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Hao Huang, Tarun Belwal, Songbai Liu, Zhenhua Duan, Zisheng Luo
PII: S0268-005X(18)31440-1
DOI: https://doi.org/10.1016/j.foodhyd.2019.02.023
Reference: FOOHYD 4949
Please cite this article as: Huang, H., Belwal, T., Liu, S., Duan, Z., Luo, Z., Novel multi-phase
nano-emulsion preparation for co-loading hydrophilic arbutin and hydrophobic coumaric acid using
hydrocolloids, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.02.023.
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5 Hao Huanga†, Tarun Belwala†, Songbai Liua, Zhenhua Duanb*, Zisheng Luoa*
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a
7 Zhejiang University, College of Biosystems Engineering and Food Science, Key
8 Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and
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9 Rural Affairs, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou, 310058,
10 People’s Republic of China
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11 Institute of Food Science and Engineering, Hezhou University, Hezhou, People’s
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12 Republic of China
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16 *Corresponding author
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17 Zisheng Luo
18 College of Biosystems Engineering and Food Science
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19 Zhejiang University
20 Hangzhou 310058, People’s Republic of China
E-mail: luozisheng@zju.edu.cn
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22 Phone: +86-571-88982175
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24 Zhenhua Duan
25 Institute of Food Science and Engineering, Hezhou University, Hezhou, People’s
26 Republic of China
27 E-mail: dzh65@126.com
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29
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30 Abstract
31 This study was designed to co-load hydrophilic arbutin and hydrophobic
32 coumaric acid in nano-sized multi-phase emulsion (W/O/W) using hydrocolloids and
33 examined for its various stability and bioaccessibility parameters. The creaming
34 stability and sensory evaluation of emulsion in Lactobacillus beverage and its release
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35 kinetics in simulated oral and gastrointestinal conditions were also studied. Results
36 revealed that multi-phase emulsion exhibited small particle size and low
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37 polydispersity index with high zeta potential. Higher encapsulation efficiency was
38 also observed. Moreover, the multi-phase emulsion showed good storage stability
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39 under different conditions. The multi-phase emulsion also showed controlled release
40 of arbutin and coumaric acid in simulated conditions with increasing bioaccessibility.
41
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Stability of these compounds under unfavorable conditions was also found to be
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42 increased. Moreover, high creaming stability and sensory evaluation score of the
43 multi-phase emulsion suggested that the formulation was capable of retaining arbutin
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44 and coumaric acid in Lactobacillus beverage without affecting its sensory properties.
45 These results indicated that the multi-phase nano-emulsion (W/O/W) could
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46 effectively co-deliver hydrophilic arbutin and hydrophobic coumaric acid and thus
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51
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52 1. Introduction
53 In recent years, research has been increased on product formulations containing
54 polyphenols and other bioactive compounds for their applications in nutraceuticals,
55 pharmaceuticals and cosmeceuticals (Belščak-Cvitanović & Komes, 2017). Moreover,
56 polyphenolics formulations as dietary supplements, showed positive clinical
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57 evidences against some of the most life-threatening diseases such as, diabetes, cancer
58 and cardiovascular diseases (Shamloo et al., 2018; Rai et al., 2018; Irondi et al., 2018).
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59 The multifarious effect of polyphenolics is largely due to its free radical scavenging
60 activity, which is considered as one the main causative agent in several diseases (Del
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61 Rio et al., 2013). Polyphenolics are present in various plants and also extracted from
62 some microorganisms and formulated as functional food. In recent years, the
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production of functional foods for providing bioactive compounds such as phenolic
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64 compounds has aroused much interest among the consumers (Lam et al., 2014).
65 However, the formulations containing polyphenolics needs special consideration to
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71 acid present in edible plants such as peanuts, tomatoes, carrots, etc. (Stojković et al.,
72 2013). Both these compounds have been found its use in food, cosmetics and
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73 medicines (Jurica et al., 2017; Pei et al., 2016). Among others, studies have shown
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74 arbutin possesses whitening and anticancer properties (Jiang et al., 2018), while
75 coumaric acid exhibits antioxidant, anti-inflammatory and anti-cancer activities
76 (Pragasam et al., 2013). However, instability of arbutin and coumaric acid hampers
77 their bioactive function. For instance, Avonto et al. (2016) examined arbutin stability
78 under low pH, high temperature and enzymatic conditions and found that the stability
79 of arbutin decreased under these conditions and finally affect the bioaccessibility.
