Development of chitosan films containing β-cyclodextrin inclusion complex for controlled release of bioactives

Journal Pre-proof Development of chitosan films containing β-cyclodextrin inclusion complex for controlled release of bioactives I. Zarandona, C. Barba, P. Guerrero, K. de la Caba, J. Maté PII:

S0268-005X(19)32273-8

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105720

Reference:

FOOHYD 105720

To appear in:

Food Hydrocolloids

Received Date: 28 September 2019 Revised Date:

3 January 2020

Accepted Date: 27 January 2020

Please cite this article as: Zarandona, I., Barba, C., Guerrero, P., de la Caba, K., Maté, J., Development of chitosan films containing β-cyclodextrin inclusion complex for controlled release of bioactives, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105720. 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.

Author statement I. Zarandona: Investigation, Writing-Original Draft C. Barba: Conceptualization, Investigation, Funding acquisition P. Guerrero: Formal analysis, Supervision, Writing-Review & Editing K. de la Caba: Supervision, Writing-Review & Editing, Funding acquisition J. Maté: Conceptualization, Resources, Writing-Review & Editing,

    60

2-phenylethanol Retention efficiency (%)

β-cyclodextrin (β-CD)

40

20

0

-CD

H3C HO

OH

NH2

HO

O HO OH

NH O O

O H2N

-CD

O

O

O

O

-CD

OH

Chitosan films with β-CD:2-phenylethanol inclusion complexes

Development of chitosan films containing β -cyclodextrin inclusion complex for controlled release of bioactives. I. Zarandonaa, C. Barbab, P. Guerreroa*, K. de la Cabaa, J. Matéb a

BIOMAT research group, University of the Basque Country (UPV/EHU), Escuela de

Ingeniería de Gipuzkoa, Plaza de Europa 1, 20018 Donostia-San Sebastián, Spain. b

Department of Agronomy, Biotechnology and Food Science, Public University of

Navarre (UPNa), Campus Arrosadía s/n, Edificio Los Olivos, Pamplona 31006, Spain.

*Corresponding author: Pedro Guerrero e-mail: [email protected] telephone number: +34943018535 1

1

Abstract

2

2-phenyl ethanol is a natural compound, which have many applications due to its nice

3

fragrance, bacteriostatic and antifungal character. However, it is difficult to keep it

4

stable and it is highly volatile. In this work, chitosan films with 2-phenyl ethanol were

5

developed and inclusion complexes with cyclodextrins (CDs) were prepared in order to

6

have a controlled release of 2-phenyl ethanol. β-CD was selected to develop the

7

inclusion complex since it showed higher retention yield (45 %, molar basis) than α- or

8

γ-CDs. Chitosan films incorporated with β-CD:2-phenyl ethanol were homogeneous,

9

transparent and colorless, and showed high mechanical resistance. Furthermore, the

10

release results of the films without the inclusion complex indicated that 2-phenyl

11

ethanol was evaporated during the film preparation, and only an 8 % of the total

12

bioactive was retained in the film, while more than 90 % of 2-phenyl ethanol was

13

retained in the films with the inclusion complex.

14

Keywords: chitosan film; inclusion complex; β-cyclodextrin; 2-phenylethanol; bioactive

15

retention; controlled release.

2

16

1. Introduction

17

The use of active compounds is a strategy to confer functional properties, such as

18

antimicrobial, antioxidant or ultraviolet (UV) light barrier properties, to films. However,

19

many of these compounds, such as vitamins, antioxidants or flavors, are not stable

20

under the preparation, storage and/or use conditions (Hosseini, Nahvi, & Zandi, 2019;

21

Li, Maldonado, Malmr, Rouf, Hua, & Kokini, 2019). Therefore, the use of encapsulation

22

agents for active compounds become necessary to protect the compound from

23

volatilization or possible reactions with external agents, and also to control a sustained

24

release (Reineccius, 2009). Among encapsulation processes, spray-drying, spray-

25

cooling, extrusion or molecular inclusion are employed in industrial processes, but only

26

molecular inclusion occurs at molecular level and, thus, one molecule of the active

27

compound is trapped in the cavity of the host molecule (Reineccius, Reineccius, &

28

Peppard, 2002). The union between the active compound and the host molecule is

29

called inclusion complex. One of these host molecules is cyclodextrin, since the Food

30

and Agriculture Organization (FAO) of the United Nations recognizes it as additive

31

(FAO, 2019).

