of carvacrol nanoemulsions formed by

AC

339 spontaneous emulsification. J Agric Food Chem, 61(37), 8906-8913.

340 Hatanaka, J., Chikamori, H., Sato, H., Uchida, S., Debari, K., Onoue, S., & Yamada, S.

341 (2010). Physicochemical and pharmacological characterization of

342 α-tocopherol-loaded nano-emulsion system. International Journal of

343 Pharmaceutics, 396(1–2), 188-193.

17
 ACCEPTED MANUSCRIPT

344 Huang, Q., Yu, H., & Ru, Q. (2010). Bioavailability and delivery of nutraceuticals

345 using nanotechnology. J Food Sci, 75(1), R50-57.

346 Kacem, N., Roumy, V., Duhal, N., Merouane, F., Neut, C., Christen, P., . . . Rhouati, S.

347 (2016). Chemical composition of the essential oil from Algerian Genista

PT
348 quadriflora Munby and determination of its antibacterial and antifungal

RI
349 activities. Industrial Crops and Products, 90, 87-93.

SC
350 Kerekes, E. B., Deak, E., Tako, M., Tserennadmid, R., Petkovits, T., Vagvolgyi, C., &

351 Krisch, J. (2013). Anti-biofilm forming and anti-quorum sensing activity of

352
U
selected essential oils and their main components on food-related
AN
353 micro-organisms. J Appl Microbiol, 115(4), 933-942.
M

354 Li, B., Zhang, C., Peng, L., Liang, Z., Yan, X., Zhu, Y., & Liu, Y. (2015). Comparison

of essential oil composition and phenolic acid content of selected Salvia

D

355
TE

356 species measured by GC–MS and HPLC methods. Industrial Crops and

357 Products, 69, 329-334.

EP

358 Makwana, S., Choudhary, R., Haddock, J., & Kohli, P. (2015). In-vitro antibacterial
C

359 activity of plant based phenolic compounds for food safety and preservation.
AC

360 LWT - Food Science and Technology, 62(2), 935-939.

361 Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B., & Graves, S. M. (2007).

362 Nanoemulsions: formation, structure, and physical properties. Journal of

363 Physics: Condensed Matter, 19(7), 079001.

364 Omar, E., Pavlović, I., Drobac, M., Radenković, M., Branković, S., & Kovačević, N.

18
 ACCEPTED MANUSCRIPT

365 (2016). Chemical composition and spasmolytic activity of Cymbopogon

366 nervatus (Hochst.) Chiov. (Poaceae) essential oil. Industrial Crops and

367 Products, 91, 249-254.

368 Parvathy, K. S., Negi, P. S., & Srinivas, P. (2009). Antioxidant, antimutagenic and

PT
369 antibacterial activities of curcumin-[beta]-diglucoside. Food Chemistry, 115(1),

RI
370 265-271.

SC
371 Peng, C. H., Ker, Y. B., Weng, C. F., Peng, C. C., Huang, C. N., Lin, L. Y., & Peng, R.

372 Y. (2009). Insulin secretagogue bioactivity of finger citron fruit (Citrus medica

373
U
L. var. Sarcodactylis Hort, Rutaceae). J Agric Food Chem, 57(19), 8812-8819.
AN
374 Quave, C. L., Plano, L. R. W., Pantuso, T., & Bennett, B. C. (2008). Effects of extracts
M

375 from Italian medicinal plants on planktonic growth, biofilm formation and

adherence of methicillin-resistant Staphylococcus aureus. Journal of

D

376
TE

377 Ethnopharmacology, 118(3), 418-428.

378 Schito, A. M., Piatti, G., Stauder, M., Bisio, A., Giacomelli, E., Romussi, G., &
EP

379 Pruzzo, C. (2011). Effects of demethylfruticuline A and fruticuline A from

C

380 Salvia corrugata Vahl. on biofilm production in vitro by multiresistant strains

AC

381 of Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus

382 faecalis. International Journal of Antimicrobial Agents, 37(2), 129-134.

383 Sheih, I. C., Wu, T.-K., & Fang, T. J. (2009). Antioxidant properties of a new

384 antioxidative peptide from algae protein waste hydrolysate in different

385 oxidation systems. Bioresource Technology, 100(13), 3419-3425.

19
 ACCEPTED MANUSCRIPT

386 Tsai, C.-H., Stern, A., Chiou, J.-F., Chern, C.-L., & Liu, T.-Z. (2001). Rapid and

387 Specific Detection of Hydroxyl Radical Using an Ultraweak

388 Chemiluminescence Analyzer and a Low-Level Chemiluminescence Emitter:

389 Application to Hydroxyl Radical-Scavenging Ability of Aqueous Extracts of

PT
390 Food Constituents. Journal of Agricultural and Food Chemistry, 49(5),

RI
391 2137-2141.

SC
392 Wianowska, D., & Dawidowicz, A. L. (2016). Can matrix solid phase dispersion

393 (MSPD) be more simplified? Application of solventless MSPD sample

394
U
preparation method for GC–MS and GC–FID analysis of plant essential oil
AN
395 components. Talanta, 151, 179-182.
M

396 Wu, Z., Li, H., Yang, Y., Zhan, Y., & Tu, D. (2013). Variation in the components and

antioxidant activity of Citrus medica L. var. sarcodactylis essential oils at

D

397
TE

398 different stages of maturity. Industrial Crops and Products, 46, 311-316.

399 Xu, S. X., Li, Y. C., Liu, X., Mao, L. J., Zhang, H., & Zheng, X. D. (2012). In vitro
EP

400 and in vivo antifungal activity of a water-dilutable cassia oil microemulsion

C

401 against Geotrichum citri-aurantii. J Sci Food Agric, 92(13), 2668-2671.