80 Also, it is unstable when exposed to UV radiation (Yang et al., 2013). Similarly,
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81 coumaric acid has lower aqueous solubility and higher sensitivity to extreme
82 environmental conditions such as pH, temperature, oxygen, light and enzymes (Altin
83 et al., 2018; Salameh et al., 2008). Very few studies have been conducted to improve
84 the stability of arbutin and coumaric acid. For instance, Li et al., (2016) and Liu et al.,
85 (2016) reported encapsulation by β-cyclodextrin could improve the heat stability of
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86 arbutin and chemical stability of coumaric acid, respectively. However, these reports
87 didn’t test the stability of arbutin and coumaric acid under different conditions.
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88 Moreover, the poor water solubility of β-CD could be another limitation factor for its
89 application in food products containing water (Qian et al., 2008).
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90 Addressing these issues related to stability and bioaccessibility of bioactive
91 compounds, various new formulation/encapsulation methods have been evolved
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92 (Gleeson et al., 2016). Also, these formulations mask the undesirable taste of
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93 bioactive compounds, which is considered to be a limitation factor for direct loading
94 into the formulation (Yao et al., 2015). However, formulating a product containing
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95 hydrophilic and hydrophobic compounds is another challenge for its stability and
96 bioaccessibility.
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98 and formulating agents, and also used as encapsulating agent (Li & Nie, 2016).
99 Among others, gelatin is the most used hydrocolloids in food applications due to its
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100 unique properties such as, natural origin, generally recognized as safe (GRAS) status
101 and versatile in applications (Williams & Phillips, 2014). As such gelatin has been
used to encapsulate various bioactive compounds such as polyphenols
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104 (Comunian et al., 2018) and many others under various formulations.
105 Nano-formulations containing multi-phase emulsion (W/O/W) is one of the most
106 widely used food delivery system combining both water-in-oil (W/O) and oil-in-water
107 (O/W) phase together. Due to its unique structure, the nano-emulsion may be
108 effectively deliver and protect both hydrophilic arbutin and hydrophobic coumaric
109 acid in a single-phase formulation. However, studies on nano-emulsion to increase the
110 stability of arbutin and coumaric acid and to co-load it in complex food products is
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111 being lacking. Thus, the present study performed to formulate multi-phase
112 nano-emulsion containing both arbutin and coumaric acid as bioactive components
113 and gelatin as gelling agent and tested for its stability, bioaccessibility and release
114 kinetics. Also food product, such as Lactobacillus beverage was used to check the
115 suitability of incorporating nano-emulsion as functional food.
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116
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118 2.1. Materials
119 Arbutin (purity, >98 %), coumaric acid (purity, >98 %), olive oil, Tween 80,
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120 polyglycerol polyricinoleate (PGPR) and gelatin were purchased from Aladdin
121 Industrial Co. (Shanghai, China). KCl, KSCN, NaCl, NaH2PO4, NaHCO3, urea, uric
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acid, α-amylase, and mucin, porcine bile extract, pepsin, lipase and pancreatin were
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123 obtained from Sangon Biotech Co. (Shanghai, China). All solvents and standards used
124 for chromatographic analyses were of HPLC grade and other chemicals were of
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128 emulsion according to Aditya et al. (2015a), with some minor modification (Fig. 1).