32

Cyclodextrins (CD) are cyclic oligosaccharides constituted of glucose molecules joined

33

together by α-1,4 bonds (Szente & Szejtli, 1999). CDs are hollow truncated cone

34

structures with an external hydrophilic character and an internal hydrophobic character

35

(Simionato, Domingues, Nerín, & Silva, 2019; Huang, Xu, Ge, & Cheng, 2019).

36

Therefore, it is a suitable molecule to host a variety of bioactives, including those that

37

are non-soluble in water since interactions between cyclodextrins and guest molecules

38

include hydrophobic forces as well as hydrogen bonding (Zhou, Lu, Zhou, & Liu, 2019).

39

Depending on the glucopyranose units conforming the molecule, the most common

40

CDs are α-, β- or γ-CDs, composed of 6, 7 or 8 glucopyranose units, respectively

41

(Prabu & Mohamad, 2020). Due to the variety of guest molecules that can host and

42

their acceptability as food additives, CDs are widely used in food and pharmaceutical 3

43

industries. Regarding food industry, CDs can be employed for different applications,

44

such as food supplement for spaghetti, adding pumpkin oil (Durante et al., 2019), as

45

flavor masking, reducing the high intensity of sourness, bitterness and astringency

46

flavor in lingonberry juice (Kalanne et al., 2019), or as thermal stabilizers (Yoshikiyo et

47

al., 2019). In the pharmaceutical industry, CDs are used to increase drug permeability,

48

solubility and stability and to enhance organoleptic characteristics (Chantasart, &

49

Rakkaew, 2019; Santana, Nadvorny, Passos, Soares, & Soares-Sobrinho, 2019; Yildiz,

50

& Uyar, 2019). Regarding preparation, there are different methods for the inclusion

51

complex preparation, such as freeze-drying, spray drying or coprecipitation (Suzuki et

52

al., 2019).

53

CDs can be used in a variety of matrixes. Morin-β-CD complexes have been

54

incorporated into gelatin to prepare antioxidant films (Yuan et al., 2019). Also

55

antimicrobials, such as citral, have been used to prepare β-CD inclusion complexes to

56

preserve bioactivity during melt extrusion of EVOH films (Chen, Li, Ma, Mcdonald, &

57

Wang, 2019). In order to provide films with both antioxidant and antibacterial

58

properties, essential oils have been encapsulated into β-CD to promote cumulative

59

release from chitosan films for food packaging applications (Adel, Ibrahim, El-Shafei, &

60

Al-Shemy, 2019). In this regard, several works have been reported in relation to the

61

use of chitosan with CD inclusion complexes for controlled release of bioactives such

62

as resveratrol (Zhang, Cao, Ma, Chen, & Li, 2017), carvacrol (Andrade-Del Olmo,

63

Pérez-Álvarez, Hernáez, Ruiz-Rubio, & Vilas-Vilela, 2019), or gallic acid (Munhuweyi,

64

Caleb, Reenen, & Opara, 2018).

65

The goal of this work was to assess the effectiveness of cyclodextrin inclusion

66

complexes as stabilizers and release controllers of 2-phenyl ethanol in chitosan films.

67

2-phenyl ethanol is naturally present in more than one hundred food products (Nijssen,

68

Ingen-Visscher, & van Donders, 2016) and it is used in food industry to enhance

69

flavour and odor. Although it has bacteriostatic and antifungal character, it is very 4

70

volatile and highly susceptible to oxidation, thus, storing and keeping it stable is a

71

troublesome (Yadav, & Lawate, 2011). In order to address this challenge, 2-phenyl

72

ethanol has been microencapsulated with methylcellulose, alginate and carboxymethyl

73

chitosan (Qiu, Tian, Yin, Zhou, & Zhu, 2019). However, to the best of our knowledge,

74

this work assesses the effectiveness of 2-phenyl ethanol trapped into α-, β-, and γ-CDs

75

for the first time. Additionally, chitosan films with β-CD:2-phenyl ethanol were

76

developed and the optical, physicochemical and mechanical properties of the films

77

were characterized. In order to assess if the addition of the inclusion complex alters the

78

properties of the film, neat chitosan and chitosan with 2-phenyl ethanol films were

79

prepared. Furthermore, the release of 2-phenyl ethanol from chitosan films into a fatty

80

food simulant was determined.

