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Nano-encapsulation of fish oil and garlic essential oil by a novel composition of wall
material: Persian gum-chitosan

Sara Raeisi, Seyed Mahdi Ojagh, Siew Young Quek, Parastoo Pourashouri, Fabien
Salaün

PII: S0023-6438(19)30836-9
DOI: https://doi.org/10.1016/j.lwt.2019.108494
Reference: YFSTL 108494

To appear in: LWT - Food Science and Technology

Received Date: 28 January 2019

Revised Date: 6 August 2019
Accepted Date: 7 August 2019

Please cite this article as: Raeisi, S., Ojagh, S.M., Quek, S.Y., Pourashouri, P., Salaün, F., Nano-
encapsulation of fish oil and garlic essential oil by a novel composition of wall material: Persian gum-
chitosan, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108494.

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1 Nano-encapsulation of fish oil and garlic essential oil by a novel composition of wall
2 material: Persian gum-chitosan

4 Running title: Nano-encapsulation of fish oil-garlic essential oil by Persian gum and Chitosan
5

6 Sara Raeisi1, Seyed Mahdi Ojagh1*, Siew Young Quek2,3**, Parastoo Pourashouri1, Fabien
7 Salaün4

8
1
9 Department of Fisheries, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan,
10 Iran.
2
11 Food Science Programme, School of Chemical Sciences, University of Auckland, 23 Symonds Street,
12 Auckland 1010, New Zealand.
3
13 Riddet Institute, Centre of Research Excellent, Palmerston North 4474, New Zealand.
4
14 ENSAIT, GEMTEX – Laboratoire de Génie et Matériaux Textiles, F-59000 Lille, France
15
16
*
17 Corresponding author
18 Dr. Seyed Mahdi Ojagh
19 Assoc. Professor, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
20 Email: mahdi_ojagh@yahoo.com
21 Phone: +989126760805

**
22 Corresponding author

23 Dr. Siew Young Quek

24 Assoc. Professor, University of Auckland
25 Email: sy.quek@auckland.ac.nz
26 Phone: +64 9 373 7599 Ext 85852
27 Fax: +64 9 373 7422
28

1
29 Abstract

30 This study investigated nano-encapsulation of fish oil (FO) and garlic (Allium sativum L.) essential oil
31 (GEO) using Persian gum (PG) and chitosan (Chi) as wall materials (at the ratio of 2:1, 1:1 and 1:2 (w/w)
32 and core/wall ratio of 10:2 (w/w)), applying the electrostatic layer-by-layer deposition method. Our
33 findings revealed that PG:Chi 2:1 ratio was the best combination of wall material, giving good zeta
34 potential (33.4±1.6 mV), maximum emulsion stability, surface tension (56 ± 0.3 mN.m-1) and viscosity
35 (1.09 ± 0.04 Pa.s) while maintaining minimum droplet size (23.19 nm) and PDI (0.120 ± 0.002).
36 Consistent with the emulsion studies, the PG:Chi 2:1 ratio gave nano-capsules with the best reconstituted
37 emulsion properties. This includes zeta potential of 34±1mV, droplet size in nano range (mean size, 65
38 nm) with good stability and low dispersibility (PDI, 0.385 ± 0.004). From scanning electron microscopy
39 (SEM), the FO-GEO nano-capsules show agglomerated morphology with spherical particles. The success
40 of FO-GEO encapsulation was confirmed by the results obtained from differential scanning calorimetry
41 (DSC), X-Ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FTIR) analyses. The
42 physicochemical characteristics and thermal stability indicated that such nano-capsules have promising
43 use in pharmaceutical and food industries for delivery of FO-GEO.
44
45 Keywords: Encapsulation; Omega-3 fatty acids; Essential oil; gum; Thermal stability
46
47 1. Introduction

48 The main food sources of Omega-3 polyunsaturated fatty acids (ω-3-PUFA) are from fish and fish oil,
49 with the latter being the most important ω-3-PUFA source (Jeyakumari, Janarthanan, Chouksey, &
50 Venkateshwarlu, 2016). ω-3-PUFA, particularly eicosapentaenoic acid (EPA, C20:5 ω-3) and
51 docosahexaenoic acid (DHA, C22:6 ω-3) are vital for human health due to their beneficial health effects.
52 Since the biosynthesis pathways of the ω-3 fatty acids in the human body are slow, the consumption of
53 fish oil that is rich in ω-3 fatty acids can be useful for human health (Sanguansri, et al., 2016). Nowadays,
54 soft capsules and oral liquids are the main fish oil supplement products, and due to short shelf life, further
55 processing of fish oil is limited. Encapsulation is a well-known technique used in food industries to
56 deliver and protect sensitive materials such as oils and fats, vitamins, aroma compounds, colorants,
57 enzymes and minerals to prevent degradation of active compounds, volatile losses, and/or physico-
58 chemical interactions with other food components (Shamaei, et al., 2016). Many studies confirmed the
59 use of encapsulation for designing functional foods as an appropriate method to obtain the desired
60 characteristics of storability, stability and delivery of nutrients (Chatterjee & Judeh, 2016). Encapsulation

2
61 is used to fabricate materials in the form of micro- and nano-particles involving either mechanical or
62 physico-chemical/ chemical process to immobilize a substance (e.g. active and/or sensitive component)
63 within a homogeneous or heterogeneous matrix called wall material (Gharsallaoui, Roudaut, Chambin,
64 Voilley, & Saurel, 2007). It is a promising method to provide stability for fish oil stable during processing
65 and to mask the fishy odour (Kolanowski, Ziolkowski, Weißbrodt, Kunz, & Laufenberg, 2006).

66 Selection and investigation of suitable combination of wall materials are important to maximize the
67 retention of the active component, and to obtain stable particles during encapsulation. Carbohydrates such
68 as gums have been used as wall material for encapsulation of a wide variety of active and/or sensitive
69 components due to their emulsifiying characteristics and high stability during processing. Recently, the
70 applications of “Persian gum” as a novel hydrocolloid for encapsulating bioactive compounds have been
71 evaluated (Abbasi, 2017). Persian gum (PG) is a polysaccharide obtained from the branches and trunk of
72 wild almond tree (Amygdalus scoparia Spach), originated from central Asia and Irano-Turanian and
73 Zagrros regions of Iran. Its unique properties make the gum an ideal material for diverse applications in
74 medical or food fields (Golkar, Taghavi, & Aghili Dehnavi, 2018).