129 For primary W/O emulsion preparation, the arbutin (500 µg/mL), gelatin (3 %, w/v)
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130 and NaCl (3 %, w/v) was dissolved in ultrapure water (Milli-Q A10, Millipore Corp.,
131 Billerica, Mass., U.S.A.) and then stirred at 55 ℃ for 10 min using a magnetic stirrer
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132 (GL-3250, Qilinbeer, Haimen, China). Simultaneously, the oil phase was prepared in
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133 another flask by dissolving coumaric acid (500 µg/mL) and polyglycerol
134 polyricinoleate (PGPR) (8 %, v/v) in olive oil and then stirred at 55 ℃ for 10 min. The
135 water phase was dropped into the oil phase with a 3:7 ratio (v/v) and stirred at 55 ℃
136 for 5 min. The mixture was sonicated to form primary W/O emulsion using a probe
137 sonicator (JY96-IIN, Scientz, Ningbo, China) at 25 kHz, 80 W power for 3 min (work
138 time, 1 s; rest time, 1 s) and stored at 4 ℃ for 30 min for sol-gel transition. For second
139 water phase, Tween 80 (2 %, v/v) and NaCl (3 %, w/v) were added into ultrapure
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140 water. For the secondary emulsification step (W/O/W), the primary W/O emulsion
141 was dropped into water phase with a 3:7 ratio and stirred at 25 ℃ for 10 min. The
142 mixture was sonicated under the same conditions for 3 min (work time, 1 s; rest time,
143 1 s) to form W/O/W emulsion. The multi-phase W/O/W emulsion was stored at 4 ℃
144 for further experiments.
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145 2.3. Confocal laser scanning microscopy (CLSM)
146 The microstructure of multi-phase emulsion and single-phase emulsion was
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147 observed using a confocal laser scanning microscope (Leica TCSSP8, Leica
148 Microsystems, Germany) according to the protocol of Aditya et al. (2015b). Briefly,
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149 50 µL of 100 µg/mL nile red was added into 2.5 mL of the emulsion and kept at room
150 temperature for 30 min. An aliquot of the stained emulsion was placed onto a glass
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151 slide and observed under CLSM within 15 min. The excitation and collecting
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152 wavelength was set at 488 nm and 570–600 nm, respectively, and the images were
153 taken at 1500 ×.
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157 emulsion was added into the aluminum pan and sealed hermetically and placed in the
158 calorimeter. Nitrogen was applied as purge gas and the measurement was taken from
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159 -40 to 100 ℃ at a heating rate of 5 °C/min. The STARe software v. 16.0 was used to
160 analyze the DSC data.
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161 2.5. Measurement of particle size, zeta potential and polydispersity index (PDI)
162 Particle size, zeta potential and PDI were measured by dynamic light scattering
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163 (DLS) method on a nano-ZS nanosize analyzer (Zetasizer Nano ZS90, Malvern,
164 Worcestershire, U.K.) at 25 ℃ (Aditya et al., 2015a). To avoid multiple scattering,
165 multi-phase emulsions were diluted by 1000 times with ultrapure water before
166 measurements. About 1 mL of the emulsion was put in the measurement cell of the
167 nano-ZS nanosize analyzer. The detector angle was set at 90 ° and wavelength was set
168 at 623 nm for measuring the particle size and PDI. For determining zeta potential, the
169 voltage in the measurement cell was kept at 150 V. All the samples were measured 10
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175 12000 rpm for 10 min at 4 °C using a refrigerated centrifuge (Eppendorf Centrifuge
176 5810R, Eppendorf AG, Hamburg, Germany). Water phase containing arbutin and
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177 coumaric acid was collected at the bottom and passed through 0.22 µm filter. The
178 samples were then analyzed by Waters HPLC-DAD 2998 system (Waters, Milford,
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179 Massachusetts, U.S.A.). The injection volume was set at 20 µL and the separation was
180 carried out at 30 °C using a ZORBAX-SB C18, 100 mm× 4.6 mm, 1.8 µm column
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181 (Agilent Technologies Co., Santa Clara, USA). Mobile phase consisted of 0.5 % (v/v)
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182 aqueous acetic acid (A) and 1 % (v/v) formic acid in acetonitrile (B). The elution
183 gradients were set as follows: 0-2 min, 2 % B; 2-20 min, 2 % to 8 % B; 20-21 min, 8 %
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186 Encapsulation efficiency was calculated using Eq. (1) for arbutin and Eq. (2) for
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No-Ns
EEcoumaric acid (%) = × 100 (2)
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188 where, Nw is the amount of arbutin and No is the amount of coumaric acid added to
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189 the emulsion. Ns is the amount of arbutin or coumaric acids measured in the water
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190 phase.
