A peppermint oil emulsion stabilized by resveratrol-zein-pectin complex particles: Enhancing the chemical stability and antimicrobial activity in combination with the synergistic effect

Journal Pre-proof A peppermint oil emulsion stabilized by resveratrol-zein-pectin complex particles: Enhancing the chemical stability and antimicrobial activity in combination with the synergistic effect Hao Cheng, Muhammad Aslam Khan, Zhenfeng Xie, Shengnan Tao, Yunxing Li, Li Liang PII:

S0268-005X(19)31278-0

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105675

Reference:

FOOHYD 105675

To appear in:

Food Hydrocolloids

Received Date: 14 June 2019 Revised Date:

14 January 2020

Accepted Date: 16 January 2020

Please cite this article as: Cheng, H., Khan, M.A., Xie, Z., Tao, S., Li, Y., Liang, L., A peppermint oil emulsion stabilized by resveratrol-zein-pectin complex particles: Enhancing the chemical stability and antimicrobial activity in combination with the synergistic effect, Food Hydrocolloids (2020), doi: https:// doi.org/10.1016/j.foodhyd.2020.105675. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

Hao Cheng: Conceptualization, Investigation, Writing- Original draft preparation, Editing. Muhammad Aslam Khan: Investigation. Zhenfeng Xie: Investigation. Shengnan Tao: Resources. Yunxing Li: Resources. Li Liang: Conceptualization, Resources, Reviewing, Supervision.

A peppermint oil emulsion stabilized by resveratrol-zein-pectin complex

particles:

Enhancing

the

chemical

stability

and

antimicrobial activity in combination with the synergistic effect Hao Cheng1,2, Muhammad Aslam Khan1,2, Zhenfeng Xie1,2, Shengnan Tao3, Yunxing Li3, Li Liang1,2* 1

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

Jiangsu, China 2

School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China

3

Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School

of Chemical and Material Engineering, Jiangnan University, Wuxi, Jiangsu, China

*Corresponding author: Li Liang Address: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China. Telephone: +86(510)8519-7367; Email: [email protected].

1

Abstract The combination of different antimicrobial agents might produce synergistic effects and has gained increasing interest. Interfacial engineering of emulsion systems has been developed to co-encapsulate and protect bioactive components with different solubility. In this study, peppermint oil and resveratrol display synergistic effect against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Salmonella Typhimurium. Partially wettable resveratrol-loaded zein-pectin complex particles with a three-phase contact angle of ~78° were fabricated via a desolvation method. Peppermint oil emulsions with the co-inclusion of resveratrol were successfully prepared by zein-pectin complex particles, showing a high encapsulation efficiency for peppermint oil (~88%) and resveratrol (~99%). Addition of pectin decreased size distribution of the emulsions, improved antimicrobial activity, physical and chemical stability and prolonged antimicrobial efficiency against Staphylococcus aureus and Salmonella Typhimurium. Overall, the current study may have a valuable contribution to develop an efficient antimicrobial system based on the synergistic effect of combined agents and a single emulsion stabilized by protein-polysaccharide complex particles. Keywords:

Complex

particle,

peppermint

antimicrobial activity, chemical stability

2

oil,

resveratrol,

co-encapsulation,

1

1 Introduction

2

Essential oils (EOs) are naturally-derived aroma compounds obtained from

3

various parts of edible and medicinal plants and exert strong antibacterial and

4

antifungal activity (Donsi & Ferrari, 2016; Seow, Yeo, Chung, & Yuk, 2014). When

5

two or more agents work together, synergism occurs to produce an effect greater than

6

the sum of individual effects, due to their function on one or more different targets in

7

a metabolic pathway (Seow, et al., 2014). For example, the combination of

8

cinnamaldehyde with carvacrol showed synergistic antibacterial effects against both

9

Escherichia coli and Staphylococcus aureus (S. aureus) (Ye, et al., 2013). A

10

synergistic antibacterial activity against S. aureus was also observed in nisin

11

combined with cinnamaldehyde in pasteurized milk (Shi, et al., 2017). The

12

combination of multiple antibacterial agents has become an important approach to

13

enhance the efficiency of antibacterial therapy and overcome resistance to

14

antibacterial agents. However, poor hydro-solubility and high volatility of EOs limit

15

their application in the pharmaceutical, food and cosmetic industries. It is thus

16

necessary to develop the carriers not only to overcome the limitations but also to

17

enhance the antibacterial activity based on the synergistic effect.

18

Oil-in-water (O/W) emulsions have been considered to be efficient delivery

19

systems for improving water dispersibility of EOs and preventing their interactions

20

with other food ingredients (Donsi, et al., 2016; McClements & Li, 2010). However,

21

the oxidative and physical stability of conventional emulsions is limited due to the

3

22

high interfacial area and a characteristic porous thin interfacial layer (Berton-Carabin,

23

Sagis, & Schroen, 2018; McClements & Decker, 2018). Recently, interfacial

24

engineering of emulsion systems has been developed to improve the oxidative

25

stability by minimizing interactions between pro-oxidants and bioactive lipids

26

(Berton-Carabin, Ropers, & Genot, 2014; McClements & Decker, 2018). A safflower

27

oil emulsion stabilized by lipid droplets coated by milk protein concentrate (MPC)

28

was prepared, showing slower lipid oxidation than conventional emulsions stabilized

29

by MPC alone. (Okubanjo, Loveday, Ye, Wilde, & Singh, 2019). Resveratrol, as an

30

antioxidant co-emulsifier, can be accumulated at the oil-water interface by interacting

31

with proteins to enhance the oxidative stability (Wan, Wang, Wang, Yuan, & Yang,

32

2014; Wang, et al., 2016a). These provide an opportunity not only to improve the

33

oxidative stability of O/W emulsions but also to co-encapsulate bioactive components

34

with different solubility in the single emulsions.

35

Zein, a major storage protein in corn, contains more than 50% hydrophobic

36

amino acid residues and is soluble in concentrated aqueous ethanol solutions (60 ~

37

90%) but not in pure water (Shukla & Cheryan, 2001). This property makes zein a

38

suitable material for the encapsulation of bioactive components, such as α-tocopherol,

39

resveratrol and epigallocatechin gallate (Davidov-Pardo, Joye, & McClements, 2015;

40

Donsi, Voudouris, Veen, & Velikov, 2017; Luo, Zhang, Whent, Yu, & Wang, 2011). A

41

Pickering O/W emulsion was successfully produced by bare zein colloidal particles

42

with droplet size in the range of 10 - 200 µm (de Folter, van Ruijven, & Velikov,

43

2012). However, the resulting emulsions were unstable against coalescence at low pH 4

44

due to the poor wettability of the protein. Surface-modified zein particles with

45

water-soluble biopolymers have been utilized to regulate the surface wettability of

46

zein particles and form stable O/W emulsions (Chen, et al., 2018; Dai, Sun, Wei, Mao,

47

& Gao, 2018; Feng & Lee, 2016). Pectin, an anionic polysaccharide, belongs to a

48

family of heterogeneous polysaccharides containing mainly α-(1→4)-linked partially

49

methyl esterified D-galacturonic acid and rhamnogalacturonan units (Synytsya,

50

Copikova, Matejka, & Machovic, 2003). Zein-pectin core-shell nanoparticles

51

overcome the aggregation problem of bare zein particles and provide better protection

52

for encapsulated molecules than bare zein particles did (Hu, et al., 2015; Huang, et al.,

53

2017). Additionally, high methoxyl pectin could strongly absorb to the interface of

54

mandarin or lemongrass oil emulsion and improve physical stability against Ostwald

55

ripening

56

Martin-Belloso, 2016).