75 Chitosan, a natural polysaccharide, is one of the most applied polymers served as an encapsulate agent for
76 food applications. This is due to its ability to control the release of active ingredient, the presence of free
77 amine groups allowing further cross-linking reaction, and the electro-potential responsible for its stability
78 (Liu, Li, Zhu, Li, & Kumar, 2014).

79 In the present study, garlic (Allium sativum L.) essential oil has been chosen for co-encapsulating with
80 fish oil. Chitosan and Persian gum were selected as polymeric wall materials due to their possibility to
81 induce the formation of ionically complex at a pH range applied in most food systems. Nano-
82 encapsulation of fish oil was previously reported (García-Moreno, et al., 2016), however, study on nano-
83 encapsulation of fish oil together with garlic essential oil as core material has not been explored.
84 Furthermore, the applicability of Persian gum in combination with chitosan as wall materials has not been
85 reported. Therefore, the aim of this work was to investigate the nano-encapsulation of fish oil (FO) and
86 garlic essential oil (GEO) using Persian gum (PG) and chitosan (Chi) as wall materials, applying the
87 electrostatic layer-by-layer deposition technique.

88 2. Material and methods

89 2.1. Materials

90 Fish liver oil from Gadus morrhua was purchased from Sigma-Aldrich (St Louis, MO, USA). GEO were
91 sourced from the Barij Essence Pharmaceutical Company, Tehran, Iran. Chitosan (Chi) with low

3
 92 molecular weight (75–85% deacetylation degree) purchased from Sigma–Aldrich (St. Louis, MO, USA)
93 and Persian gum (PG), sourced from a local herbal store (Shiraz, Iran), were used as wall material. The
94 gum was ground and sieved; the collected gum powder (size < 0.25 mm) was placed in a sealed container
95 and stored at a cool dry place. Tween 80, a surfactant, was purchased from Sigma–Aldrich (St. Louis,
96 MO, USA). Deionized water was utilized for preparation of all the solutions.

97 2.2. Preparation of wall material dispersions

98 PG powder was gradually added to distilled water and mixed slowly for 2 h. The mixture was kept at 4 ±
99 1°C overnight to ensure complete hydration. To separate the soluble and insoluble fractions in the gum,
100 the suspension was centrifuged at 20379 g (14,000 rpm) for 45 min and the soluble fraction was gathered.
101 The fraction was then dried at room temperature, powdered and dissolved in distilled water to prepare the
102 PG stock solution of 20 g/L in concentration. For preparing chitosan (Chi) wall material dispersion,
103 chitosan powder was separately added to the 1% (v/v) glacial acetic acid solution to achieve concentration
104 of 20 g/L.

105 2.3. Preparation of emulsion and nano-capsules

106 For preparation of emulsions using the layer-by-layer deposition technique, a primary emulsion was
107 firstly produced by combining the FO-GEO (75-25 % w/w) and the chitosan dispersion (20g/L). Tween
108 80 (0.1%) was slowly added to the emulsion followed by mixing using a magnetic stirrer (at 400 rpm for
109 5 min). The mixture was pre-homogenized by a rotor-stator homogenizer (WiseTis Homogenizer, HG-
110 15D, Wertheim, Germany) at 4000 and 10000 rpm, for 3 and 1 min respectively, followed by sonication
111 at amplitude of 100% for 4 min (UP 400S, Hielscher, Teltow, Germany). At the next step, PG solution
112 was gradually added to the above emulsion, and the pH was adjusted to 3.6 using 1N acetic acid, before
113 mixing with a magnet stirrer at 300 rpm for 90 min. The solution obtained was again, homogenized by a
114 rotor-stator homogenizer (at 3000 rpm for 0.5 min and 10000 rpm for 0.5 min) followed by sonication (at
115 amplitude 80% for 0.5 min). Emulsions were prepared to have the PG:Chi ratios of 1:1, 2:1 and 1:2 (w/w)
116 and wall-core (FO-GEO) ratio of 10:2 (w/w). Finally, the emulsions were frozen at –70 °C overnight, and
117 dried in a freeze dryer at −51°C and 12 Pa (Beta 1–8 LSCplus, Martin Christ Gefriertrocknungsanlagen
118 GmbH, Harz, Germany) for 72 h. After drying, the samples obtained were kept in moisture–impermeable
119 plastic bags and stored at –18 °C in dark.

120 2.4. Physical stability of emulsions

121 To determine the emulsion stability, 10 mL of each emulsion was introduced into a test tube maintained at
122 4 ±1°C for 14 days in a refrigerator. Emulsion stability index (ESI) was calculated using equation 1.

4
 HE − HS
123 ESI (%) = × 100 (1)
HE

124 where HE and HS are the total emulsion and serum layer heights (mm), respectively (Gu, Decker, &
125 McClements, 2007).

126 2.5. Droplet size distribution and polydispersity index (PDI) of emulsions

127 The average droplet size and polydispersity index (PDI) of the various emulsions were determined by
128 dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd., Malvern, UK) at
129 a fixed detector angle of 90° after 24 h. Before performing measurement, sample solutions were diluted
130 using buffer solution having the same pH value than the analyzed sample to avoid multiple scattering
131 effects and to reach the droplet concentration of approximately 0.005 % w/w (Klinkesorn & McClements,
132 2009).

133 2.6. Zeta potential of emulsions

134 The emulsions were diluted using buffer solution at the same pH prior to analysis. The obtained
135 emulsions were then poured into the measurement chamber of a particle electrophoresis instruments.
136 Zetasizer Nano-ZS90 (Malvern Instruments Ltd., Malvern, UK) was used to determine the zeta-potential
137 of the droplets, by measuring the direction and velocity of droplet movement in the used electric field. For
138 each analyzed sample, ten measurements were carried out. The results were presented as the mean value
139 of the measurement.
140

141 2.7. Surface tension of emulsions

142 Surface tension of the different PG:Chi emulsions was measured using a Krüss K9-Mk1 tensiometer
143 (Krüss GmbH, Hamburg, Germany) according to the standard Wilhelmy's plate method (Pareta &
144 Edirisinghe, 2006). All measurements were performed in triplicate at 22 ± 1 °C for 45 minutes until a
145 constant value was achieved.