191 2.7. In vitro release
192 In vitro release experiment of arbutin and coumaric acid from multi-phase
193 emulsion was conducted using the simulated oral fluid (SOF), simulated gastric fluid
194 (SGF) and simulated intestinal fluid (SIF) as per the protocol of Marefati et al. (2017)
195 and Park et al. (2018). SOF consisted of KCl (1.2 %, w/v), KSCN (0.3 %, w/v), NaCl
196 (0.16 %, w/v), NaH2PO4 (1.5 %, w/v), NaHCO3 (2.8 %, w/v), urea (0.3 %, w/v), uric
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197 acid (0.03 %, w/v), α-amylase (0.48 %, w/v) and mucin (0.04 %, w/v), and the pH
198 was adjusted to 6.8 using phosphate buffer. SGF was comprised of pepsin (0.32 %,
199 w/v), NaCl (0.2 %, w/v) and HCl (0.7 %, v/v), and the pH was adjusted to 2.0 using
200 HCl. SIF consisted of lipase (0.4 mg/mL), pancreatin (0.5 mg/mL), bile extract
201 (0.7 mg/mL) and 1 mL of 750 mM CaCl2 solution, and the pH was adjusted to 7.0
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202 using NaOH. Before studying the in vitro release profile under different conditions,
203 the dialysis bags were soaked in ultrapure water for 6 h. For oral release, the dialysis
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204 bags were filled with multi-phase emulsion and SOF digestive media. While for
205 gastrointestinal release, SOF was replaced with SGF digestive media. After incubated
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206 in SGF, the bags were placed in absolute methanol and incubated at 37 °C, 50 rpm for
207 6 h and then again incubated in SIF medium for similar time and conditions.
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208 Concurrently, during incubation, 1 mL of the sample was collected at different time
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209 intervals (i.e., 60, 120, 180, 240, 300 and 360 min) to determine the percent release of
210 arbutin and coumaric acid using HPLC.
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213 digestion assay according to Aditya et al. (2013). The samples were incubated in SGF
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214 at 37 °C and shaken at 50 rpm for 2 h. After incubation, the samples were transferred
215 to SIF for intestinal digestion for 2 h. Thereafter, the digested mixture was centrifuged
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216 at 12000 rpm for 30 min at 4 °C. The supernatant was diluted with methanol at 1:10
217 ratio (v/v) and then centrifuged at 12,000 rpm for another 30 min. Finally, the
supernatant was collected for HPLC analysis and bioaccessibility was calculated
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225 methanol at 1:10 ratio (v/v) and centrifuged at 12000 rpm for 30 min at 4 °C. The
226 supernatant was collected for HPLC analysis.
227 2.10. Creaming stability of multi-phase emulsion in Lactobacillus beverage
228 Multi-phase emulsion was added to Lactobacillus beverage (Weiquan, Hangzhou,
229 China) with a ratio of 1:10 (v/v) and the mixture was homogenized using a vortex
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230 homogenizer (Shupeilab, Shanghai, China). The sample was placed in a transparent
231 tube and the creaming stability of emulsion in Lactobacillus beverage was estimated
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232 visually after storage at 4 °C and 23 °C for 35 days (Xiong et al., 2018). The creaming
233 stability of emulsion was set to be good if no separation between the phases occurs in
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234 the formulated beverage The creaming height (Hc, cm) and total height (Ht, cm) was
235 recorded at 0, 7, 14, 21, 28, 35 days of interval and the creaming stability was
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236 calculated using Eq. (4):
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Hc
Creaming stability (%) = (1 − ) × 100 (4)
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237 2.11. Stability of arbutin and coumaric acid in Lactobacillus beverage
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238 For testing storage stability, one of the Lactobacillus beverage containing free
239 arbutin and coumaric acid and other containing multi-phase emulsion were stored at
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240 30 ℃ for 25 d. The sample from each beverage was collected at 0, 5, 10, 15, 20, 25 d
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241 and thoroughly mixed with equal volume of methanol. Before centrifuged at 12000
242 rpm for 30 min, a vortex homogenizer was used to break the emulsion and solubilize
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243 arbutin and coumaric acid. The supernatant was used to determine the concentration
244 of arbutin and coumaric acid by HPLC analysis. The antioxidant activity during the
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245 storage was also measured using DPPH method (Zhao et al., 2017). Briefly, for DPPH
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246 antioxidant assay, the supernatant was mixed with DPPH solution (200 µg/mL) at a
247 volume ratio of 1:1. After 30 min in the dark, the absorbance was measured at 517 nm.