AC

402 Zhang, Y., Shen, Y., Zhu, Y., & Xu, Z. (2015). Assessment of the correlations between

403 reducing power, scavenging DPPH activity and anti-lipid-oxidation capability

404 of phenolic antioxidants. LWT - Food Science and Technology, 63(1), 569-574.

405 Ziani, K., Chang, Y., McLandsborough, L., & McClements, D. J. (2011). Influence of

406 surfactant charge on antimicrobial efficacy of surfactant-stabilized thyme oil

20
 ACCEPTED MANUSCRIPT

407 nanoemulsions. J Agric Food Chem, 59(11), 6247-6255.

408

409

410

PT
RI
U SC
AN
M
D
TE
C EP
AC

21
 ACCEPTED MANUSCRIPT

411 Table 1

412 The main components and their relative contents (%) of essential oil of Citrus medica

413 L. var. sarcodactylis

RT Peak Area Identification

Peak

PT
(min) Area (%) of the compounds

1 4.07 1.598E06 0.65 Trifluoroacetic Acid, TBDMS derivative

RI
2 4.32 1.146E06 0.47 Cyclotrisiloxane, hexamethyl-

3 6.18 2.866E06 1.17 à-Phellandrene

SC
4 6.32 6.727E06 2.75 à-Pinene

5 7.01 8.134E05 0.33 3-Carene

U Bicyclo[3.1.1]heptane,
AN
6 7.08 5.943E06 2.43
6,6-dimethyl-2-methylene-, (1S)-

Bicyclo[3.1.1]heptane,
M

7 7.29 2.976E06 1.22

6,6-dimethyl-2-methylene-, (1S)-
D

8 7.75 1.753E06 0.72 1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)-

9 7.95 1.224E08 50.04 D-Limonene

TE

10 8.07 3.983E06 1.63 (1R)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene

11 8.25 4.003E06 1.64 1,3,6-Octatriene, 3,7-dimethyl-, (Z)-

EP

12 8.45 7.855E07 32.11 ç-Terpinene

13 8.96 3.191E06 1.30 Cyclohexene, 1-methyl-4-(1-methylethylidene)-

C

14 14.69 1.503E06 0.61 Germacrene D

AC

Androst-5-ene-3,17-diol, 17-methyl-, diacetate,

15 14.96 1.241E06 0.51
(3á,17á)-

16 17.17 1.250E06 0.51 Glycocholic acid

2-Methyl-3,5-dinitrobenzyl alcohol, TBDMS

17 17.28 1.513E06 0.62
derivative

18 17.34 1.059E06 0.43 Fluoren-9-ol, 3,6-dimethoxy-9-(2-phenylethenyl)-

22
 ACCEPTED MANUSCRIPT

19 18.03 1.236E06 0.51 Methyl cholate

414

415

PT
RI
U SC
AN
M
D
TE
C EP
AC

23
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
416

417 Figure 1 The effect of oil phase composition (the percentage of essential oil in oil
M

418 phase) on mean particle diameter of emulsions and nanoemulsions. Emulsions and
D

419 nanoemulsions were parepared using 10% (w/w) oil (containing essential oil and
TE

420 MCT, w/w), 20% (w/w) surfactant and 70% (w/w) aqueous phase (citrate buffer, 5
EP

421 mM).

422
C

423
AC

24
 ACCEPTED MANUSCRIPT

424

PT
RI
U SC
AN
425
M

426 Figure 2 The iron reducing power of pure essential oil and nanoemulsified essential
D

427 oil. Values were presented as means ± standard deviation.

TE

428
EP

429
C
AC

25
 ACCEPTED MANUSCRIPT

430

PT
RI
U SC
AN
431
M

432 Figure 3 The hydroxyl radicals scavenging activity of pure and nanoemulsified
D

433 essential oils. Values were presented as means ± standard deviation.

TE

434
EP

435
C
AC

26
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
436

437 Figure 4 The DPPH radicals scavenging ability of pure and nanoemulsified essential
M

438 oils. Values were presented as means ± standard deviation.

D

439
TE
C EP
AC

27
 ACCEPTED MANUSCRIPT

440

PT
RI
U SC
AN
441
M

442 (a)
D
TE
EP
C
AC

443

444 (b)
28
 ACCEPTED MANUSCRIPT

445 Figure 5 The inhibitive activity of pure and nanoemulsified essential oils on S. aureus

446 (a) and E. coli (b). Values were presented as means ± standard deviation.

447

PT
RI
U SC
AN
M
D
TE
C EP
AC

29
 ACCEPTED MANUSCRIPT

448

PT
RI
U SC
AN
449
M

450 Figure 6 The inhibitive effect of pure and nanoemulsified essential oils on the
D

451 formation of biofilm. Values were presented as means ± standard deviation.

TE

452
C EP
AC

30
 ACCEPTED MANUSCRIPT

453

PT
RI
U SC
AN
454
M

455 Figure 7 The antibacterial activity of pure and nanoemulsified essential oils in tofu.
D

456 Values were presented as means ± standard deviation.

TE
C EP
AC

31
 ACCEPTED MANUSCRIPT

The nanoemulsion of essential oil (EO) was prepared.

Nanoemulsification improved the antioxidant activity of EO.

Nanoemulsification enhanced the antibacterial and antibiofilm ability of EO.

PT
Nanoemulsification enhanced the antibacterial efficiency of EO in tofu.

RI
It introduces an effective way to improve the activity and application of EO.

U SC
AN
M
D
TE
C EP
AC