(Guerra-Rosas,

Morales-Castro,

Ochoa-Martinez,

Salvia-Trujillo,

&

57

Peppermint (Mentha piperita) oil is one of the most widely produced and used

58

essential oils in food, flavorings, and pharmaceutical products. Peppermint oil

59

possesses antimicrobial, antiviral and antifungal activities against various types of

60

bacteria and yeasts (Iscan, Kirimer, Kurkcuoglu, Baser, & Demirci, 2002; Mahboubi

61

& Haghi, 2008). Resveratrol, a natural polyphenol, is produced in plants in response

62

to injury and fungal attack (Summerlin, et al., 2015). Resveratrol exhibits a broad

63

spectrum of antimicrobial activity across a wide range of microorganisms (Ma, et al.,

64

2018), mainly due to the generation of reactive oxygen species causing DNA damage

65

(Subramanian,

Soundar,

&

Mangoli,

2016), 5

oxidative

membrane

damage

66

(Subramanian, Goswami, Chakraborty, & Jawali, 2014), and metabolic enzyme

67

inhibition (Dadi, Ahmad, & Ahmad, 2009). In this study, resveratrol-fortified

68

zein-pectin particles were prepared to stabilize a peppermint oil emulsion.

69

Physiochemical property of the colloidal particles and emulsions was characterized.

70

Furthermore, antimicrobial efficiency of peppermint oil and resveratrol combination

71

was evaluated against food-borne pathogens.

72

2 Materials and methods

73

2.1 Materials

74

Zein (~98%) was purchased from J&K Chemical Co., Ltd. (Shanghai, China).

75

Pectin (50~300 kDa, degree of esterification ≥47.9%) and resveratrol (trans-isomer,

76

≥98%) were purchased from Sango Biotech Co. (Shanghai, China). Peppermint (M.

77

piperita) oil was obtained from Shanghai Orinno International Business Co., Ltd.

78

(Shanghai, China). The composition of peppermint oil was reported in Table S1.

79

Menthol (≥98%, GC), menthone (≥97%, GC) and Nile red dye were obtained from

80

Sigma-Aldrich Co. (St. Louis, MO, USA). Other reagents of analytical grade were

81

purchased from SinoPharm CNCM Ltd. (Shanghai, China).

82

2.2 Preparation of resveratrol-loaded zein-pectin complex particles

83

Zein colloidal nanoparticles were prepared following the desolvation method

84

(Zhong & Jin, 2009). Briefly, 5% (w/v) zein and/or resveratrol at various

85

concentrations [0.10%, 0.25% and 0.50% (w/v)] were dissolved in 80% (v/v) ethanol

86

aqueous solution under stirring at 600 rpm for 30 min and then added into the 6

87

anti-solvent (aqueous phase) at a volume ratio of 1:9 under stirring at 1,200 rpm for 5

88

min. Ethanol in the dispersions was removed using a RE-52C rotary evaporator

89

(Shanghai Tianheng Instrument Co. Ltd, Shanghai, China) at 40°C for 30 min. The

90

pH of the resulting dispersion was 4.0(±0.1). Pectin at 0.5% (w/v) was dispersed in

91

Milli-Q water under stirring for 6 h to allow complete hydration. The pH of the

92

polysaccharide solution was adjusted to 4.0 with 0.6 M HCl or NaOH. Then zein (Z)

93

and resveratrol-loaded zein (R/Z) nanoparticle dispersions were poured into the pectin

94

solution under stirring at 700 rpm for 10 min. Freshly-prepared zein-pectin (ZP) and

95

resveratrol-loaded zein-pectin (R/ZP) nanoparticles contained 0.20% (w/v) zein and

96

0.05%, 0.10% and 0.20% (w/v) pectin. The final concentration of resveratrol in R/ZP

97

particles was 0.02% (w/v).

98

2.3 Preparation of peppermint oil emulsions

99

Emulsions were prepared according to our previous method with some

100

modifications (Fan, et al., 2017). Peppermint oil was slowly added to the aqueous

101

phase of Z, R/Z, ZP or R/ZP colloidal particles at pH 4.0 to obtain coarse emulsions

102

with a final weight of 100 g by using a high-speed blender (Ultra Turrax T25, IKA,

103

Germany) operating at 8,000 rpm for 2 min. Oil droplet size was further reduced by

104

passing the coarse emulsions for four times through an ATSAH2100 high-pressure

105

homogenizer (ATS Engineering Ltd., ON, Canada) at a pressure of 25 MPa and 10°C.

106

The final concentration of zein was 0.20% (w/w), while the concentrations of pectin

107

were 0, 0.05%, 0.10% and 0.20% (w/w). The final content of peppermint oil was 5%

108

(w/w). 7

109

2.4 Size and zeta-potential measurements

110

Size distribution and ζ-potential of zein-based colloidal particles and peppermint

111

oil emulsions were analyzed by a NanoBrooker Omni Particle Sizer and ζ-Potential

112

Analyzer (Brookhaven Instruments Ltd, New York, USA) with a He/Ne laser (λ = 633

113

nm). Samples were diluted with water at pH 4.0 by 100 times before measurement at

114

25°C and at a scattering angle of 173°. Size distribution by the intensity and

115

ζ-potential were obtained by using a NNLS mode analysis and the Smoluchowski

116

model, respectively.

117

2.5 Encapsulation efficiency of the whole peppermint oil, menthol, and menthone

118

Fresh emulsions were centrifuged at 13,000 × g for 30 min at 4°C using a 5804 R

119

centrifuge (Eppendorf Co. Ltd, Hamburg, Germany). The amount of the whole

120

peppermint oil, menthol, and menthone in the whole emulsion (Aw) and in the

121

subnatant (Asub) was determined by using a method of liquid-liquid extraction (Donsi,

122

Annunziata, Vincensi, & Ferrari, 2012; Wang, et al., 2016a). In a brief, 1 mL of

123

emulsion or subnatant was mixed with 1 mL ethanol under vortexing for 1 min.

124

Peppermint oil was then extracted by adding 4-mL hexane under vortexing for 90 s

125

followed by centrifuging at 3,500 × g at 4°C for 5 min. The content of the whole

126

peppermint oil in hexane supernatants was determined by a UV-visible

127

spectrophotometer (Shimadzu Co., Tokyo, Japan) based on a standard curve

128

(absorbance at 240 nm) of peppermint oil at 0.025 - 0.250 mg/mL in hexane (Chen &

129

Zhong, 2015). The content of menthol and menthone in hexane supernatants was

8

130

determined by gas chromatograph (Shimadzu Co., Kyoto, Japan) according to the

131

procedure described in section 2.13. Encapsulation efficiency was calculated from the

132

difference between Aw and Asub divided by Aw.

133

2.6 Quantitation of resveratrol using high performance liquid chromatography

134

(HPLC)

135

Exactly 0.2 mL sample was added into 1.6 mL polydatin (0.01 mg/mL, internal

136

standard) in methanol under vortexing and centrifuged at 13,000 × g for 10 min. The

137

methanol extract was passed through a 0.22-µm filter and then analyzed by an HPLC

138

system (Waters, Milford, MA, USA) according to our previous method (Cheng, Fang,

139

Liu, Gao, & Liang, 2018).

140

2.7 Encapsulation efficiency of resveratrol within zein-based nanoparticles and

141

partition of resveratrol within peppermint oil emulsions

142

Freshly-prepared colloidal dispersions were centrifuged at 150,000 × g at 4°C for

143

30 min by using a CP70ME ultra-centrifuge (Hitachi Co. Ltd, Tokyo, Japan) to

144

separate any particles (Fan, et al., 2017). The amounts of resveratrol in the whole

145

dispersions (Rd) and the supernatants (Rsup) were determined by using HPLC.