248 Ascorbic acid as a standard antioxidant compound was used to develop a calibration
249 curve and the results were expressed as microgram ascorbic acid equivalent per
250 milliliter (µg AAE/mL).
251 For enzymatic stability, the samples were incubated in SGF for 2 h and SIF for
252 another 2 h. Then, the digested mixture was centrifuged at 12000 rpm for 30 min at
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253 4 °C and the supernatant was collected to determine the concentration of arbutin and
254 coumaric acid using HPLC analysis.
255 2.12. Sensory evaluation
256 For sensory evaluation, color, taste, odor and overall acceptability of
257 Lactobacillus beverage with or without adding the multi-phase emulsion was
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258 compared by panelists (10 male and 10 female volunteers, aged 20 to 35) from the
259 department of food science and nutrition of Zhejiang University, based on a nine point
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260 hedonic scale (9 = extremely like, 5 = neither like nor dislike, 1 = extremely dislike)
261 (Alozie & Ene-Obong, 2018). The sensory evaluation procedure was carried out
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262 according to Agarwal et al. (2018). Throughout, 10 mL of each sample was placed in
263 a well-lit and ventilated Sensory Science Center of Zhejiang University at 20 ± 1 °C
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264 for evaluation. For each test, the panelists need to evaluate the color and odor of
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265 samples before taste. Then, the panelists need to put the whole sample into mouth and
266 let the sample coat the roof of their mouth for at least 5 s before swallowing. The
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267 panelists were requested to cleanse their palate with pure water (Wahaha, China)
268 before evaluating for the next sample. The panelists were instructed to score the test
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269 samples. Forced-choice mode was applied in the test, so the panelists were asked to
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270 give scores even if the perceived difference was negligible. All the experiments were
271 performed in compliance with ISO standards (ISO 5495: 2005) and in accordance
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272 with the institutional framework and practices established by the Ethics Committee of
273 department of food science and nutrition of Zhejiang University. All panelists
received written details about the research before giving their informed consent.
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275 Average scores for color, taste, odor and overall acceptability were calculated as
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276 results.
277 2.13. Statistical analysis
278 All experiments were performed in triplicate. The data was statistically analyzed
279 using the SAS 9.4 program and the results were reported as means ± SD. Significant
280 differences were calculated using ANOVA and p< 0.05 was considered as significant.
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287 O/W emulsions. Therefore, the CLSM image illustrated the typical microstructure of
288 double emulsion, which confirmed that secondary emulsification retained primary
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289 W/O emulsion.