146 2.8. Viscosity of emulsions

147 Viscosities of the emulsions were determined using a Brookfield DV-II + Pro viscometer (Brookfield
148 Engineering Labs Inc., Middleboro, MA, USA) at 25 °C with a sample volume of 50 mL operating at 100
149 rpm by using a spindle set of S61.

150 2.9. Encapsulation efficiency (EE)

5
151 Encapsulation efficiency (EE) was determined as described by Bae and Lee (2008). For surface oil
152 extraction, 15 mL of hexane were added to 1.5 g of each powder sample in a glass jar with a lid, followed
153 by shaking for 2 min at room temperature. The mixtures were filtered through Whatman filter paper no. 1
154 and the collected powders were rinsed for three times using 20 mL hexane each time into a flask. Then,
155 the solvent was evaporated in a first step at room temperature and in a second step at 60 °C, until reaching
156 a constant weight. The un-encapsulated oil was calculated by mass difference between the initial clean
157 flask and the weight of the flask after the oil residue was extracted. The total oil was oil content in the
158 emulsion. Encapsulation efficiency (EE) was calculated using equation 2.

OilTotal -OilSurface
159 EE (%) = ×100 (2)
OilTotal

160 where OilTotal and OilSurface are the total and surface oil contents (g) based on the dry matter of the final
161 emulsion, respectively.

162 2.10. Droplet size, zeta potential and PDI of the reconstituted emulsions

163 Particle size, PDI and zeta potential of the encapsulated fish oil were measured using the Zetasizer Nano-
164 ZS90 (Malvern Instruments Ltd., Malvern, UK). Measurements were performed using aqueous diluted
165 samples (2:1 ratio) as described above.

166 2.11. Morphology of nano-capsules

167 The morphology of the freeze-dried nano–capsules was analyzed using the Scanning Electron Microscopy
168 (SEM) (Leo EVO–40 VPX, Carl Zeiss SMT, Cambridge, UK). Samples were placed on the SEM stubs
169 using a two-sided adhesive tape, and were then coated with gold using a magnetron sputter coater. Coated
170 samples were analyzed at an accelerating voltage of 26 kV.

171 2.12. Thermal properties of nano-capsules

172 A Differential Scanning Calorimetry (DSC) instrument (Mettler-Toledo DSC model 822, Urdorf,
173 Switzerland) was used to investigate the thermal stability of the nano-encapsulated FO-GEO.
174 Experiments were conducted at 10°C/min heating rate from 0 to 300 °C.

175 2.13. Crystalline properties of nano-capsules

176 The crystalline and amorphous behaviors of the nano-capsules were analyzed by X-Ray Diffraction
177 (XRD). The XRD patterns were recorded over a 2θ range of 5–80◦ using an X-ray diffractometer
178 (Siemens D5000, Munich, Germany) with a step size of 0.026◦.

6
179 The crystallinity index was determined as proposed by Focher, Beltrame, Naggi, and Torri, (1990). This
180 involved measuring the maximum intensity, I110, at 2θ = 20° of the (110) lattice diffraction and that of the
181 amorphous diffraction, Iam, at 2θ = 16°. The crystallinity index was calculated using the following
182 formula:

183 CrI peak = (I110-Iam)/ I110 (3)

184 2.14. Interactions of wall materials

185 The interaction of wall materials was investigated by Fourier-Transform Infrared Spectroscopy (FTIR).
186 The FO-GEO and encapsulated FO-GEO in PG:Chi spectrum were recorded using a Bruker Equinox 55
187 spectrometer (Bruker Optics, Banner Lane, Coventry, UK) within the wavenumber range of 4000 to 400
188 cm−1.

189 2.15. Statistical Analysis

190 The encapsulation experiments were carried out in duplicate and each analysis was conducted three times.
191 Data for each sample was calculated as mean value ± standard deviation. One-way analysis of variance
192 (ANOVA) was performed using SPSS version 11.5 (IBM SPSS, New York, USA) and differences
193 between samples means were compared using the Duncan’s multiple range test, taking statistical
194 significant at p < 0.05.

195 3. Results and discussion

196 3.1. Physical stability of emulsions

197 The highest and lowest ESI values were observed in samples containing PG:Chi 2:1 and PG:Chi 1:2 with
198 100 and 80 % ESI, respectively (Table 1). Increasing PG concentration has improved stability of
199 emulsion with no phase separation observed during storage of 14 days. Abbasi and Mohammadi (2013)
200 have reported that PG could enhance emulsion stability as it increased the viscosity of solution. At higher
201 concentrations, polysaccharides macromolecules can form an interacting three-dimensional network to
202 retain oil droplets and/ or trap oil droplets by the entangled polymer chains, which blocks their movement
203 (Dickinson, 1992).

204 3.2. Droplet size distribution and polydispersity index (PDI) of emulsions

205 The influence of different PG:Chi ratios on droplet size and polydispersity index (PDI) of emulsions is
206 presented in Fig. 1 and Table 1, respectively. Minimum and maximum droplet size were observed in
207 PG:Chi 2:1 and PG:Chi 1:2 samples with particle size of 23 nm and 152 nm, respectively (Fig. 1).