146

Encapsulation efficiency was calculated from the difference between Rd and Rsup

147

divided by Rd. Partition of resveratrol in emulsions was determined according to the

148

procedure described by Fan et al. (2017). Emulsions were centrifuged at 13,000 × g

149

for 30 min at 4°C. The amount of resveratrol in the whole emulsion (Rw), in the

150

subnatant (Rsub) and in the precipitation (Rp) was determined using HPLC. The 9

151

percentage of resveratrol in the emulsified oil droplets (Pe), encapsulated (Pp) and free

152

(Pf) in the aqueous phase was calculated using the formula as follow,

153

Pe (%) = (Rw - Rsub - Rp)/Rw ×100

(1)

154

Pp (%) = Rp/Rw ×100

(2)

155

Pf (%) = Rsub/Rw ×100

(3)

156

2.8 Determination of zein and pectin at the oil-water interface

157

The content of zein and pectin at the oil-water interface was determined

158

according to the method described by Wan, Wang, Wang, Yang, & Yuan. (2013) and

159

Fan et al. (2017) with some modifications. Freshly-prepared emulsions were

160

centrifuged using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg, Germany) at

161

13,000 × g for 30 min at 4°C. Content of zein and pectin in the subnatant (Csub),

162

precipitation (Cp) and in the whole emulsion (Cw) was determined using the Kjeldahl

163

method and phenol-sulfuric method (Dubois, Gilles, Hamilton, Rebers, & Smith,

164

1956; Ye, Flanagan, & Singh, 2006), respectively. The interfacial protein and

165

polysaccharide percentage (%) was calculated by Eq. (4).

166

167

Percentage % =





× 100

(4)

2.9 Morphology

168

The morphological structure of zein-based particles and emulsions was

169

visualized on a scan electron microscopy (SEM) SIGMA HD (ZEISS, Jena, Germany)

170

or SU8020 (Hitachi, Tokyo, Japan). Zein colloidal particles and emulsions were 10

171

freeze-dried using Benchtop Freeze Dryers (Freezone, 2.5, Labconco, MO, USA).

172

Emulsions were also spray-dried using a commercial Buchi B-290 mini Spray-dryer

173

(Buchi Labortechnik AG, Flawil, Switzerland). The emulsion was fed into the

174

spray-dryer at an inlet temperature of 150℃, aspiration 100% and %pump of 15%,

175

which was equivalent to a mass flow of emulsion of 5.0 mL/min. The outlet

176

temperature was 80℃. The emulsion was sprayed through a nozzle with an inner

177

diameter of 0.7 mm and dried under an airflow of 35 m3/h. Samples were

178

gold-sputtered and observed with magnifications of 50,000× for freeze-dried particles,

179

10,000× for freeze-dried emulsions and 2,000× for spray-dried emulsions.

180

2.10 Wettability measurement

181

The oil-water three-phase contact angle of zein-based particles and pectin was

182

measured using an OCA 15EC (Dataphysics Instruments GmbH, Germany).

183

Freeze-dried powders of zein-based particles and pectin were compressed into thin

184

tablets, which were then immersed in peppermint oil. A droplet (2 µL) of Milli-Q

185

water was placed on the surface of the tablets. After equilibrium was reached, the

186

shape of droplets was recorded by a camera, and contact angles were calculated using

187

the instrumental software based on the LaPlace-Young equation (Dai, et al., 2018).

188

2.11 Fluorescence microscopy

189

Fluorescence microscopy images of emulsions were recorded on a Zeiss Axio

190

Vert.A1 inverted microscope (Leica, Heidelberg, Germany) at a magnification of 40×.

191

Exactly 20 µL of Nile red (1 mg/mL) was added into 1 mL of emulsions in order to 11

192

stain the oil phase. The stained emulsions were placed on concave slides and covered

193

with coverslips without trapping air bubbles. Nile red was excited at 634 nm.

194

2.12 Antimicrobial assays

195

2.12.1 Microorganisms and growth conditions

196

The Gram-positive bacteria Staphylococcus aureus (S. aureus) CGMCC 1.1861

197

and the Gram-negative bacterial Salmonella Typhimurium (S. Typhimurium) CMCC

198

50115 were obtained from China General Microbiological Culture Collection Center

199

(Beijing, China) and National Center Medical Culture Collections (Beijing China),

200

respectively. Both of the strains were kept at 4°C on their appropriate slant. S. aureus

201

and S. Typhimurium were incubated in nutrient broth (beef extract 5 g/L, NaCl 5 g/L,

202

peptone 10 g/L, pH7.4) at 37°C for 20 h, in BPY broth (beef extract 5 g/L, NaCl 5 g/L,

203

yeast extract 5 g/L, glucose 5 g/L, peptone 10 g/L, pH7.0) at 37°C for 10 h,

204

respectively, to retain early stationary growth phase. The culture solutions with a final

205

cell concentration of ~108 CFU/mL for S. aureus and ~109 CFU/mL for S.

206

Typhimurium were used as working solutions.

207

2.12.2 Determination of minimal inhibitory concentration

208

The minimal inhibitory concentration (MIC) values of peppermint oil, resveratrol,

209

zein particles, and pectin were determined by a broth dilution method as described in

210

previous studies with some modifications (Shi, et al., 2017; Zhao, et al., 2014). A 100

211

µL of antimicrobial agents after being serially diluted was added in each tube

212

containing 9.8 mL of broth. A 100-µL suspension of tested microorganism was added 12

213

to each tube with a final concentration of 1 × 105 CFU/mL and incubated at 37°C for

214

24 h. The MIC was defined as the lowest concentration of antimicrobials that

215

inhibited > 90% of the microorganism's growth by visual reading and optical density

216

(OD) at 600 nm using a UV-1800 UV-Vis spectrophotometer (Shimadzu Co., Tokyo,

217

Japan) (Andrews, 2001; Oo, Cole, Garthwaite, Willcox, & Zhu, 2010).

218

2.12.3 Checkerboard synergy testing.

219

In vitro interactive inhibition between resveratrol and peppermint oil was

220

measured with the broth dilution checkerboard assay with some modifications (Oo, et

221

al., 2010; Shi, et al., 2017). Briefly, peppermint oil was diluted two-fold in vertical

222

orientation, while resveratrol was diluted two-fold in horizontal orientation. Their

223

concentrations correspond to 1/2, 1/4 and 1/8 of the MIC values, respectively.

224

Subsequently, 100-µL suspension of the indicator strain was added to each tube with a

225

final concentration of 1 × 105 CFU/mL. The inoculated tubes were incubated

226

overnight at 37°C for the evaluation of microbial growth. Fractional inhibitory

227

concentration index (FICI) was then calculated to assess the antimicrobial effects of

228

combinations. FICI was calculated using the following equations:

229

FICI = FICIA + FICIB

(5)

230

FICIA = MICA of the combination/MICA alone

(6)

231

FICIB = MICB of the combination/MICB alone

(7)

232

The results were interpreted as synergistic effects (FICI < 0.9), addictive effects (0.9 <

233

FICI < 1.1), and antagonistic effects (FICI > 1.1) (Romano, Abadi, Repetto, Vojnov, 13

234

& Moreno, 2009; Santiesteban-Lopez, Palou, & Lopez-Malo, 2007).

235

2.12.4 Antimicrobial activity during storage

236

Peppermint oil emulsions were stored in an incubator at 25°C and antimicrobial

237

activity was characterized by standard plate count method after storage for 7, 14, 28

238

and 42 days (Donsi, et al., 2012). In brief, 100-µL emulsion was added into 9.8 mL

239

0.85% physical saline and then mixed with 100 µL of culture solution containing 1 ×

240

104 ~ 1 × 108 CFU/ml S. aureus or S. Typhimurium. Exactly 1.0 mL of the mixture

241

was plated onto nutrient agar and BPY agar for S. aureus or S. Typhimurium,

242

respectively, and incubated at 37°C for 24 h for enumeration. Antimicrobial activity of

243

emulsions was calculated by the following equation:

244

Reduction = log (N/N0) log CFU/mL

(8)

245

Where, N0 and N were the initial and final viable colony counts, respectively.

246

2.13 Quantitation of menthol and menthone using gas chromatography

247

Menthol and menthone in peppermint oil emulsion during storage were

248

determined by using Shimadzu capillary gas chromatograph model GC-2010 (Kyoto,

249

Japan) fitted with a Rtx-Wax silica capillary column (30 m × 0.32 mm, 0.25 µm film

250

thickness) and a flame ionization detector (FID). Exactly 1.0 mL of the emulsion was

251

mixed with 1 mL ethanol under vortexing. Exactly 4-mL hexane was added to extract

252

menthol and menthone. After centrifuging at 3,500 × g and 4°C for 5 min, the

253

supernatants were collected and passed through a 0.22-µm filter. The temperature

254

program began with 5 min at 40°C and ramped at 10°C/5 min to 150°C. The injector 14

255

temperature was 200°C, injection volume was 1 µL，injection time was 0.04 min, split

256

mode (1:1) was used, and the detector temperature was 250°C. The carrier gas was

257

nitrogen at a flow rate of 1.5 mL/min and the assisting gas (air) flow rate was 400

258

mL/min. Quantification of menthol and menthone was based on their standard curve.