81

2. Materials and methods

82

2.1. Materials

83

Chitosan (CHI), with a molecular weight of 375 kDa and a deacetylation degree above

84

75 %, was supplied by Sigma-Aldrich, Spain. Acetic acid (1 N) and glycerol (GLY, 99.0

85

% purity), used as solvent and plasticizer, respectively, were supplied by Panreac,

86

Spain. 2-phenyl ethanol and α- and β-cyclodextrins (CAVAMAX® w6 and w7,

87

respectively), used for the development of inclusion complexes, were food grade and

88

supplied by Wacker Chemical, Spain; while γ-cyclodextrins were provided by Roquette,

89

France.

90

2.2. Inclusion complex preparation

91

The inclusion complexes of 2-phenyl ethanol were prepared according to Barba,

92

Eguinoa, & Maté (2015) with some modifications. Firstly, 10 % (w/w) cyclodextrins

93

were hydratated for 10 min; then, 2-phenyl ethanol was added in equimolar ratio and

94

the mixture was mechanically stirred for 24 h. In order to obtain the dry powder, a B-

95

191, Büchi spray-dryer was used at the following conditions: 100 % aspirator capacity, 5

96

inlet temperature of 180 ºC, and pump at 4 mL/min. The powder was collected and

97

stored in vials at -20 ºC.

98

2.3. FTIR analysis of α-, β-, and γ-cyclodextrin complexes

99

Fourier-transform infrared (FTIR) spectroscopy was carried out with a Nicolet Avatar

100

260. Inclusion complexes (α-CD:2-phenyl ethanol, β-CD:2-phenyl ethanol, and γ-CD:2-

101

phenyl ethanol), cyclodextrins (α-CD; β-CD and γ-CD) and 2-phenyl ethanol were

102

milled with anhydrous KBr and the pellet was formed by compression. The spectra

103

were recorded between 4000-800 cm-1 with 32 scans and a resolution of 4 cm-1.

104

2.4. Retention of 2-phenyl ethanol cyclodextrin

105

2-phenyl ethanol was extracted from cyclodextrins by liquid-liquid extraction following

106

the method of Charve & Reineccius (2009) with some modifications. The analysis was

107

carried out one week after the inclusion complex was formed. 0.15 g of the inclusion

108

complex was weighed and added into a centrifuge glass tube and 5 mL hexane and 10

109

mL distilled water were added. The mixture was shaken energetically for 2 min and

110

then, vortexed for 2 min. The samples were put into a bath at 85 ºC and shacked for 30

111

min. The organic phase was gathered into a 50 mL flask. For each sample, three

112

extractions were carried out, adding the organic phase into the 50 mL flask. A fourth

113

extraction was done to confirm that the extraction was completed. From a 1:10

114

solution, 1 µL was injected into a Hewlett-Packard 5890 series II gas chromatography

115

spectrometer (Agilent Technology, Barcelona, Spain), equipped with a flame ionization

116

detector (FID) and a Supra Wax 280 column (1.0 µm x 0.53 mm, 30 m). The gas carrier

117

employed was helium, the injection temperature was 250 ºC, the oven temperature

118

program was started at 40 ºC with a temperature ramp of 5 ºC/min up to 220 ºC (1

119

min), and the detector temperature was set at 300 ºC. In order to avoid the fluctuation

120

of the signal, an internal standard (1-heptanol) was added to the calibration standards

6

121

and samples. The retention of 2-phenyl ethanol was calculated as the ratio of

122

experimental concentration over the theoretical content.

123

2.5. Films preparation

124

Chitosan films were prepared by solution casting. 1 wt % chitosan was dissolved in 1

125

wt % acetic acid solution under stirring for 45 min. Then, 10 wt % 2-phenyl ethanol or

126

10 wt % β-CD:2-phenyl ethanol (based on chitosan) was added and stirring was

127

continued for other 30 min. Finally, 15 wt % glycerol (based on chitosan) was added

128

into some solutions as plasticizer and other 30 min of stirring were needed until total

129

homogenization of the mixture. The solution was casted into Petri dishes and dried at

130

room temperature for 48 h. In total, six compositions were analyzed: control films,

131

named as CHI0GLY and CHI15GLY as a function of glycerol content; chitosan films

132

with 2-phenyl ethanol, named as CHI0GLY2PE and CHI15GLY2PE; and chitosan films

133

with β-CD:2-phenyl ethanol inclusion complex, named as CHI0GLYCD:2PE and

134

CHI15GLYCD:2PE.

135

2.6. Optical properties of films

136

Color and gloss parameters of the films were analyzed. Color measurements were

137

recorded with a CR-400 Minolta Chroma Meter colorimeter (Konica Minolta, Tokyo,

138

Japan). Ten replicates were carried out for each sample. For the determination of color

139

parameters CIELAB scale was used: L* from 0 to 100 (from black to white), a* from –

140

to + (from greenness to redness), and b* from – to + (from blueness to yellowness).