290 3.2. Thermal characteristics
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291 Fig. 3 showed the DSC thermogram of the multi-phase emulsion with onset
292 temperature, peak temperature and melting enthalpy. No freezing signal between
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293 −40 °C and 0 °C was recorded, which is an excellent characteristic feature of an
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294 emulsion (Clausse et al., 2018). During the heating process; there was a major
295 endothermic event with peak temperature at around 4.52 °C. The peak was attributed
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296 to melting of the aqueous phase of emulsions (Zhu et al., 2017). These results
297 indicated that olive oil do not crystallizes in emulsion droplets at -40 °C and thus the
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301 preparation and after storage for 30 days at 25 ℃. As presented in Table 1, particle
302 size of blank emulsion was larger than emulsion containing phenolic compounds by
10 to 13 % from day 0 to day 30, respectively. The decrease in particle size after
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304 encapsulating arbutin and coumaric acid might be due to the surface activity of the
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305 phenolic compounds, which reduced the interfacial tension at the interface of oil and
306 water (Zembyla et al., 2018; Aditya et al., 2017; Gomes et al., 2016; Akhtar et al.,
307 2014). These results showed that arbutin and coumaric acid might also work as
308 emulsifiers and form stable emulsion. After storage for 30 days at 25 ℃, the particle
309 size was relatively stable in emulsion containing phenolic compounds (1.67 %
310 increase) as compared to blank emulsion (4.83 % increase). Zeta potential played an
311 important role in the physical stability of the emulsions (Wu et al., 2018). Higher
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312 absolute value of zeta potential indicated stable emulsions (Zimmermann and Müller,
313 2018). The multi-phase emulsion containing arbutin and coumaric acid showed higher
314 zeta potential value as compared to the blank emulsion, which reveal that the phenolic
315 compounds significantly promote higher stability of emulsion and overall the
316 formulation achieved greater stability. PDI is also an important emulsion criteria
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317 reflecting the overall stability of the emulsion (Katsouli et al., 2017). For multi-phase
318 emulsion containing phenolic compounds, PDI value was less than 0.3, which suggest
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319 monodisperse droplet size distribution (Baccarin & Lemos-Senna, 2017). In the
320 present study, the presence of arbutin and coumaric acid decreased the PDI value of
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321 emulsion compared to the blank emulsion Moreover, there was no noticeable change
322 in PDI after storage for 30 days, which demonstrated a good storage stability of
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323 multi-phase emulsion containing arbutin and coumaric acid.
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324 3.4. Encapsulation efficiency
325 The encapsulation efficiency of gelatin and olive oil for for arbutin (90 %) and
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326 coumaric acid (80 %) in W/O and O/W emulsion, respectively showed good
327 percentage. Also, in the multi-phase emulsion (W/O/W), the encapsulation efficiency
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328 was found higher for both arbutin (91 %) and coumaric acid (82 %). However, no
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331 the encapsulation efficiency is quite optimized and the formulation has excellent
332 ability to co-load arbutin and coumaric acid.
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335 gastrointestinal condition (SGF and SIF) is an important performance index for
336 delivery systems (Marefati et al., 2017). The stability and release of multi-phase
337 emulsion containing arbutin and coumaric acid in oral and gastrointestinal system was
338 checked and it is expected that the formulation protect these phenolics in SOF, SGF
339 and release them in SIF. Oral release profiles of of arbutin and coumaric acid were
340 shown in Fig. 4A. Only around 5 % for arbutin and 3 % for coumaric acid of the
341 release were recorded in 360 min of incubation in SOF. Gastrointestinal release
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342 profiles were shown in Fig. 4B. For arbutin, around 50 % of the release was recorded
343 in first 120 min, which increased to 86% in 360 min of incubation in SIF. For
344 coumaric acid, similar kinetics was observed. These results indicated that the
345 multi-phase emulsion (W/O/W) was effective in controlling the release of arbutin and
346 coumaric acid under simulated gastrointestinal conditions. The sustained release was
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347 mainly attributed to gelatin hydrogel in the out water phase, which might delay the
348 release process (Gómez-Mascaraque et al., 2016). At the end of 360 min of incubation,
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349 around 86 % of arbutin and 58 % coumaric acid were released. Moreover, compared
350 to coumaric acid more arbutin was released in SIF, which may be due to its higher
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351 hydrophilicity (Aditya et al., 2015a). Chen et al. (2018) also reported that hydrophilic
352 (-)-epigallocatechin-3-gallate was released more quickly in W/O/W emulsion as
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353 compared to hydrophobic quercetin. These results supported that the multi-phase
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354 emulsion (W/O/W) was an effective delivery system to control the release of arbutin
355 and coumaric acid under oral and gastrointestinal conditions.