259

2.14 Chemical stability during storage

260

Samples were stored in an incubator at 25°C and analyzed after storage for 7, 14,

261

28 and 42 days. The retention of resveratrol, menthol, and menthone in the whole

262

emulsion during storage was expressed as a percentage relative to the initial

263

concentration.

264

2.15 Statistical analysis

265

All the measurements were conducted at least in triplicate and data were

266

presented as mean ± standard deviation (SD). The significant differences were

267

evaluated by the one-way analysis of variance (ANOVA), and the subsequent

268

Duncan’s test was set at the 5% level (p < 0.05) to compare the means (SPSS 20.0

269

statistical software, SPSS Inc., Chicago, USA).

270

3 Results and discussion

271

3.1 Characterization of resveratrol/zein-pectin complex particles

272

3.1.1 Size and ζ-potential

273

The size distribution of bare zein particles had a peak around 80 nm (Fig. S1A),

274

which was consistent with a previous report that zein nanoparticles prepared by 15

275

desolvation method had an average size of 50 - 200 nm (Kasaai, 2018). The presence

276

of resveratrol at 0.004%, 0.010%, and 0.020% had no impact on the size of zein

277

particles (Fig. S1A). However, a further increase of resveratrol concentration to 0.040%

278

resulted in precipitation to naked eyes. The polyphenol concentration of 0.020% was

279

thus used for further study. The size of the resveratrol-loaded zein particles gradually

280

increased from about 140 to 240 nm as the concentration of pectin increased from

281

0.05 to 0.20% (Fig. 1A).

282

Zein has an isoelectric point around 6.2 (Shukla, et al., 2001). The ζ-potential of

283

bare zein particles was about +33 mV at pH 4.0 (Fig. S1B). The ζ-potential was

284

similar in the absence and presence of resveratrol. Together with size distribution (Fig.

285

S1A), these results suggest that the polyphenol did not affect the formation and

286

surface property of zein particles. The carboxyl groups on pectin have a dissociation

287

constant (pKa) around 3.5 (Jones, Lesmes, Dubin, & McClements, 2010). The

288

ζ-potential of resveratrol-loaded zein particles changed to -27 mV upon addition of

289

0.05% pectin (Fig. 1B). Absolute values of ζ-potential increased as the polysaccharide

290

concentration increased, reaching a constant of -35 mV at 0.10% and 0.20% pectin.

291

These results suggest that anionic pectin molecules absorbed by electrostatic

292

attraction onto the surface of cationic zein nanoparticles to form core-shell particles

293

(Hu, et al., 2015). The formation of pectin shell structure contributes to the increase in

294

the zein particle size (Fig. 1A).

295

3.1.2 Morphological observation

16

296

Resveratrol-loaded zein particles were observed as tightly packing of spherical

297

particles with the diameter smaller than 100 nm (Fig. 2A). Zein particles coated with

298

pectin at 0.05% also produced spherical particles (Fig. 2B). However, irregular

299

geometry shape particles were observed at the pectin concentrations of 0.10% and

300

0.20% (Fig. 2C and D). It has been reported that zein particles coated by gum Arabic

301

(GA) showed an irregular geometry shape and larger size than zein particles and was

302

hard to see individual particles (Dai, et al., 2018). The SEM images (Fig. 2)

303

confirmed the results of size distribution based on dynamic light scattering (Fig. 1A).

304

3.1.3 Wettability

305

The intermediate wettability [oil-water three-phase contact angles (θow) close to

306

90°] of particulate emulsifier is necessary to stabilize O/W emulsion against

307

coalescence. The wetting property of zein-based particles was detected through

308

investigating the θow of particle tablets immersed in peppermint oil (Fig. 3). The θow of

309

bare zein particles was 132.07°, which was similar with the θow ~ 134° for

310

medium-chain triglyceride oil (Dai, et al., 2018) but different from the θow ~ 107° for

311

corn oil (Zou, Guo, Yin, Wang, & Yang, 2015). The presence of resveratrol had no

312

impact on the wettability of the particles (Fig. 3). In the presence of 0.05% pectin,

313

R/ZP particles had near-neutral wettability (θow ~ 78.30°), suggesting that the particles

314

could favor interfacial particle adsorption and formation of O/W Pickering emulsion

315

(Wang, et al., 2016b). Further increase in the pectin concentration resulted in a

316

gradual decrease in the θow of R/ZP particles, with 68.27° and 63.53° at the

317

polysaccharide concentrations of 0.10% and 0.20%, respectively. These results proved 17

318

that the surface coating with hydrophilic pectin (θow ~ 58.65°) through electrostatic

319

interaction can tune the wettability of R/ZP particles (Zhou, et al., 2018).

320

3.1.4 Encapsulation efficiency of resveratrol

321

The encapsulation efficiency of resveratrol in zein particles was ~70%, which

322

was independent of the polyphenol concentrations (Table 1). The similar result was

323

previously observed for the encapsulation of resveratrol in sodium caseinate-coated

324

zein particles (Davidov-Pardo, et al., 2015). The encapsulation efficiency of

325

resveratrol was about 74% in zein-pectin complex particles (Table 1). The increase in

326

the encapsulation efficiency might be due to the fact that pectin could interact with

327

resveratrol through hydrogen bonding (Buchweitz, Speth, Kammerer, & Carle, 2013;

328

Cheng, et al., 2018; Davidov-Pardo, et al., 2015). Loading efficiency of resveratrol

329

was greater in β-lactoglobulin-pectin complex particles than in β-lactoglobulin alone

330

(Cheng, et al., 2018).

331

3.2 Particle-stabilized peppermint oil emulsions

332

3.2.1 Emulsion formation

333

In the emulsions stabilized by resveratrol-loaded zein and zein-pectin particles,

334

peppermint oil droplets were visualized using an inverted fluorescence microscope. In

335

the emulsions stabilized by R/Z particles, both large (~1 - 2 µm) and small (< 0.5 µm)

336

oil droplets were observed and some droplets aggregated together (Fig. 4A). The

337

percentage of zein adsorbed at the surface of peppermint oil droplets was only 45%

338

(Fig. 5). The emulsions in the presence of pectin at 0.05% showed small (< 0.5 µm) 18

339

and homogeneous oil droplets with no sign of flocculation (Fig. 4B). The interfacial

340

protein percentage increased to 73%, while the interfacial pectin percentage was 21%

341

(Fig. 5). These results might be attributed to the fact that the near-neutral wettability

342

(Fig. 3) could promote R/ZP particles to absorb on the oil-water interface against

343

aggregation and coalescence by electrostatic repulsion and steric hindrance (Dai, et al.,

344

2018; Feng & Lee, 2016). Most oil droplets were smaller than 0.2 µm at higher pectin

345

concentrations (Fig. 4C and D). The sum of the interfacial protein and polysaccharide

346

percentages kept constant as the pectin concentration increased (Fig. 5). However, the

347

interfacial protein percentage decreased to 70% and 64%, while the interfacial pectin

348

percentage increased to 24% and 30% at 0.10% and 0.20% pectin, respectively. In the

349

case of canola oil emulsion stabilized by zein-sodium caseinate (SC) particles, surface

350

protein concentration decreased when the zein/SC mass ratio decreased from 10:2 to

351

10:4, due to that the adsorption of SC occupied the interface and hindered the further

352

adsorption of zein-SC particles (Feng & Lee, 2016). Methyl groups and/or acetyl

353

groups in pectin molecules enhance the hydrophobic nature, make it exhibit surface

354

activity (Dickinson, 2009). Competitive adsorption between zein-pectin particles and

355

excessive pectin molecules occurred when the polysaccharide concentration was

356

above 0.10% (Fig. 5), contributing to a zein-pectin particles and pectin molecules

357

mixed layer formation.