7
208 According to the results, the increase of PG amount decreases the emulsion droplet size. Golkar et al.,
209 (2018) have compared the emulsifying properties of Persian gum (Amygdalus scoparia Spach) and gum
210 Arabic. Their results indicated that increasing the amount of PG from 0.5 to 1.5 % (w/v) decreased the
211 emulsion droplet size, supporting the findings of current study. Current study also shows that increasing
212 the chitosan concentration causes the increase in emulsion droplet size. This is because increasing
213 chitosan concentration in solution would lead to an increase in the NH3+ group, which induce a higher
214 repulsion force, and therefore, increasing the emulsion droplet diameter (Tan, Selig, & Abbaspourrad,
215 2018). Furthermore, an increase of PG concentration leads to a narrower emulsion droplet size
216 distribution (Table 1). The PG:Chi 2:1 sample has the lowest PDI (0.120 ± 0.002) suggesting a
217 homogeneous droplet size distribution of this particular emulsion compared to others. Given the fact that
218 PG is a non-absorbing polymer and has a high molecular weight, the decrease in the mean droplet size
219 and droplet size distribution could be due to the increase of viscosity in the continuous phase and the
220 creation of a three-dimensional network by the polymer. These phenomena could reduce the droplets
221 movement and hence, their collisions number (Golkar, et al., 2018).

222 3.3. Zeta potential of emulsions

223 Table 1 presents the zeta potential of the emulsions studied. All the emulsions had positive zeta potential
224 values ranging from 33.4 to 40.3 mV. The maximum zeta potential were measured in the PG:Chi 1:1
225 sample (40.3 ± 2.4mV). At the selected pH value of 3.6, chitosan solutions gave positive zeta potential
226 values between +30 mV to +60 mV. This is related to the presence of acid that causes the protonation of
227 the NH2 groups in the chitosan molecules to the positively charged NH3+ groups (Mounsey, O’Kennedy,
228 Fenelon, & Brodkorb, 2008). After addition of PG, electrostatic interactions may take place in the system
229 between the NH3+ groups and the COO- groups. Mixing these two polymers gave positive zeta potential
230 values lower than that of the chitosan alone. In the PG:Chi 1:2 sample, less gum polyanions was present
231 in the mixed dispersion, the increased of positive charge was therefore related to the present of more
232 residual amine groups on the surface of chitosan macromolecule (Tan, et al., 2018). According to the
233 results, the zeta potential values were in agreement with the observation in emulsion stability. From
234 literature, an emulsion with good physical stability can be obtained when the zeta potential value is higher
235 than 30 mV, as the presence of strong electrostatic repulsions prevent the contact of emulsion droplets
236 with each other, and decrease the probability of flocculation in the emulsion system (Guzey &
237 McClements, 2006).
238
239 3.4. Surface tension of emulsions

8
240 Surface tensions of the different emulsions were presented in Table 1. Emulsions with a low surface
241 tension are less stable than those with a higher one. The presence of oil released in the system could lead
242 to a decrease in the surface tension of emulsions (Almeida, Larentis, & Ferraz, 2015), since the surface
243 tension of the oil is lower than those showed by the combination of Persian gum and chitosan. An
244 increase in PG concentration has a significant influence on the surface tension of the emulsions (P <
245 0.05). The maximum and minimum surface tension were recorded in the PG:Chi 2:1 and PG:Chi 1:2
246 samples with the values of 56 ± 0.3 mN.m-1 and 53 ± 0.3 mN.m-1, respectively.

247 3.5. Viscosity of emulsions

248 The apparent viscosity of the emulsions (Table 1) varied from 0.19 ± 0.02 to 1.09 ± 0.04 Pa·s (P < 0.05).
249 PG concentration appears to be the main factor affecting the viscosity of the emulsions. When the PG
250 concentration in the emulsions increased, the apparent viscosity increased. This is due to the high
251 molecular weight of PG (Weiss, Kanjanapongkul, Wongsasulak, & Yoovidhya, 2012). At higher
252 concentrations of polysaccharide, creaming is retarded because even if emulsion droplets are aggregated,
253 they are not able to move freely due to the high viscosity or formation of the 3-dimentional gel network
254 by the polysaccharides (McClements, 2000).
255

256 3.6. Encapsulation efficiency (EE)

257 The encapsulation efficiency was in the range of 63 ± 2 to 86 ± 1% for the nano-encapsulated particles
258 (Table 2). The results was significantly influenced by the wall material ratio (p < 0.05). The lowest EE
259 was obtained in the PG:Chi 1:2 sample while the highest EE was achieved in the PG:Chi 2:1 samples.
260 According to the results from emulsion studies, the combination of PG:Chi with 2:1 ratio showed the best
261 emulsifying characteristics at the oil-water interface, and therefore could bring a better nano-
262 encapsulation entrapment for the core materials. On the other hand, viscosity of the system could be
263 another reason. Tonon et al., (2011) have underlined the existence of a positive relationship between
264 encapsulation efficiency and high viscosity values, which reduced the diffusion of oil to the nano-capsule
265 surface (Tonon, Grosso, & Hubinger, 2011). Similarly, in the present study, PG:Chi 2:1 sample has
266 higher viscosity than others. The high EE value is one of the most desirable properties in the
267 encapsulation technique as it increases the loading of core material in the particles while giving shelf life
268 extension.

269 3.7. Droplet size, zeta potential and PDI of the reconstituted emulsions

9
270 Current results showed that the use of different amount of biopolymers influence the droplet mean
271 diameter of the reconstituted emulsion, ranging from 65 to 271 nm for different formulations (Fig. 2). FO-
272 GEO encapsulated by PG:Chi 2:1 wall materials has a smallest droplet size as compared to other
273 formulations. Many studies have shown that small emulsion droplet sizes enhanced the retention of
274 entrapped compounds (Donsì, Annunziata, Sessa, & Ferrari, 2011). Similar trend was observed in the PDI
275 data for the PG:Chi 2:1 sample where minimum PDI of 0.385 was obtained among the three formulations
276 (P < 0.05). In contrast, the PG:Chi 1:2 sample showed the maximum value of 0.483 (Table 2). In
277 agreement with previous studies, the droplet size distribution in this study is appropriate for use in the
278 pharmaceutical and food industries. Gonçalves et al., (2012) expressed that the PDI value close to 0.4 was
279 acceptable for these kinds of system (Gonçalves, Mertins, Pohlmann, Silveira, & Guterres, 2012).