141

The films were laid on a standard white plate with color parameter values of L*= 97.39,

142

a*= 0.03 and b*= 1.77.

143

Multi Gloss 268 Plus (Konica Minolta, Tokyo, Japan) was used for the determination of

144

gloss with an incidence angle of 60º according to ASTM D523 (ASTM, 2018). For each

145

composition, ten samples were assessed at room temperature.

7

146

Light-barrier capacity of films was measured by using a UV-VIS-NIR Shimadzu 3600

147

spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan) in the range of

148

200-800 nm.

149

2.7. Physicochemical properties of films

150

A Nicolet Nexus FTIR spectrometer (Thermo Fisher Scientific, Messachusetts, USA)

151

with a Golden Gate ATR sampling accessory was employed for collecting FTIR

152

spectra. The spectra were acquired between 4000 and 800 cm-1 with 32 scans for each

153

sample and a resolution of 4 cm-1.

154

A PANalytic Xpert Pro (PANalytical, Almelo, The Netherlands) X-ray diffraction (XRD)

155

equipment was employed with a diffraction unit at 40 kV and 40 mA. A Cu-Kα source

156

(λ= 1.5418 Å) was employed as radiation source and the data were collected between

157

2º and 34º (step size = 0.026, time per step = 118 s).

158

2.8. Mechanical and barrier properties of films

159

In order to determine the tensile strength (TS), elongation at break (EAB) and Young’s

160

modulus (E), an Instron 5967 electromechanical testing system (Instron,

161

Massachusetts, USA) was used. Samples were cut into dog bone-shaped of 4.75 mm

162

× 22.25 mm and tests were carried out according to ASTM D1708-13 (ASTM 2013). A

163

tensile load cell of 500 N was used and tensile tests were carried out with a crosshead

164

rate of 1 mm/min. Five replicates were tested for each sample.

165

Water vapor permeability (WVP) was tested with a PERME™ W3/0120 chamber

166

(Labthink Instruments Co. Ltd., Shandong, China) in a controlled humidity environment,

167

according to ASTM E96-00 (ASTM, 2000). Film discs were cut with a diameter of 7.40

168

cm and a test area of 33 cm2. The temperature and relative humidity were set up at 38

169

ºC and 90 %, respectively. Water vapor transmission rate (WVTR) was calculated as:



=

· 8

170

where G is the weight change (g), t is the time (s), and A is the film area that was

171

tested (cm2).

172

WVP was calculated by the following equation:



=

∆

·

173

where L is the film thickness (cm) and ∆P is the partial pressure difference of the water

174

vapor across the film (Pa).

175

Three replicates for each sample were reported for the WVP analysis.

176

2.9. Bioactive release

177

Film pieces (1 cm x 2 cm) were submerged into 8 mL of 95 % ethanol solution, used as

178

a fatty food simulant (Liang et al., 2017), for 4 days under continuous stirring (200 rpm)

179

at room temperature. Aliquots were collected at different times (30 min, 1 h, 2 h, 4 h, 8

180

h, 1 d, 2 d, 3 d, and 4 d). The bioactive release was analyzed by using a UV-VIS-NIR

181

Shimadzu 3600 spectrophotometer (Shimadzu Scientific Instrument, Kyoto, Japan). 2-

182

phenyl ethanol concentration was calculated by means of a calibration curve from 0.1

183

to 10.0 µg/ mL at the maximum wavelength absorbance (207 nm). The accumulative

184

release percentage was calculated by the ratio of the release determined by the

185

calibration curve and the theoretical total release.

186

2.10. Statistical analysis

187

In order to determine significant differences among the samples, analysis of variance

188

(ANOVA) was carried out with SPSS software (SPSS Statistics 25.0). For multiple

189

comparisons, Tukey’s multiple range test was used with a statistically significance at

190

the P < 0.05 level.