358

3.2.2 Emulsion characterization

359

3.2.2.1 Size distribution and ζ-potential

19

360

The emulsions stabilized by resveratrol-loaded zein particles were distributed in

361

three peaks around 80, 434 and 1,890 nm (Fig. 6A). The inclusion of resveratrol had

362

no impact on the emulsion size distribution (Fig. S2). After centrifugation, size

363

distribution in the subnatant was similar to that of corresponding colloidal particles

364

(Fig S1A) and the smallest peak of corresponding emulsions (Fig. S2). These results

365

indicate that the peak around 80 nm was attributed to zein-based colloidal particles,

366

while the larger peaks were for the particle-stabilized peppermint oil droplets. Similar

367

results were observed in sunflower oil emulsions stabilized by whey protein microgel

368

particles (Fan, et al., 2017; Sarkar, et al., 2016). Addition of pectin resulted in a

369

uniform size distribution around 709, 583 and 529 nm at the polysaccharide

370

concentrations of 0.05%, 0.10%, and 0.20% pectin, respectively (Fig. 6A), which

371

shows a similar trend with the inverted fluorescence microscope (Fig. 4). The sizes of

372

the emulsified oil droplets are approximately equal to the sum of two-fold zein-pectin

373

particle size (Figs. 1 and 2) and oil droplet size (Fig. 4). At pH 4.0, ζ-potential of

374

freshly-prepared emulsions stabilized by R/Z particles was +61 mV and changed to

375

-38, -41 and -44 mV in the presence of 0.05%, 0.10% and 0.20% pectin (Fig. 6B).

376

3.2.2.2 Morphology

377

Surface morphology of freeze-dried and spray-dried powders was observed using

378

SEM (Fig. 7). The zein-stabilized emulsions after freeze-drying showed irregular

379

shape with a porous network structure, and zein particles agglomerated and formed

380

clusters (Fig. 7A). A similar structure was also observed in the cyclohexane emulsion

381

stabilized by melamine-based microporous organic polymer particles after 20

382

freeze-drying (Lee & Chang, 2018). The porous structure formation might be due to

383

the fact that essential oils are susceptible to volatilization and loss during the

384

freeze-drying process. The addition of pectin resulted in the formation of film (Fig.

385

7B), which became denser as pectin concentration increased (Fig. 7C and D). A sheet

386

form with an irregular geometry has been reported for freeze-dried powders of

387

thymol-carvacrol or chia-essential oil emulsions stabilized by SC-lactose mixture

388

(Gursul, Karabulut, & Durmaz, 2019; Rodriguez, et al., 2019) and fish oil emulsion

389

stabilized by octenyl-succinic-anhydride modified starch (OSA-starch) (Melgosa,

390

Benito-Roman, Sanz, de Paz, & Beltran, 2019).

391

The powder prepared from zein-stabilized emulsions displayed irregular shapes

392

(Fig. 7E). It seems that the microcapsules formed agglomeration after the

393

spray-drying process, possibly due to the high surface hydrophobicity of zein (Zhang,

394

Luo, & Wang, 2011). A wide range of microcapsules with a dimension from 0.5 to 5

395

µm was observed for the emulsion stabilized by zein plus pectin at 0.05% (Fig. 7F).

396

Further increase in the pectin concentration resulted in the formation of bigger

397

microcapsules (5 - 10 µm, Fig. 7G and H), possibly due to an increase in the viscosity

398

at such high concentrations of pectin (Bai, et al., 2017; Fernandes, et al., 2016). All

399

the dimensions of spray-dried powders are bigger than those of individual zein

400

particles and oil droplets (Figs. 2, 4 and 6), possibly due to the fact that multiple

401

particles and oil droplets are present in one atomized droplet during spraying (Hogan,

402

McNamee, O'Riordan, & O'Sullivan, 2001). It is noteworthy that more and more

403

holes on the surface of microcapsules were observed as the pectin concentration 21

404

increased, which is consistent with the structure of OSA-starch-stabilized caraway

405

essential oil emulsions (Baranauskiene, Rutkaite, Peciulyte, Kazernaviciute, &

406

Venskutonis, 2016). Similarly, some holes were observed at the surface of spray-dried

407

microcapsules from caprylic capric glycerid oil emulsions stabilized by pectin

408

(Benjasirimongkol, Piriyaprasarth, Moribe, & Sriamornsak, 2019). The formation of

409

holes might be associated with competitive adsorption between zein-pectin particles

410

and pectin molecules (Fig. 5) and the interstitial space between particles coverage by

411

excessive pectin molecules. Fig. 8 illustrates the formation and interfacial structures

412

of peppermint oil emulsions at various concentrations of pectin.

413

3.2.2.3 Physical stability

414

The emulsions stabilized by R/Z particles had the peaks around 74, 324 and 3085

415

nm after storage for 42 days (Fig. 6A). ζ-Potential of the emulsions stabilized by R/Z

416

particles gradually decreased during storage and was +20 mV after 42 days (Fig. 6B).

417

The decrease in the magnitude of the ζ-potential may destabilize zein-stabilized

418

emulsions during storage, due to the lack of strongly repulsive forces against

419

aggregation. Additionally, a cream layer also appeared during storage, in agreement

420

with a previous report on zein-particle-stabilized soybean oil emulsions (de Folter, et

421

al., 2012). It is thus suggested that a significant increase in the largest peak (Fig. 6A)

422

is due to Ostwald ripening and flocculation (Dickinson, 2009). Creaming was not

423

observed in the presence of pectin. The size distribution of the emulsions stabilized by

424

R/ZP particles at 0.05% pectin remained unchanged for 14 days (data not shown) but

425

changed to two peaks around 434 and 1714 nm after 42 days (Fig. 6A). These results 22

426

suggest that pectin at 0.05% could not maintain long-term stability of the emulsions.

427

At 0.10% and 0.20% pectin, the size distribution of the emulsions increased to 709

428

and 643 nm after 42 days, respectively (Fig. 6A). At 0.05%, 0.10% and 0.20% pectin,

429

ζ-potential of the emulsions gradually changed to -33, -36 and -40 mV after 42 days,

430

respectively (Fig. 6B). On the whole, the addition of pectin could improve physical

431

stability of the emulsions against aggregation, creaming and Ostwald ripening during

432

storage, which is essentially dependent on the polysaccharide concentration. Pectin

433

could reportedly improve physical stability of orange beverage emulsions containing

434

GA (Mirhosseini, et al., 2008), lemongrass and mandarin essential oil emulsions

435

stabilized by Tween 80 (Guerra-Rosas, et al., 2016) against cream and phase

436

separation, due to strong adsorption of pectin at the oil surface to provide effectively

437

repulsive forces.

438

3.3 Co-encapsulation and protection of peppermint oil and resveratrol

439

3.3.1 Encapsulation

440

3.3.1.1 Encapsulation of peppermint oil

441

The encapsulation efficiency of peppermint oil in zein-stabilized emulsions was

442

74% (Table. 2). The encapsulation efficiency of peppermint oil increased gradually as

443

pectin concentration increased, reaching about 88% at 0.10% and 0.20% pectin. The

444

enhanced encapsulation efficiency might be due to efficient adsorption of partially

445

wettable R/ZP particles and packing of both the particles and pectin molecules on the

446

surface of oil droplets (Figs. 3 and 5). The encapsulation efficiency of peppermint oil 23

447

in zein-GA nanoparticles reportedly varied from 89% to 54% as the zein/peppermint

448

oil mass ratio decreased from 4:1 to 1:1 (Chen, et al., 2015). The encapsulation

449

efficiency of menthol and menthone was consistent with that of the whole peppermint

450

oil (Table 2). The efficiency of thymol oil encapsulated in SC-stabilized emulsions

451

decreased from 98% to 72% with the protein/oil mass ratio from 10:1 to 10:4 (Pan,

452

Chen, Davidson, & Zhong, 2014). The efficiency of clove bud oil encapsulated by

453

whey protein concentrate-GA mixtures was about 80% at the emulsifier/oil mass ratio

454

of 1:1 (Luo, et al., 2014). In the present study, the zein-pectin particles showed similar

455

encapsulation efficiency for peppermint oil even though at the emulsifier/oil mass

456

ratio lower than 1:10.