280 The zeta potential values for the reconstituted emulsions of FO-GEO were between 34 ± 1 and 44 ± 1 mV
281 (Table 2). The measured zeta potentials are higher than 30 mV, therefore the colloidal suspension
282 obtained could be considered as stable (Sherman, 1970) since the charged particles neutralize each other
283 to overcome their tendency to aggregation. The results of zeta potential were in agreement with emulsion
284 stability as discussed above.

285 3.8. Morphology of nano-capsules

286 The SEM images of the freeze-dried FO-GEO nano-capsules are shown in Fig. 3. The FO-GEO nano-
287 capsules have an agglomerated structure with spherical shape particles ranging from 65 to 271 nm.
288 Furthermore, the agglomerated particles were observed for the PG:Chi 1:2 sample, whereas nanoparticles
289 from the PG:Chi 1:1 and PG:Chi 2:1 samples are more individual, which is in accordance to the zeta
290 potential values. The particles from the PG: Chi 1:1 and PG: Chi 2:1 formulations are smaller compared
291 to those from the PG:Chi 1:2 formulation. This indicated that the increase of chitosan in the formulation
292 increase the particle size, similar results were also reflected in the droplet size of the reconstituted
293 emulsion as mentioned above.
294

295 3.9. Thermal properties of nano-capsules

296 The DSC thermograms of FO-GEO nano-capsules and different ratios of polyelectrolyte materials are
297 shown in Fig 4. Two thermal events were observed for the samples. The first one was from 50 to 150 °C,
298 corresponding to the loss of water (both free and bound molecules) associated with the hydrophilic groups
299 of the polymer and the dehydration of the polymeric shell (Cortés-Camargo, et al., 2017). The second
300 event was in the regions of 250 to 270 °C for different combinations of wall materials and wall/core
301 materials related to thermal degradation. The peaks were at: 260°C for FO-GEO nano-capsules with

10
302 PG:Chi 1:1 ratio; 259°C for FO-GEO nano-capsules with PG:Chi 2:1 ratio; 272°C for FO-GEO nano-
303 capsules with PG:Chi 1:2 ratio; 265°C for PG:Chi 1:1ratio; 257°C for PG:Chi 2:1 ratio; and 270 °C for
304 PG:Chi 1:2 ratio. Shifts in the peak position indicated the interactions between the wall and the core
305 materials which led to the formation of new chemical structural. When the chitosan amount increased, the
306 temperature of the thermal degradation shifted to higher temperature, leading to an increase in thermal
307 stability, as reported by previous studies (Mounsey, et al., 2008). This phenomena can be related to
308 formation of strong hydrogen bonds in the PG:Chi complex. The endothermic peak between 120 to 200
309 °C may be associated to the cleavage of the electrostatic interactions between the two oppositely charged
310 wall materials (Pendekal & Tegginamat, 2012). On the other hand, the presence of FO-GEO in the
311 nanoparticles only gave a small effect on the thermal transitions, since the temperatures were slightly
312 decreased from 2 to 5 °C.
313

314 3.10. Crystalline properties of nano-capsules

315 The results of X-Ray Diffraction (XRD) analysis are presented in Fig.5. The XRD patterns of PG:Chi
316 exhibit broad diffraction peaks at 2θ = 10° and 19.5° relate to the typical fingerprints of semi-crystalline
317 chitosan, corresponding to crystalline I (Bangyekan, Aht-Ong, & Srikulkit, 2006). Nevertheless, it can be
318 seen that the height of both peaks are in the following order: PG:Chi 1:2 > PG:Chi 1:1 > PG:Chi 2:1. The
319 decrease of crystallinity indicated a decreased in the intermolecular interactions (Ravi, Song, Wang,
320 Nadimicherla, & Zhang, 2016). The presence of two peaks at 10 and 20° is linked to the existence of
321 crystalline structures of chitosan. The first one corresponds to the hydrated form with the presence of
322 adsorbed water molecules in the network, and the second one relates to the more regular anhydrous form
323 (Ogawa, Yui, & Miya, 1992). The peak intensity decreased with the increase of PG in the wall material,
324 indicating that PG addition gave rise to more amorphous structure. The estimated crystallinity index was
325 found to be decreased from 48.8% (for PG:Chi 1:2) to 42.7% (for PG:Chi 2:1) as shown in Table 2.
326 Accordingly, it is clear that the decreasing in the crystallinity index can be attributed to the increasing of
327 the PG in the wall material. These results imply that the presence of PG can enhance the mobility of
328 polymeric chains, reducing the crystalline structures by interfering with the molecular chain arrangement
329 in chitosan. Previous study has reported that the presence of gum could prevent the formation of hydrogen
330 bonds between the amino and hydroxyl groups of chitosan (Li, Xie, Lin, Xie, & Ma, 2009) and this could
331 be a reason for the reduced crystallinity when PG was added in the wall material.
332

333 3.11. Interactions of wall materials

11
334 Table 3 presents the FTIR analysis of FO-GEO, FO-GEO encapsulated with different ratios of PG:Chi
335 and the wall materials without incorporation of FO-GEO. For the FO-GEO samples, a broad
336 transmittance band was observed at 3472 cm-1, relating to the stretching vibration of –OH groups. C-H
337 stretching of aliphatic =CH, due to un-conjugated cis-double bonds of FO-GEO, and unsaturated double
338 bond (-C=C-) were observed at 3011 cm-1 and 1655 cm-1, respectively. Asymmetrical and symmetrical C-
339 H stretching and bending vibrations of the methyl and methylene groups were detected at 2929, 2854,
340 1464, and 1377 cm−1, and with the presence of a shoulder for the asymmetric C-H stretching of CH3 on
341 2926 cm-1 at 2956 cm-1 (Grehk, Berger, & Bexell, 2008). The in-plane rocking vibration of the CH2
342 groups was found at 721 cm−1. The strong transmittance band at 1747 cm-1 corresponded to the
343 characteristic bond of the ester carbonyl functional group of the triglyceride (Osorio, Haughey, Elliott, &
344 Koidis, 2015). The stretching vibrations of C-O and CO ester groups were also present at 1234, 1160 and
345 1098 cm-1, and at 1118 cm-1, respectively.