191

3. Results and discussion

192

3.1. Characterization of α-, β-, and γ-cyclodextrin inclusion complexes 9

193

FTIR spectroscopy was used in order to characterize α-, β-, and γ-cyclodextrin

194

inclusion complexes with 2-phenyl ethanol. As can be seen in Figure 1, the O-H

195

stretching vibration band is observed around 3390 cm-1, indicative of the hydroxyl

196

groups of cyclodextrin (Salih et al., 2020). The C-O-C stretching vibrations are

197

observed at 1158 cm-1, associated to the oligosaccharide structure, and the bands at

198

1080 and 1030 cm-1 correspond to the C-C stretching vibrations of the cyclodextrin ring

199

carbons (Han, Zhang, Shen, Zheng & Zhang, 2019). Additionally, the characteristic

200

band of the α-pyranyl vibration in cyclodextrin appeared at 943 cm−1 and the

201

characteristic band of α-(1,4) glucopyranose in cyclodextrin appeared at 890 cm−1

202

(Yuan, Ye, Gao, Yuan, Lan, Lou, Wang, 2013). As can be seen, there was no chemical

203

interaction between the cyclodextrin and 2-phenyl ethanol. However, some

204

characteristic bands of cyclodextrin were shifted in the inclusion complex spectra. The

205

O-H stretching vibration displaced from 3383 cm-1 for β-cyclodextrin to 3393 cm-1 for

206

the inclusion complex. Furthermore, C-O-C stretching vibration band moved slightly to

207

lower wavenumbers, from 1158 cm-1 for β-cyclodextrin to 1157 cm-1 for the inclusion

208

complex. The shift of these bands suggested physical interactions between

209

cyclodextrins and 2-phenyl ethanol (Xiao, Hou, Kang, Niu, & Kou, 2019).

γ-CD α-CD β-CD

Transmittance (%)

γ-CD:2-PE α-CD:2-PE β-CD:2-PE

890 943 1158

3390

4000

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

210 211

Figure 1. FTIR spectra of α-, β-, and γ-cyclodextrin (CD) inclusion complexes. 10

212

3.2. Retention yield of 2-phenyl ethanol

213

The retention yield of 2-phenyl ethanol for α-, β-, and γ-cyclodextrin inclusion

214

complexes was determined. The highest value was observed for β-clyclodextrin

215

inclusion complex (45 %), followed by γ-cyclodextrin (40 %) and finally, α-cyclodextrin

216

(32 %). These relative low retention values can be related to the fact that 2-phenyl

217

ethanol has only one hydroxyl group to get attached to the cyclodextrin and, thus, the

218

interactions could be weaker. Regarding the differences in retention values, those

219

differences can be associated to the cavity size of CDs. While α-cyclodextrin cavity is

220

the smallest (5.7 Å) to host 2-phenyl ethanol, γ-cyclodextrin cavity is too big (9.5 Å)

221

and, therefore, the interactions between the host and the guest were weaker. However,

222

β-cyclodextrin cavity (7.8 Å) was big enough to host 2-phenyl ethanol and small

223

enough to facilitate physical interactions between them (Ciobanu, Mallard, Landy,

224

Brabie, Nistor & Fourmentin, 2013; Decock, Landy, Surpateanu, & Fourmentin, 2008).

225

Taking the above into consideration, chitosan films were prepared with the inclusion

226

complex with the highest retention yield; therefore, β-cyclodextrin:2-phenyl ethanol

227

inclusion complexes were incorporated into chitosan film forming solutions.

228

3.3. Optical properties of films

229

All films were transparent and colorless. Regarding CIELab color parameters (Table 1),

230

all films presented L* values close to 100, indicating high lightness of the films, a*

231

parameter had slightly negative values, while b* parameter showed positive values, as

232

also shown in other works for chitosan films (Pereira, Queiroz de Arruda, & Stefani,

233

2015). Considering the ∆E* value referred to CHI0GLY, the films did not show

234

differences for the naked eye, since ∆E* values were lower than 1 (Uranga et al.,

235

2019). Furthermore, statistical analysis concluded that there were no significant

236

difference among samples for L*, a* and ∆E* parameters, only a slight difference for b*

237

value, which was not relevant. Therefore, the addition of glycerol, 2-phenyl ethanol, or 11

238

the inclusion complex did not affect the film color. Regarding gloss values (Table 1),

239

there was a slight increase (P < 0.05) with the addition of 2-phenyl ethanol or the

240

inclusion complex, but there was no significant (P > 0.05) difference among the films

241

with 2-phenyl ethanol, regardless the presence of CDs. Gloss and surface roughness

242

are inversely correlated; therefore, lower values of gloss are related to rougher

243

surfaces (Luchese, Uranga, Spada, Tessaro, & de la Caba, 2018; Valencia-Sullca et

244

al., 2016). For an incidence angle of 60º, values greater than 70 gloss units (G.U.) are

245

considered glossy surfaces (Villalobos et al. 2005); therefore, the values measured in

246

this work indicated that chitosan film surface was rough.