457

3.3.1.2 Partition of resveratrol

458

The total encapsulation efficiency of resveratrol in all the emulsions stabilized by

459

zein and zein-pectin particles was about 99% (Table. 2). The polyphenol

460

encapsulation efficiency in the peppermint oil emulsions was significantly greater

461

than that in the corresponding lipid-free system of colloidal particles (Table. 1). When

462

the concentration of pectin increased from 0.05% to 0.10%, the percentage of

463

resveratrol encapsulated by zein colloidal particles in the aqueous phase decreased

464

from 33% to 18%, while the polyphenol content in the emulsified oil droplets

465

increased from 66% to 80%. However, the percentage of resveratrol in zein colloidal

466

particles and in the emulsified oil droplets was 26% and 72% at 0.20% pectin,

467

respectively. A significant positive correlation (r = 0.963, p = 0.037) between the

468

percentage of resveratrol in the emulsified oil droplets and the interfacial zein 24

469

percentage was observed, while the percentage of encapsulated resveratrol in the

470

aqueous phase was negatively correlated with the protein interfacial percentage (r =

471

-0.971, p = 0.029). Similar trends were also reported in sunflower oil emulsions

472

stabilized by whey protein isolate-resveratrol complexes (Fan, et al., 2017) and corn

473

oil emulsions stabilized by soy protein isolate-resveratrol complexes (Wan, et al.,

474

2014). It has been reported that resveratrol has limited solubility both in aqueous

475

solution (30 µg/mL) (Summerlin, et al., 2015) and vegetable oils (e.g. ~ 86 µg/mL in

476

corn oil and below 0.1 µg/mL in sunflower oil) (Filip, et al., 2003; Hung, Chen, Liao,

477

Lo, & Fang, 2006). The total encapsulation efficiencies of resveratrol in peppermint

478

oil emulsions (Table 2) were greater than that (~ 70% - 80%) in sunflower oil

479

emulsions (Fan, et al., 2017). Matos et al. (2018) reported that about 98% resveratrol

480

was encapsulated in miglyol and orange oil emulsions stabilized by OSA-starch

481

particles, as a result of the polyphenol high solubility in flavor oils. It is thus

482

speculated that resveratrol co-existed in the inner oil phase and at the surface of

483

emulsified peppermint oil droplets. The less than 2% of free resveratrol (~ 4 µg/mL)

484

in the aqueous phase was lower than its solubility (30 µg/mL) (Summerlin, et al.,

485

2015). Therefore, peppermint essential oil emulsions stabilized by zein-based

486

particles could be a suitable carrier for the encapsulation of resveratrol.

487

3.3.2 Storage stability of bioactive compounds

488

3.3.2.1 Menthol and menthone

489

Peppermint oil contained about 27% menthol and 29% menthone as shown in the

25

490

composition reported in Table S1. Loss of menthol and menthone in zein-stabilized

491

emulsions was fast, with 18% and 26% remaining after 42 days, respectively (Fig. 9A

492

and B). Inclusion of resveratrol in zein-stabilized emulsions significantly delayed the

493

decomposition of both menthol and menthone, with 37% and 51% remaining after 42

494

days, respectively. This is in agreement with a previous report that oxidative stability

495

of corn oil emulsion was enhanced by using soy protein-resveratrol complexes as

496

stabilizers with reduced lipid hydroperoxides and volatile hexanal (Wan, et al., 2014).

497

A further decrease in the peppermint oil decomposition was observed as the

498

concentration of pectin increased. At 0.2% pectin, approximately 70% of menthol and

499

76% of menthone remained after 42 days.

500

3.3.2.2 Resveratrol

501

Resveratrol is labile to isomerization, oxidation, and degradation, which was

502

dependent on light, pH, and temperature (Zupancic, Lavric, & Kristl, 2015). The

503

content of resveratrol in the whole zein-stabilized emulsions decreased slowly to 93%

504

after storage for 14 days, which then accelerated to 52% remaining after 42 days (Fig.

505

10A). The decomposition was faster than that in soy-lecithin-stabilized peanut oil

506

emulsions with about 80% remaining after 30 days (Sessa, Tsao, Liu, Ferrari, & Donsi,

507

2011). The stability of resveratrol was significantly improved in the whole emulsions

508

with pectin, with 95% of the polyphenol remaining after 28 days. Then, the

509

resveratrol decomposition was dependent on the concentration of pectin, with 70%,

510

82% and 83% remaining at the polysaccharide concentrations of 0.05%, 0.10% and

511

0.20% after 42 days, respectively (Fig. 10A). Similar degradation patterns were 26

512

observed in the aqueous phase (Fig. 10B) and the emulsified oil droplets (Fig. 10C).

513

However, loss of resveratrol was faster in the emulsified oil droplets than in the

514

continuous phase.

515

Addition of pectin facilitated higher surface coverage of both oil droplets and

516

resveratrol-loaded zein particles (Figs 2 and 5) to further shield peppermint oil and

517

resveratrol from environmental factors, thus improving their storage stability (Figs. 9

518

- 10). It has been reported that resveratrol at the oil-water interface could provide the

519

protective effect on α-tocopherol dissolved in the inner oil phase of sunflower oil

520

emulsions against decomposition by loss of the polyphenol itself (Wang, et al., 2016a).

521

Therefore, resveratrol in the emulsified oil droplets could provide better protection of

522

peppermint oil against decomposition compared to the polyphenol in the aqueous

523

phase.

524

3.4 Antimicrobial activity

525

3.4.1 Antimicrobial activity and synergism testing

526

The MIC value of peppermint oil against S. aureus and S. Typhimurium was

527

1000 and 1250 µg/mL, respectively (Table. 3). These results are in agreement with the

528

finding of Iscan et al. (2002) that peppermint oil showed moderate inhibitory effects

529

for S. aureus (MIC 625 - 2500 µg/mL) and S. Typhimurium (MIC 1250 - 2500 µg/mL).

530

Resveratrol displayed a higher inhibitory effect compared to peppermint oil, which is

531

evident from the lower MIC value against S. aureus (100 µg/mL) and S. Typhimurium

532

(150 µg/mL). It has been reported that the MIC of resveratrol against Gram-positive 27

533

and Gram-negative bacteria was 16.5 - 260 µg/mL and 0.625 - 521 µg/mL,

534

respectively (Ma, et al., 2018). The more effective antimicrobial activity against S.

535

aureus than S. Typhimurium was due to the difference in the outer layers of

536

Gram-negative and Gram-positive bacteria (Seow, et al., 2014). Peppermint oil and

537

resveratrol displayed a synergistic effect with the FICI values of 0.750 and 0.625 for S.

538

aureus and S. Typhimurium, respectively (Table 3). Phenolic compounds exhibit the

539

highest antimicrobial activity among the plant phytochemicals (Burt, 2004). Menthol

540

and menthone were reportedly the major compounds contributing to antimicrobial

541

activity of peppermint oil (Iscan, et al., 2002). Synergistic effect in antimicrobial

542

activity is believed to be the fact that the combined compounds with distinct

543

molecular structures simultaneously challenge the resistance of target microorganisms

544

at multiple different sites (Burt, 2004; Seow, et al., 2014; Shi, et al., 2017).

545

3.4.2 Effect of resveratrol on antimicrobial activity of peppermint oil emulsions

546

The MIC values of zein and pectin were higher than 5000 µg/mL (data not

547

shown), indicating that both materials had no significant antimicrobial activity. The

548

MIC values of peppermint oil emulsions stabilized by bare zein particles for S. aureus

549

and S. Typhimurium in Table 4 were similar to those of bulk peppermint oil. Liang et

550

al. (2012) reported that peppermint oil emulsions stabilized by modified starch had

551

the same value of MIC as bulk oil for L. monocytogenes and S. aureus. A significant

552

enhancement in the antimicrobial activity of zein-stabilized emulsions was observed

553

upon the addition of pectin. In the case of 0.05% pectin, the MIC values against S.