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358 (Pei et al., 2016; Helal et al., 2014) limited their applications in nutraceutical and
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359 medicinal products, which might be resulted from the chemical instability in the
360 gastrointestinal digestion. As shown in Fig. 4B, the multi-phase emulsion exhibited a
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361 high bioaccessibility for arbutin ( 65 ± 2 %) compared to 53 ± 2 % for single-phase
362 emulsion and 22 ± 1 % for free solution. Similarly, higher bioaccessibility for
coumaric acid (60 ± 3 %) was recorded in multi-phase compared to single-phase
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363
364 emulsion (56 ± 2 %) and its free solution (18 ± 2 %). The increase of bioaccessibility
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365 might be due to several reasons. For instance, the multi-phase emulsion provided a
366 protective layer against acidic environment and enzymatic (pepsin) activity (Kim et
367 al., 2016). Also, it has been reported that tween 80 act as a hydrophilic stabilizer and
368 protected the emulsion during gastric digestion (Van Aken et al., 2011). Moreover, the
369 use of gelatin hydrogel in sustained release of multi-phase emulsion further protected
370 arbutin and coumaric acid from gastrointestinal degradation. Also the formed micelles
371 after the digestion could solubilize and protect the arbutin and coumaric acid, which
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372 was reported in earlier study (Ortega et al., 2009). Overall, these results revealed that
373 multi-phase emulsion led to sustained release and higher bioaccessibility, which
374 exhibited the superiority to co-deliver arbutin and coumaric acid in formulation.
375 3.7. Stability study of arbutin and coumaric acid under different condition
376 Instability of arbutin and coumaric acid at low pH, high temperature and UV-C
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377 exposure limits their utilization (Avonto et al., 2016; Altin et al., 2018). Degradation
378 of bioactive compounds when exposed to unfavorable conditions during processing
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379 and storage might result in loss and/or conversion of compounds (Rothwell et al.,
380 2015; Wang et al., 2008). Thus, it is necessary for any formulation to provide stability
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381 to its active compounds, while providing better bioaccessibility. In the present study,
382 the multi-phase emulsion formulation was found to decrease the degradation of
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383 arbutin and coumaric acid under different adverse conditions (Fig. 5). For instance,
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384 multi-phase emulsion as compared to single-phase emulsion or solution provided
385 better protection to arbutin and coumaric acid under low pH, high temperature and
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386 UV-C exposure (Fig. 5A and 5B). In earlier reports, multi-phase emulsion was
387 reported to protect the co-loaded nutraceuticals from degradation by providing a
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388 protective screen (Zou et al., 2015). The unfavorable condition could prevail during
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389 storage and transportation conditions, which degrade or change the chemical
390 composition of product and thus lower down its activity. The protecting results clearly
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391 showed that the multi-phase emulsion was effective in protecting arbutin and
392 coumaric acid during unfavorable conditions.
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393 3.8. Creaming stability of emulsion and stability of arbutin and coumaric acid in
394 Lactobacillus beverage
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395 Recently, the International Scientific Association for Probiotics and Prebiotics
396 (ISAPP) expanded the concept of prebiotics and included phenolics in their list
397 (Gibson et al., 2017). This statement suggested the potential of phenolic compounds
398 in modulating beneficial microorganisms and acts as prebiotics to confer a health
399 benefit. In order to find out the interaction between multi-phase emulsion containing
400 arbutin and coumaric acid and prebiotics, the emulsion was mixed into a food product
401 (Lactobacillus beverage). Also the creaming ability of emulsion and stability of
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407 Similar reports are available elsewhere (Aditya et al., 2015b; Hosseini et al., 2015;
408 Mwangi et al., 2016).
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409 Fig. 6B showed the stability of free arbutin, free coumaric acid and multi-phase
410 emulsion containing both in Lactobacillus beverage after storage at 30 ℃ for 25 d. As
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411 it has been seen that, the stability of arbutin and coumaric acid in multi-phase
412 emulsion was higher than arbutin and coumaric acid solution in the beverage (Fig.