247

Table 1. Color (L*, a*, b* and ∆E* parameters) and gloss values for chitosan films. Film

L*

a* a

b* a

a

∆E*

Gloss60 (G.U.)

CHI0GLY

96.9 ± 0.5

-0.03 ± 0.06

2.5 ± 0.2

---

14 ± 2a

CHI15GLY

96.6 ± 0.4a

-0.11 ± 0.08a

3.1 ± 0.3b

0.6a

16 ± 1a

CHI0GLY2PE

96.7 ± 0.3a

-0.08 ± 0.09a

2.8 ± 0.2a

0.3a

18 ± 2a,b

CHI15GLY2PE

96.8 ± 0.3a

-0.11 ± 0.07a

2.9 ± 0.2b

0.4a

20 ± 2b

CHI0GLYCD:2PE

96.4 ± 0.3a

-0.10 ± 0.04a

2.9 ± 0.2b

0.6a

20 ± 1b

CHI15GLYCD:2PE

96.4 ± 0.7a

-0.11 ± 0.08a

2.9 ± 0.2b

0.6a

20 ± 1b

248

Two means followed by the same letter in the same parameter are not significantly

249

(P > 0.05) different through the Tukey's multiple range test.

250

Additionally, ultraviolet-visible (UV-vis) spectroscopy was carried out and results are

251

shown in Figure 2. As can be seen, films were transparent since there was no

252

absorption at 600 nm (Uranga et al., 2019). Furthermore, there was no absorption in

253

the visible range from 400 to 800 nm, although chitosan films absorbed UV light,

254

especially below 250 nm, probably due to some chromophores present in chitosan

255

(Leceta, Guerrero, Ibarburu, Dueñas, & de la Caba, 2013). In this regard, UV light

256

barrier properties provide films with value-added properties, which can reduce lipid

257

oxidation in food products and extend food shelf life (Fasihi, Noshirvani, Hashemi,

258

Fazilati, Salavati, & Coma, 2019).

12

Absorbance (a.u.) 200

300

400

500

600

700

800

Wavelength (nm)

259 260

Figure 2. UV-vis light absorption of chitosan films: CHI0GLY, blue line; CHI15GLY, red

261

line; CHI0GLY2PE, green line; CHI15GLY2PE, pink line; CHI0GLYCD:2PE, orange

262

line; and CHI15GLYCD:2PE, black line.

263

3.4. Physicochemical properties of films

264

In order to assess the interactions among the components of the films, FTIR analysis

265

was carried out and the FTIR spectra of chitosan films are shown in Figure 3. The

266

characteristic bands of chitosan appeared at 1630 cm-1 (amide I band), associated to

267

C=O stretching; at 1530 cm-1 (amide II band), related to N-H bending; and at 1310 cm-1

268

(amide III band) assigned to C-N stretching (Mauricio-Sánchez, Salazar, Luna-

269

Bárcenas, & Mendoza-Galván, 2018). Moreover, O-H stretching band and C-O-C

270

absorption band were observed at 3250 cm-1 and around 1080 cm-1, respectively

271

(Branca et al., 2016). No new band was observed between the control spectra and the

272

spectra corresponding to the films with 2-phenyl ethanol, with or without CDs,

273

indicating that no chemical reaction occurred. However, some band displacements

274

were observed when β-CD:2-phenyl ethanol was incorporated into chitosan films; in

275

particular, for O-H and N-H stretching band from 3264 to 3214 cm-1, for amide II band

276

from 1546 to 1538 cm-1, and for amide III band from 1324 to 1332 cm-1. This suggests

13

physical interactions, such as hydrogen bonding or electrostatic interactions among the

278

components of the films (Roy & Rhim, 2020). Additionally, a similar behavior was

279

observed with the addition of glycerol since O-H and N-H stretching band shifted from

280

3214 to 3243 cm-1 and amide II band from 1538 to 1540 cm-1 due to hydrogen bonding

281

with glycerol.

Transmittance (%)

277

Amide III Amide I Amide II

4000

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

282 283

Figure 3. FTIR spectra of chitosan films: CHI0GLY, blue line; CHI15GLY, red line;

284

CHI0GLY2PE, green line; CHI15GLY2PE, pink line; CHI0GLYCD:2PE, orange line;

285

and CHI15GLYCD:2PE, black line.