554

aureus and S. Typhimurium were 750 and 875 µg/mL, respectively. However, a further 28

555

increase in pectin concentration resulted in a decrease in antimicrobial activity. The

556

co-inclusion of resveratrol further improved the antimicrobial activity of all the

557

emulsions.

558

The emulsion-based delivery of EOs is likely to improve antimicrobial activity

559

(Zahi, El Hattab, Liang, & Yuan, 2017), which is related to oil droplet size and surface

560

charge (Donsi, et al., 2016). This influences the transport of EOs to the cell membrane,

561

as well as their interaction with the multiple molecular sites at the microbial cell

562

membrane. A significant decrease in droplet size as a result of pectin addition (Fig. 6)

563

might contribute to bringing the peppermint oil molecules in contact with their action

564

sites for Gram-positive bacteria (Nazzaro, Fratianni, De Martino, Coppola, & De Feo,

565

2013). Furthermore, the emulsion droplets with hydrophilic surfaces might pass

566

through the cell membrane of Gram-negative bacteria (Majeed, et al., 2016). However,

567

the decrease in the antimicrobial activity at higher pectin concentrations (Table 4)

568

might be due to high encapsulation efficiency (Table. 2), leading to less content of

569

antimicrobial components dispersed into the aqueous phase (Donsi, et al., 2012).

570

3.4.3 Antimicrobial activity of peppermint oil emulsions during storage

571

The reductions of S. aureus and S. Typhimurium with regard to freshly-prepared

572

emulsions stabilized by bare zein particles was 3.33 and 3.17 Log CFU/mL (Fig. 11A

573

and B), respectively. The emulsions stabilized by resveratrol-loaded zein-pectin

574

particles exhibited the highest inhibitory effect against both targeted microorganisms

575

when the pectin concentration was 0.05%. A decrease in the inhibitory effect was

29

576

observed at higher pectin concentrations. The results were consistent with those

577

obtained by MIC determination (Table. 4). Resveratrol-loaded zein and zein-pectin

578

colloidal particles reduced about 0.38 and 0.22 Log CFU/mL for S. aureus and S.

579

Typhimurium, respectively (Table. S2). The peppermint oil emulsions stabilized by

580

R/Z and R/ZP particles had a greater reduction of both targeted microorganisms than

581

the sum of corresponding colloidal particles and peppermint oil emulsions. These

582

results indicate that the combination of peppermint oil and resveratrol showed a

583

synergistic antimicrobial effect against the two tested microorganisms.

584

Antimicrobial activity of all peppermint oil emulsions decreased significantly

585

over time (Fig. 11). The reductions of S. aureus and S. Typhimurium always ranked in

586

the order R/ZP-stabilized emulsions > R/Z-stabilized emulsions > Z-stabilized

587

emulsions. After storage for 42 days, the reduction of S. aureus was 0.68, 1.00 and

588

1.78 Log CFU/mL, while the reductions of S. Typhimurium were 0.54, 0.78 and 1.54

589

Log CFU/mL, respectively. Further increases in the pectin concentration resulted in

590

the improvement of retention of antimicrobial activity during storage, with about 2.92

591

Log CFU/mL reduction for S. aureus and 2.75 Log CFU/mL reduction for S.

592

Typhimurium remaining after 42 days at 0.20% pectin. In general, the higher the

593

pectin concentration, the higher was the retention of antimicrobial activity. Although

594

pectin at 0.10% and 0.20% impaired antimicrobial activity of freshly-prepared

595

emulsions slightly (Fig. 11 and Table 4), pectin at such concentrations could

596

significantly improve chemical stability of both resveratrol and peppermint oil (Figs.

597

9 - 10), contributing to a long-term antimicrobial activity in the whole emulsions 30

598

during storage (Fig. 11). Additionally, the sustained release over time of the EOs from

599

the emulsion droplets to the aqueous phase may contribute to prolonged antimicrobial

600

activity (Donsi, et al., 2016). An increase in the pectin concentration resulted in the

601

formation of more and more holes on the surface of oil droplets (Fig. 7F-H), which

602

may facilitate the release of antimicrobial components from the emulsified oil

603

droplets to the aqueous phase during storage.

604

4 Conclusions

605

In

the

present

study,

peppermint

oil

emulsions

stabilized

by

606

resveratrol-zein-pectin ternary complex particles have been successfully prepared,

607

showing a good encapsulation performance for both resveratrol and peppermint oil.

608

This system has combined the synergistic effect of two antibacterial agents and

609

emulsion-based carrier, which contributes to the improvement of antimicrobial

610

efficiency and chemical stability. These results obtained here should provide the

611

possibility of co-encapsulating multiple antimicrobial agents with different

612

physicochemical properties within single-emulsion-based carrier systems.

613

Declaration of Competing Interest

614

615

The authors declare no conflict of interest. Acknowledgements

616

This work received supports from the National Natural Science Foundation of

617

China (NSFC Project 31571781), the Fundamental Research Funds for the Central

618

Universities (JUSRP51711B) and the Postgraduate Research & Practice Innovation 31

619

Program of Jiangsu Province (KYCX17_1411).

32

620

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621

Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal

622

of Antimicrobial Chemotherapy, 48, 5-16.

623

Bai, L., Liu, F. G., Xu, X. F., Huan, S. Q., Gu, J. Y., & McClements, D. J. (2017).

624

Impact of polysaccharide molecular characteristics on viscosity enhancement

625

and depletion flocculation. Journal of Food Engineering, 207, 35-45.

626

Baranauskiene, R., Rutkaite, R., Peciulyte, L., Kazernaviciute, R., & Venskutonis, P.

627

R. (2016). Preparation and characterization of single and dual propylene oxide

628

and

629

microencapsulation of essential oils. Food & Function, 7(8), 3555-3565.

630

Benjasirimongkol, P., Piriyaprasarth, S., Moribe, K., & Sriamornsak, P. (2019). Use of

631

risk assessment and plackett-burman design for developing resveratrol

632

spray-dried emulsions: A quality-by-design approach. Aaps Pharmscitech,

633

20(1), p. 14

octenyl

succinic

anhydride

modified

starch

carriers

for

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830

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834

Ye, A. Q., Flanagan, J., & Singh, H. (2006). Formation of stable nanoparticles via

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838

Synergistic interactions of cinnamaldehyde in combination with carvacrol

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840

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epsilon-polylysine. Food Chemistry, 221, 18-23.

843

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structural, rheological, and antioxidant properties of alpha-zein. Food

845

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846

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847

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852

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853

Q. (2018). Fabrication of zein/pectin hybrid particle-stabilized Pickering high

854

internal phase emulsions with robust and ordered interface architecture.

855

Journal of Agricultural and Food Chemistry, 66(42), 11113-11123.

856

Zou, Y., Guo, J., Yin, S. W., Wang, J. M., & Yang, X. Q. (2015). Pickering emulsion

857

gels prepared by hydrogen-bonded zein/tannic acid complex colloidal particles.

858

Journal of Agricultural and Food Chemistry, 63(33), 7405-7414.

859

Zupancic, S., Lavric, Z., & Kristl, J. (2015). Stability and solubility of

860

trans-resveratrol are strongly influenced by pH and temperature. European

861

Journal of Pharmaceutics and Biopharmaceutics, 93, 196-204. 43

862

Figure captions

863

Fig. 1 Size distribution by the intensity (A) and ζ-potential (B) of resveratrol-loaded

864

zein particles in the absence and presence of pectin at various concentrations. The

865

concentration of zein was 0.2% (w/v).

866

Fig. 2 SEM images of resveratrol-loaded zein particles (A) and zein-pectin particles at

867

the polysaccharide concentrations of 0.05% (B), 0.10% (C) and 0.20% (w/v) (D). The

868

concentration of zein was 0.2% (w/v).