346 Characteristic bands of polysaccharides were detected between 3500 cm−1 and 2800 cm−1 for PG:Chi
347 (without FO-GEO). Thus, the broad band at 3348 cm−1 was assigned to the presence of hydroxyl and
348 amino strectchings. Furthermore, this broad band underlines hydrogen bonding involved in the interaction
349 between PG and Chi (Fan, Rong, Zhang, & Chen, 2018). The C-H stretching vibrations were also
350 observed at 2926 cm−1. The stretching vibrations of carbonyl group were present between 1800 and 1500
351 cm-1. In this spectral region, the bands at 1745 and 1630 cm-1 were assigned to the methyl ester group
352 (COOCH3) and the undissociated carboxylic acid (COOH), and to the asymmetric stretching vibration of
353 the carbonyl group of the carboxylate anion (-COO-) (G. Zhao, Dai, Wang, & Zhao, 2015), respectively.
354 The C=O stretching vibration of the amide group (amide I) of the acetylated units of chitosan cuold be
355 found at 1645 cm-1. Interaction between amine group and carbonyl group of carboxylate of the
356 polyelectrolytes was detected with the presence broad band between 1600 and 1500 cm-1 (Maciel,
357 Yoshida, & Franco, 2015). The C-O-C linkage in the glycosidic structure was also detected between
358 1150–1040 cm−1. Furthermore, the presence of the carboxylic acid (-COOH) moieties at 1255-1260 cm−1,
359 indicated that some acid groups did not interact with the NH3+ of the chitosan. Concerning the spectra of
360 PG: Chi nano-capsules (with FO-GEO), the characteristic bands of the wall materials and FO-GEO as
361 observed, there is good evidence showing that FO-GEO have been entrapped in the nano-capsules.

362 4. Conclusion

363 This work reveled a novel combination of wall materials (Persian gum- chitosan) for nano-capsulating
364 FO-GEO. Overall, the physicochemical characteristics of FO-GEO nano-capsules are affected by the ratio
365 of Persian gum (PG) and chitosan (Chi) as wall materials. Higher concentration of PG increased emulsion

12
366 stability, surface tension and viscosity while decreased droplet size and PDI of emulsion droplets, leading
367 to smaller nano-capsules and better encapsulation efficiency. The results of DSC, XRD and FTIR
368 confirmed that the FO-GEO were successfully encapsulated by the wall materials. The FO-GEO nano-
369 capsules produced were shown to have excellent thermal stability of > 250 °C, implying that the
370 combination of Persian gum and chitosan was an excellent wall material for encapsulating these oils. This
371 property is very important as many food processing methods involve the application of high temperature.
372 Good thermal stability indicates enhanced protection and stability of the encapsulated compounds, and
373 this aspect is critically important for highly oxidized core material such as fish oil. Based on current
374 results, the use of Persian gum and chitosan mixture for encapsulation could be a promising move in
375 delivery of FO-GEO. For practical application, other important aspects should be taken into
376 consideration, including stability of nano-capsules during storage, effect of GEO on the oxidative stability
377 of FO and nano-capsules, application in food products, sensory evaluation and product shelf life. We shall
378 address these aspects in our future work to confirm the applicability of this novel combination of wall
379 material for food and pharmaceutical applications.

380

381 Conflict of interest

382 The authors declare no financial or other conflicts of interest.

383

384 References
385 Abbasi, S. (2017). Challenges towards characterization and applications of a novel hydrocolloid: Persian
386 gum. Current Opinion in Colloid & Interface Science, 28, 37-45.
387 Abbasi, S., & Mohammadi, S. (2013). Stabilization of milk–orange juice mixture using Persian gum:
388 Efficiency and mechanism. Food Bioscience, 2, 53-60.
389 Almeida, T. C. A., Larentis, A. L., & Ferraz, H. C. (2015). Evaluation of the stability of concentrated
390 emulsions for lemon beverages using sequential experimental designs. PloS one, 10(3),
391 e0118690.
392 Bae, E., & Lee, S. (2008). Microencapsulation of avocado oil by spray drying using whey protein and
393 maltodextrin. Journal of Microencapsulation, 25(8), 549-560.
394 Bangyekan, C., Aht-Ong, D., & Srikulkit, K. (2006). Preparation and properties evaluation of chitosan-
395 coated cassava starch films. Carbohydrate Polymers, 63(1), 61-71.
396 Chatterjee, S., & Judeh, Z. M. (2016). Impact of encapsulation on the physicochemical properties and
397 gastrointestinal stability of fish oil. LWT-Food Science and Technology, 65, 206-213.

13
398 Cortés-Camargo, S., Cruz-Olivares, J., Barragán-Huerta, B. E., Dublán-García, O., Román-Guerrero, A.,
399 & Pérez-Alonso, C. (2017). Microencapsulation by spray drying of lemon essential oil:
400 Evaluation of mixtures of mesquite gum–nopal mucilage as new wall materials. Journal of
401 Microencapsulation, 34(4), 395-407.
402 Dickinson, E. (1992). Introduction to food colloids: Oxford University Press, Oxford, UK, pp. 1–207.
403 Donsì, F., Annunziata, M., Sessa, M., & Ferrari, G. (2011). Nanoencapsulation of essential oils to
404 enhance their antimicrobial activity in foods. LWT-Food Science and Technology, 44(9), 1908-
405 1914.
406 Fan, L. F., Rong, M. Z., Zhang, M. Q., & Chen, X. D. (2018). A Very Simple Strategy for Preparing
407 External Stress‐Free Two‐Way Shape Memory Polymers by Making Use of Hydrogen Bonds.
408 Macromolecular Rapid Communications, 1700714.
409 Focher, B., Beltrame, P. L., Naggi, A., & Torri, G. (1990). Alkaline N-deacetylation of chitin enhanced
410 by flash treatments. Reaction kinetics and structure modifications. Carbohydrate Polymers,
411 12,405-418.
412 García-Moreno, P. J., Stephansen, K., van der Kruijs, J., Guadix, A., Guadix, E. M., Chronakis, I. S., &
413 Jacobsen, C. (2016). Encapsulation of fish oil in nanofibers by emulsion electrospinning: Physical
414 characterization and oxidative stability. Journal of Food Engineering, 183, 39-49.
415 Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., & Saurel, R. (2007). Applications of spray-
416 drying in microencapsulation of food ingredients: An overview. Food Research International,
417 40(9), 1107-1121.
418 Golkar, A., Taghavi, S. M., & Aghili Dehnavi, F. (2018). Emulsifying properties of Persian gum
419 (Amygdalus scoparia Spach) as compared with gum Arabic. International Journal of Food
420 Properties, 21, 416-436.