286

In order to determine the structure of the films, XRD analysis was carried out. As can

287

be observed in Figure 4, films without the inclusion complex present two broad peaks,

288

one at 11.7º and another one around 21.3º, characteristic of chitosan (Pang &

289

Zhitomirsky, 2005). These broad peaks indicate the amorphous character of chitosan

290

films. Regarding the films with the inclusion complex, two sharp peaks appeared at 6.2º

291

and 12.3º, characteristic of β-CD (Campos et al., 2019; Menezes et al., 2016). This

292

indicates that CDs maintained certain degree of crystallinity after the incorporation into

293

chitosan matrix. However, the intensity of these peaks was different for the films with

294

and without glycerol, which could be attributed to the change in molecular organization

14

of the cyclodextrin (Campos et al., 2018). Since glycerol is a small molecule, it can

296

penetrate between chitosan chains, decreasing intramolecular interactions among

297

chitosan chains and facilitating the interactions of chitosan with the additives

298

incorporated into the film forming formulation (Zarandona, Puertas, Dueñas, Guerrero,

299

& de la Caba, 2020). As shown by FTIR results, the intermolecular interactions with

300

cyclodextrin molecules occurred by hydrogen bonding, which changed the chitosan film

301

structure due to the heterogeneous nucleation effect between the inclusion complex

302

and chitosan (Li, & Zhen, 2017) and, as a consequence, the film mechanical behavior

303

also changed.

Intensity (a.u.)

295

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 34

2θ (º)

304 305

Figure 4. XRD diffractograms of chitosan films: CHI0GLY, blue line; CHI15GLY, red

306

line; CHI0GLY2PE, green line; CHI15GLY2PE, pink line; CHI0GLYCD:2PE, orange

307

line; and CHI15GLYCD:2PE, black line.

308

3.5. Mechanical and barrier properties

309

In order to assess the effect of structure changes in the mechanical behavior, tensile

310

tests were performed and TS and EAB values were measured and shown in Table 2.

311

As expected, films plasticized with glycerol showed higher EAB values and lower TS

312

and EM values, indicating more flexible and less rigid films due to the increase of free 15

313

volume (Rivero, Damonte, Garcia, & Pinotti, 2016). Regarding the addition of 2-phenyl

314

ethanol to the film forming solution, it was observed that this compound did not

315

significantly (P > 0.05) change TS or EAB values with respect to the corresponding

316

control film, probably due to the 2-phenyl ethanol evaporation during film preparation

317

when CDs were not used; however, an increase of EM values was noticed, indicating a

318

higher rigidity, probably due to the interactions among chitosan and the remaining

319

amount of 2-PE. The most significant change was observed for TS values when the

320

inclusion complex was incorporated into formulations; in particular, TS value

321

significantly (P < 0.05) increased from 34.5 to 48.8 MPa for the chitosan films with the

322

inclusion complex but without glycerol. In accordance, EM values also increased and

323

EAB values decreased. This behavior can be related to the interactions among

324

chitosan and CD, as shown by FTIR analysis, which would lead to a more compact

325

network, increasing resistance and rigidity, but decreasing flexibility (Siripatrawan &

326

Vitchayakitti, 2016; Sun et al., 2014). It is worth noting that TS values above 40 MPa

327

are higher values than those found for commercial packaging films, such as PP or

328

LDPE (Honarvar et al., 2017, Lomate, Dandi, & Mishra, 2018).

329

Table 2. Tensile strength (TS, MPa), elongation at break (EAB, %), Young’s Modulus

330

(EM, MPa) and water vapor permeability (WVP, g·cm-1·s-1·Pa-1) of chitosan films.

Film

TS

EAB

EM

WVP -12

(MPa)

(%)

(MPa)

(10 g·cm-1·s-1·Pa-1)

CHI0GLY

34.5 ± 2.2a

9.2 ± 2.2b

1511 ± 163a,b

1.08 ± 0.04a

CHI15GLY

32.4 ± 3.1a 16.4 ± 3.1a

1374 ± 125a

1.04 ± 0.02a

CHI0GLY2PE

39.5 ± 3.6a

7.8 ± 1.6b

2205 ± 114c

0.89 ± 0.04b

CHI15GLY2PE

33.7 ± 3.4a 12.3 ± 3.4a

1687 ± 72b

0.82 ± 0.01b

CHI0GLYCD:2PE

48.8 ± 3.2b

7.4 ± 2.2b

2639 ± 173d

0.83 ± 0.03b

CHI15GLYCD:2PE 37.5 ± 4.1a

8.1 ± 2.3b

1960 ± 84c

0.85 ± 0.02b

331

Two means followed by the same letter in the same parameter are not significantly

332

(P > 0.05) different through the Tukey's multiple range test.