869

Fig. 3 Three-phase contact angles of bare zein (Z) particles, resveratrol-loaded zein

870

(R/Z) particles, resveratrol-loaded zein-pectin (R/ZP) particles with various

871

concentrations of pectin and pure pectin (P). The concentration of zein was 0.2%

872

(w/v).

873

Fig.

874

resveratrol-loaded zein particles (A) and zein-pectin particles at pectin concentrations

875

of 0.05% (B), 0.10% (C) and 0.20% (w/w) (D). The concentration of zein was 0.2%

876

(w/w).

877

Fig. 5 Interfacial zein or pectin percentages of the emulsions stabilized by

878

resveratrol-loaded zein particles at various pectin concentrations. The concentration of

879

zein was 0.2% (w/w).

880

Fig. 6 Size distribution by the intensity [(—), storage for 0 day; (- - -), storage for 42

881

days] and ζ-potential (B) of the emulsions stabilized by resveratrol-loaded zein

882

particles at various pectin concentrations. The concentration of zein was 0.2% (w/w).

883

Fig. 7 SEM images of the emulsions stabilized by resveratrol-loaded zein particles (A,

4

Fluorescent

microscopic

images

44

of

the

emulsions

stabilized

by

884

E) and zein-pectin particles at pectin concentrations of 0.05% (B, F), 0.10% (C, G)

885

and 0.20% (w/w) (D, H). A-D, powder obtained by freeze-drying; E-G, powder

886

obtained by spray-drying. The concentration of zein was 0.2% (w/w).

887

Fig. 8 Schematic diagram of peppermint oil emulsions formation and interfacial

888

structures at various concentrations of pectin. The concentration of zein was 0.2%

889

(w/w).

890

Fig. 9 Retention of menthol (A) and menthone (B) in emulsions prepared by bare zein

891

particles (Z), resveratrol-loaded zein (R/Z) and zein-pectin (R/ZP) particles at various

892

pectin concentrations and stored up to 42 days. The concentration of zein was 0.2%

893

(w/w).

894

Fig. 10 Retention of resveratrol in peppermint oil emulsions made with zein-based

895

particles at various pectin concentrations and stored up to 42 days; (A), the whole

896

emulsion; (B), the aqueous phase; (C), the emulsified oil droplets. The concentration

897

of zein was 0.2% (w/w).

898

Fig. 11 Reductions of S. aureus (A) and S. Typhimurium (B) treated with emulsions

899

stabilized by bare zein particles (Z), resveratrol-loaded zein (R/Z) and zein-pectin

900

(R/ZP) particles at various pectin concentrations and stored up to 42 days. The

901

concentration of zein was 0.2% (w/w).

45

Figure 1

(A)

0.20%

0.10%

0.05%

0 1

2

10

3

10

10

Size (d, nm)

45

(B) 30

ζ -Potential (mV)

15

0

-15

-30

-45

0.00

0.05

0.10

Pectin (%)

0.15

0.20

Figure 2

Figure 3

Figure 4

Figure 5 100 Pectin Zein

Percentage (%)

80

60

40

20

0

0.00

0.05

0.10

Concentration of pectin (%)

0.20

Figure 6

(A)

0.00%

0.05% 0.10% 0.20% 1

2

10

3

10

4

10

10

Size (d, nm)

80

0.00% 0.05% 0.10% 0.20%

60

ζ -Potential (mV)

40 20 -30 -35 -40 -45

(B) -50

0

7

14

28

Storage time (day)

42

Figure 7

Figure 8

Figure 9

(A)

Content of menthol (%)

100

80

60

40 Z R/Z R/ZP (0.05%) R/ZP (0.10%) R/ZP (0.20%)

20

0

0

7

14

21

28

35

42

Storage time (day)

(B)

Content of menthone (%)

100

80

60

40 Z R/Z R/ZP (0.05%) R/ZP (0.10%) R/ZP (0.20%)

20

0 0

7

14

21

28

Storage time (day)

35

42

Figure 10

Content of resveratrol (%)

100

(A)

80

60

0.00% 0.05% 0.10% 0.20%

40 0 0

7

14

21

28

35

42

49

Storage time (day)

Content of resveratrol (%)

100

(B)

80

60

0.00% 0.05% 0.10% 0.20%

40 0 0

7

14

21

28

35

42

49

Storage time (day)

Content of resveratrol (%)

100

(C)

80

60

0.00% 0.05% 0.10% 0.20%

40 0 0

7

14

21

28

Storage time (day)

35

42

49

Figure 11 5

Reduction (Log CFU/mL)

(A) 4

3

2 Z R/Z R/ZP (0.05%) R/ZP (0.10%) R/ZP (0.20%)

1

0

0

7

14

21

28

35

42

Storage time (day) 5

Reduction (Log CFU/mL)

(B) 4

3

2

Z R/Z R/ZP (0.05%) R/ZP (0.10%) R/ZP (0.20%)

1

0 0

7

14

21

28

Storage time (day)

35

42

Table 1 Encapsulation efficiency of resveratrol at various concentrations in zein colloidal particles and of resveratrol at 0.020% in zein-pectin colloidal particles at various pectin concentrations.

Resveratrol concentration

Pectin concentration

Encapsulation efficiency

(w/v%)

(w/v%)

(%)

0.004

70.8±1.1a

0.010

69.0±1.5a

0.020

69.0±2.6a

0.020

0.05

72.4±1.8ab

0.020

0.10

74.5±1.2b

0.020

0.20

75.6±2.2b

Different letters in the same column represent statistically significant differences (p < 0.05).

Table 2 Encapsulation efficiency of the whole peppermint oil (PO), menthol, and menthone and partition of resveratrol in the emulsions stabilized by resveratrol-loaded zein and zein-pectin particles at various pectin concentrations.

Resveratrol (%) Aqueous Pectin (%)

PO (%)

Menthol (%)

Menthone (%)

Emulsified phase

Total oil droplet

Particle 0.00

74.1±2.1a

72.2±3.0a

73.7±2.9a

33.3±1.8a

65.7±2.9a

99.0±0.9a

0.05

83.1±1.3b

84.5±2.6b

82.8±2.0b

17.7±1.8b

81.5±2.1b

99.2±0.3a

0.10

88.4±1.8c

89.9±1.1c

88.3±2.1c

19.2±2.2b

79.2±1.8b

98.4±0.8a

0.20

87.4±1.8c

89.1±1.9c

88.9±2.8c

26.2±1.7c

72.3±2.5c

98.5±0.7a

Different letters in the same column represent statistically significant differences (p < 0.05).

Table 3 MIC and fractional inhibitory concentration index (FICI) of resveratrol and peppermint oil (PO) against the two microorganisms tested.

MIC (µg/mL) Strains

Alone

Combination

FICI

Resveratrol

PO

Resveratrol

PO

S. aureus

100

1000

25

500

0.750

S. Typhimurium

150

1250

18.75

625

0.625

Table 4 Effect of resveratrol (Res) on the MIC values of peppermint oil emulsions stabilized by bare zein (Z) and zein-pectin (ZP) particles at various pectin concentrations against S. aureus and S. Typhimurium. The MIC values were expressed as real contents of Res and peppermint oil on the emulsion.

MIC (µg/mL, S. aureus)

MIC (µg/mL , S. Typhimurium)

Emulsion PO

PO, Res

PO

PO, Res

Z

1000

875, 3

1250

1000, 4

ZP (0.05%)

750

625, 2

875

625, 2.5

ZP (0.10%)

875

750, 2.5

1000

750, 3

ZP (0.20%)

875

750, 2.5

1000

750, 3

Highlights 1. Resveratrol was loaded in zein-pectin particles with partial wettability. 2. Peppermint oil and resveratrol were co-encapsulated in zein-pectin O/W emulsions. 3. Mixture of particles and pectin at the interface decreased size of emulsions. 4. Peppermint oil and resveratrol display synergistic antimicrobial activity. 5. Addition of pectin improved antimicrobial and physicochemical stability.