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413 6B). Throughout the period the stability of encapsulated arbutin and coumaric acid
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414 was found to be much higher compared to its free form. For instance, at the end (25 d),
415 the stability was recorded as 47 % and 37 % higher for arbutin and coumaric acid,
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416 respectively, compared to its free solution. Polyphenolic compounds are known to
417 exert antioxidant activity and this has been highlighted as its signature feature in most
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418 of the formulations. Arbutin and coumaric acid in multi-phase emulsion was also
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419 tested for its in-vitro antioxidant potential and found to exhibit significantly higher
420 antioxidant activity compared to its free solution (Fig. 6C). After 25 days of storage,
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421 the DPPH scavenging activity of arbutin and coumaric acid in multi-phase emulsion
422 was significantly higher (53 %) than that in solution. Due to the lower pH of
Lactobacillus beverage, free arbutin and coumaric acid in solutions might degrade and
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424 resulted in lower stability. The enzymatic stability of arbutin and coumaric acid in
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425 multi-phase emulsion was much higher than in solution after added into the beverage
426 (Fig. 6D). The enzymatic stability in multi-phase emulsion was 216 % and 287 %
427 higher for arbutin and coumaric acid, respectively, than in solutions. Therefore, in the
428 present study encapsulate these phenolic compounds in multi-phase emulsion would
429 increase their stability in the food product. These results also provided the foundation
430 to realize the potential of utilizing phenolic compounds as encapsulated formulation
431 in Lactobacillus beverage and other food products.
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437 was accepted well by the participants. Therefore, the multi-phase emulsion didn’t
438 affect the sensory properties of Lactobacillus beverage.
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439 4. Conclusion
440 The current study showed a novel multi-phase nano-emulsion formulation that
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441 showed an effective delivery system to co-load hydrophilic arbutin and hydrophobic
442 coumaric acid. The multi-phase emulsion (W/O/W) exhibited excellent encapsulating
443
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ability with relatively high encapsulation efficiency (91 ± 3 % for arbutin and 82 ± 2 %
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444 for coumaric acid). Moreover, the greater physical stability after long-time storage
445 was also achieved. The encapsulated arbutin and coumaric acid showed sustained
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446 release due to gelatin hydrogel, which resulted in the controlled release in simulated
447 oral condition, the increase of their bioaccessibility under simulated gastrointestinal
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448 condition and stability under unfavorable conditions (low pH, high temperature and
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451 showed an increased stability of arbutin and coumaric acid meanwhile, providing a
452 better antioxidant activity. Moreover, the multi-phase emulsion didn’t affect the
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459 Acknowledgements
460 The research was financially supported by the National Key Research and
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673 Fig. 1. Simplified flowchart for the preparation of primary single-phase (W/O) and
674 secondary multi-phase (W/O/W) emulsions.
675
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676 Fig. 2. Visual appearance of W/O/W multi-phase emulsion (A) and microstructure
677 images of W/O/W multi-phase emulsion (B), W/O emulsion (C) and O/W emulsion
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678 (D) using CLSM (CLSM operated at 1500 × using Nile red dye).
679
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680 Fig. 3. DSC thermogram between heat flow and temperature showing thermal
681 stability of W/O/W emulsion with onset temperature, peak temperature and melting
682 enthalpy.
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684 Fig. 4. Release kinetics of arbutin and coumaric acid under simulated oral (A) and
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685 gastrointestinal conditions (B), and in vitro bioaccessibility of arbutin and coumaric
686 acid in different formulations under simulated gastrointestinal conditions (C) (means
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690 emulsions) and conditions (pH, temperature and UV-C treatment) on the degradation
691 of arbutin (A) and coumaric acid (B).
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692
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Table 1 Comparison between particle size, zeta potential and polydispersity index
(PDI) of emulsions within different time.
Blank W/O/W emulsion
Time Variables (without adding arbutin W/O/W emulsion
and coumaric acid)
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Particle size (nm) 395.52 ± 15.21 355.98 ± 10.44
Day 0 Zeta potential (mV) -35.25 ± 1.80 -44.10 ± 0.62
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PDI 0.18 ± 0.03 0.12 ± 0.01
Particle size (nm) 415.63 ± 8.32 362.06 ± 12.35
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Day 30 Zeta potential (mV) -32.43 ± 1.76 -39.86 ± 0.98
PDI 0.20 ± 0.03 0.13 ± 0.02
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• Hydrophilic arbutin and hydrophobic coumaric acid were co-loaded in W/O/W
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acid.
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• Sustainable release of arbutin and coumaric acid in gastrointestinal fluid.
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• W/O/W emulsion retained phenolics in Lactobacillus beverage as functional
food.
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