421 Gonçalves, M. C., Mertins, O., Pohlmann, A. R., Silveira, N. P., & Guterres, S. S. (2012). Chitosan
422 coated liposomes as an innovative nanocarrier for drugs. Journal of Biomedical Nanotechnology,
423 8(2), 240-250.
424 Grehk, T. M., Berger, R., & Bexell, U. (2008). Investigation of the drying process of linseed oil using
425 FTIR and ToF-SIMS. Journal of Physics: Conference Series, 100, 012019.
426 Gu, Y. S., Decker, E. A., & McClements, D. J. (2007). Application of multi-component biopolymer
427 layers to improve the freeze–thaw stability of oil-in-water emulsions: β-lactoglobulin–i-
428 carrageenan–gelatin. Journal of Food Engineering, 80, 1246–1254.
429 Guzey, D., & McClements, D. J. (2006). Formation, stability and properties of multilayer emulsions for
430 application in the food industry. Advances in Colloid and Interface Science, 128, 227-248.

14
431 Jeyakumari, A., Janarthanan, G., Chouksey, M., & Venkateshwarlu, G. (2016). Effect of fish oil
432 encapsulates incorporation on the physico-chemical and sensory properties of cookies. Journal of
433 Food Science and Technology, 53(1), 856-863.
434 Klinkesorn, U., & McClements, D. J. (2009). Influence of chitosan on stability and lipase digestibility of
435 lecithin-stabilized tuna oil-in-water emulsions. Food Chemistry, 114(4), 1308-1315.
436 Kolanowski, W., Ziolkowski, M., Weißbrodt, J., Kunz, B., & Laufenberg, G. (2006). Microencapsulation
437 of fish oil by spray drying--impact on oxidative stability. Part 1. European Food Research and
438 Technology, 222(3-4), 336-342.
439 Li, X., Xie, H., Lin, J., Xie, W., & Ma, X. (2009). Characterization and biodegradation of chitosan–
440 alginate polyelectrolyte complexes. Polymer Degradation and Stability, 94(1), 1-6.
441 Liu, D., Li, Z., Zhu, Y., Li, Z., & Kumar, R. (2014). Recycled chitosan nanofibril as an effective Cu (II),
442 Pb (II) and Cd (II) ionic chelating agent: adsorption and desorption performance. Carbohydrate
443 Polymers, 111, 469-476.
444 Maciel, V. B. V., Yoshida, C. M., & Franco, T. T. (2015). Chitosan/pectin polyelectrolyte complex as a
445 pH indicator. Carbohydrate Polymers, 132, 537-545.
446 McClements, D. (2000). Comments on viscosity enhancement and depletion flocculation by
447 polysaccharides. Food Hydrocolloids, 14(2), 173-177.
448 Mounsey, J. S., O’Kennedy, B. T., Fenelon, M. A., & Brodkorb, A. (2008). The effect of heating on β-
449 lactoglobulin–chitosan mixtures as influenced by pH and ionic strength. Food Hydrocolloids,
450 22(1), 65-73.
451 Ogawa, K., Yui, T., & Miya, M. (1992). Dependence on the preparation procedure of the polymorphism
452 and crystallinity of chitosan membranes. Bioscience, Biotechnology, and Biochemistry, 56(6),
453 858-862.
454 Osorio, M., Haughey, S., Elliott, C., & Koidis, A. (2015). Identification of vegetable oil botanical
455 speciation in refined vegetable oil blends using an innovative combination of chromatographic
456 and spectroscopic techniques. Food Chemistry, 189, 67-73.
457 Pareta, R., & Edirisinghe, M. J. (2006). A novel method for the preparation of biodegradable
458 microspheres for protein drug delivery. Journal of the Royal Society Interface, 3(9), 573–582.
459 Pendekal, M. S., & Tegginamat, P. K. (2012). Development and characterization of chitosan-
460 polycarbophil interpolyelectrolyte complex-based 5-fluorouracil formulations for buccal, vaginal
461 and rectal application. DARU Journal of Pharmaceutical Sciences, 20(1), 67.
462 Ravi, M., Song, S., Wang, J., Nadimicherla, R., & Zhang, Z. (2016). Preparation and characterization of
463 biodegradable poly (ε-caprolactone)-based gel polymer electrolyte films. Ionics, 22(5), 661-670.

15
464 Sanguansri, L., Udabage, P., Bhail, S., Ying, D., Cheng, L., Shen, Z., & Augustin, M. (2016).
465 Microencapsulated Fish Oil Powder Formulation with Improved Resistance to Oil Leakage
466 During Powder Compression. Journal of the American Oil Chemists' Society, 93(5), 701-710.
467 Shamaei, S., Kharaghani, A., Seiiedlou, S. S., Aghbashlo, M., Sondej, F., & Tsotsas, E. (2016). Drying
468 behavior and locking point of single droplets containing functional oil. Advanced Powder
469 Technology, 27(4), 1750-1760.
470 Sherman, P. (1970). Rheology of dispersed systems. Industrial Rheology, 97-183.
471 Tan, C., Selig, M. J., & Abbaspourrad, A. (2018). Anthocyanin stabilization by chitosan-chondroitin
472 sulfate polyelectrolyte complexation integrating catechin co-pigmentation. Carbohydrate
473 Polymers, 181, 124-131.
474 Tonon, R. V., Grosso, C. R., & Hubinger, M. D. (2011). Influence of emulsion composition and inlet air
475 temperature on the microencapsulation of flaxseed oil by spray drying. Food Research
476 International, 44(1), 282-289.
477 Weiss, J., Kanjanapongkul, K., Wongsasulak, S., & Yoovidhya, T. (2012). Electrospun fibers: fabrication,
478 functionalities and potential food industry applications. In Nanotechnology in the food, beverage
479 and nutraceutical industries (pp. 362-397): Elsevier.
480 Zhao, G., Dai, C., Wang, S., & Zhao, M. (2015). Synthesis and application of nonionic polyacrylamide
481 with controlled molecular weight for fracturing in low permeability oil reservoirs. Journal of
482 Applied Polymer Science, 132(11), 41637.
483