16

333

Regarding water vapor permeability (WVP), there was no significant difference (P >

334

0.05) between the chitosan films without 2-phenyl ethanol. However, the addition of the

335

active compound significantly (P < 0.05) decreased WVP values, probably due to the

336

interactions with chitosan (Salami, Rezaee, Askari, & EmamDjomeh, 2020), as shown

337

by FTIR, and also due to the increase of crystallinity, as observed by XRD. Although

338

WVP values found in this work are lower than those measured for chitosan films in

339

other works (Priyadarshi, Sauraj, Kumar, Deeba, Kulshreshtha, & Negi, 2018), these

340

values are still higher than those shown by commercial films (Honarvar et al., 2017,

341

Lomate, Dandi, & Mishra, 2018).

342

3.6. Release of 2-phenyl ethanol

343

The release of 2-phenyl ethanol from chitosan films with 2-phenyl ethanol and from

344

chitosan films with β-cyclodextrin:2-phenyl ethanol was carried out in order to compare

345

the delivery trend during the immersion into 95 % ethanol for 4 days (Figure 5).

346

Regarding CHI0GLY2PE films, it is worth noting that some bioactive content was lost

347

before the analysis, probably during film preparation due to the high volatility of the

348

compound and, as a result, a maximum release of 8 % was achieved. However,

349

chitosan films with the inclusion complex retained the bioactive inside β-cyclodextrin

350

after film preparation. An improvement of 2-PE retention through its encapsulation with

351

methylcellulose, alginate sodium and carboxymethyl chitosan has also been observed

352

in other works (Qiu, Tian, Yin, Zhou, & Zhu, 2019). In this work, 2-phenyl ethanol was

353

released after 4 h of immersion into 95 % ethanol. The fast release of 2-phenyl ethanol

354

could be explained due to the weak interactions with β-cyclodextrin, since 2-pheyl

355

ethanol has only one hydroxyl group.

17

100

Release (%)

80

60

40

20

0 0

10

20

30

40

50

60

70

80

90

100

Time (h)

356 357

Figure 5. Bioactive release from chitosan films: CHI0GLY2PE, green line;

358

CHI0GLYCD:2PE, orange line.

359

4. Conclusions

360

This work showed the good capacity of cyclodextrins to form inclusion complex with 2-

361

phenyl ethanol. Specifically, β-cyclodextrin showed the highest retention yield. The

362

films obtained were homogeneous, transparent and colorless. Moreover, the addition of

363

the inclusion complex into chitosan film forming solutions led to chitosan films with

364

improved properties. In particular, tensile strength reached values up to 48 MPa. The

365

tensile strength increase was related to the physical interactions between chitosan and

366

the inclusion complex, which were corroborated by FTIR and XRD analyses, which

367

indicated the crystalline structure of chitosan films with the inclusion complex. Finally,

368

the release of 2-phenyl ethanol into 95 % ethanol was carried out in films with and

369

without β-cyclodextrin. Results suggested that the bioactive was evaporated during film

370

preparation for the films without the inclusion complex. In contrast, the films with the

371

inclusion complex preserved the bioactive inside the inclusion complex. These results

372

indicated that CDs were effective to avoid the loss of bioactive compounds during film

373

preparation. 18

374

Acknowledgments

375

Authors thank the Ministry of Science, Innovation and Universities (RTI2018-097100-B-

376

C22), the University of the Basque Country (GIU18/154), and the Basque Government

377

(Department of Quality and Food Industry) for their financial support. Iratxe Zarandona

378

thanks the Quality and Food Industry Department of the Basque Government for her

379

fellowship (22-2018-00078). Carmen Barba thanks Public University of Navarre for the

380

financial support for young researcher projects (UPNA2018-04-INC). Also thanks are

381

due to the Advanced Research Facilities (SGIker) from the UPV/EHU. Finally, authors

382

would like to thank Roquette and Wacker Chemie AG for providing cyclodextrin

383

samples.

384

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27

Highlights •

Cyclodextrins (CDs) formed inclusion complexes with 2-phenyl ethanol.

•

β-CDs showed higher retention yield than α- or γ-CDs.

•

Films with β-CD:2-phenyl ethanol were homogeneous, transparent and colorless.

•

Tensile strength of chitosan films reached values up to 48 MPa.

•

Films with inclusion complex preserved the bioactive inside the inclusion complex.

Conflict of interest Authors declare no conflict of interest