484

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485 Table 1 Properties of emulsions containing different ratios of Persian gum (PG) and chitosan (Chi)

PG: Chi Emulsion stability Surface tension Viscosity polydispersity Zeta potential
-1
ratio index (%) (mNm ) (Pa.s) index (mv)
1:1 98±0.5b 53±0.4b 0.31±0.01b 0.362±0.003b 40.3±2.4a
2:1 100±0.0a 56±0.3a 1.09±0.04a 0.120±0.002c 33.4±1.6b
1:2 80±1c 53±0.3b 0.19±0.02c 0.644±0.005a 38.6±0.9a

486 Different superscript letters indicate significant differences (P < 0.05) according to Duncan’s multiple
487 range test. Values are expressed as means ± SD (n=6)

488

489
490 Table 2 Encapsulation efficiency (%), reconstituted emulsion properties and crystallinity index of the fish
491 oil (FO)-garlic essential oil (GEO) nano-capsules produced from different ratios of Persian gum (PG):
492 Chitosan (Chi)

PG: Chi ratio Encapsulation Reconstituted emulsion properties Crystallinity index

efficiency (%) Zeta potential Polydispersity (%)
(mv) index
1:1 79±1b 44±1a 0.453±0.002b 46
a b c
2:1 86±1 34±1 0.385±0.004 42.7
c a a
1:2 63±2 43±1 0.483±0.001 48.8

493 Different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range test.
494 Values are expressed as means ± SD (n=6)
495

496

497

498

17
499 Table 3 FTIR analysis of Persian gum-chitosan wall material, fish oil (FO)-garlic essential oil (GEO)
500 sample and FO-GEO nano-capsules

Wavenumber (cm-1) Vibration modes

PG:Chi wall material FO-GEO sample FO-GEO nano-capsules

3700-3000 3472 3700-3000 ν(O-H) (overlapped to the νs(N-H))*

ν(C–H) of cis-alkene −HC=CH− of

3011 3011
unsaturated fatty acids

νas(C–H) from methyl (−CH3) groups

2970 2970
of lipids

2926 2926 2926 νas(C–H) from methylene (−CH2)

2858 2854 2856 νs(C–H) from methylene (−CH2)

ν(C=O) of ester functional groups

1747 1747
primarily from lipids and fatty acids

1715 1715 ν(-C=O)

1645, 1630 ν(-C=O), ν(-COO-)

1655 1655 ν(-C=C-)

1575, 1426 δ(N-H), ν (C-N)

δas(CH2)scissor from methylene

1464 1460
(−CH2) groups of lipids

δas(CH3) and δas(CH2) of lipids and

1450-1410 1450-1410
proteins

1417, 1375 δ(C-H)

δs(CH3) and δs(CH2) of lipids and

1380 1380
proteins

1260-1255 νs(C-O-H)

1160 1160-1150 γ(CH2) of lipids

1150, 1086, 1040 1160, 1098 1160-1150, 1098-1090 νas(C-O-C) and νs(C-O-C)

Overlapping of the CH2 rocking

720 720 vibration and the out-of-plane
vibration of cis-disubstituted olefins

501 ν : stretching, νas : asymmetric stretching, νs : symmetric stretching, δas : asymmetric deformation (bend),
502 δs : symmetric deformation (bend) and γ : out-of-plane deformation.

503

18
504 Captions for Figures

505 Fig 1. Droplet size distribution of emulsions prepared with different Persian gum (PG): chitosan (Chi)
506 ratios: PG: Chi 2:1 ( ), PG: Chi 1:1 ( ) and PG: Chi 1:2 ( )

507 Fig. 2. Particle size distribution of the fish oil (FO)-garlic essential oil (GEO) nano-capsules at (a) Persian
508 gum (PG): chitosan (Chi) 1:1 ratio; (b) PG: Chi 2:1 ratio; and (c) PG: Chi 1:2 ratio

509 Fig 3. SEM images (×20000) of fish oil (FO)-garlic essential oil (GEO) nano-capsules produced from
510 different ratio of wall materials: (a) Persian gum (PG): chitosan (Chi) 1:1; (b) PG: Chi 2:1; (c) PG: Chi
511 1:2

512 Fig. 4. DSC thermograms of: (i) fish oil (FO)-garlic essential oil (GEO) nano-capsules with Persian gum
513 (PG): chitosan (Chi) 1:1 wall material; (ii) FO-GEO nano-capsules with PG: Chi 2:1 wall material; (iii)
514 FO-GEO nano-capsules with PG: Chi 1:2 wall material; (iv) PG: Chi 1:1 wall material; (v) PG: Chi 2:1
515 wall material; and (vi) PG: Chi 1:2 wall material
516
517 Fig. 5. X-Ray Diffraction patterns of different fish oil (FO)-garlic essential oil (GEO) nano-capsules: PG:
518 Chi 2:1 ( ), PG: Chi 1:1 ( ) and PG: Chi 1:2 ( ).

519

520

19
521

522 Fig. 1.

20
523

524 (a)

21
525

526 (b)

22
527

528 (c)

529

530 Fig. 2.

23
531

532 (a)

533

534

535 (b)

536

24
537

538 (c)

539 Fig. 3.

540

25
541

542

543 Fig. 4.

544

26
545

546 Fig. 5.

547

548

27
Highlights

Nano-capsules with Fish oil-Garlic essential oil (FO-GEO) were successfully produced

Nano-capsules produced have good physicochemical properties indicating good stability

Persian gum-chitosan of ratio 2:1 is recommended for FO-GEO encapsulation

FTIR, DSC and X-XRD confirmed the wall material interactions and encapsulation of FO-GEO

Thermal stability of nano-capsules justified their suitability for food processing

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