Accepted Manuscript

Incorporation of hydroxypropyl-β-cyclodextrins into chitosan films to tailor loadingcapacity for active aroma compound carvacrol

Laura Higueras, Gracia López-Carballo, Rafael Gavara, Pilar Hernández-Muñoz

PII: S0268-005X(14)00260-4

DOI: 10.1016/j.foodhyd.2014.07.017

Reference: FOOHYD 2668

To appear in: Food Hydrocolloids

Received Date: 3 March 2014

Accepted Date: 15 July 2014

Please cite this article as: Higueras, L., López-Carballo, G., Gavara, R., Hernández-Muñoz, P.,Incorporation of hydroxypropyl-β-cyclodextrins into chitosan films to tailor loading capacity for activearoma compound carvacrol, Food Hydrocolloids (2014), doi: 10.1016/j.foodhyd.2014.07.017.

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High or intermediate

relative humidity

Release of volatile

carvacrol

Chitosan blended with hydroxypropyl-β-cyclodextrins and hydrophilic coadjuvants to tailor carvacrol sorption

properties of film

Film loaded with carvacrol

% Relative humidity

0 53 75

Sor

ptio

n eq

uilib

rium

of c

arva

crol

(%

dry

wei

ght)

0,01

0,1

1

10

100

1000CS:CD-0G CS:CD-20G CS:CD-35G

Time (hours)

0 100 200 300 400 500

Res

idua

l car

vacr

ol (

mg)

0

20

40

60

80

100

120

140

160

Inhi

bitio

n zo

ne (

mm

)

0

20

40

60

80

100Carvacrol content in the filmE. coliS. aureus

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Incorporation of hydroxypropyl-β-cyclodextrins 1

into chitosan films to tailor loading capacity for 2

active aroma compound carvacrol 3

Laura Higueras, Gracia López-Carballo, Rafael Gavara, Pilar Hernández-4

Muñoz* 5

Instituto de Agroquímica y Tecnología de Alimentos. CSIC. Avenida Agustín 6

Escardino, 7. 46980 Paterna, Valencia, Spain. 7

*Corresponding author: 8

Pilar Hernandez-Munoz: Tel: +34 96 3900022 – Fax: +34 96 3636301 9

phernan@iata.csic.es 10

11

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ABSTRACT 12

Chitosan incorporating hydroxypropyl-β-cyclodextrins and glycerol films capable 13

of modulating loading capacity and release of carvacrol have been developed. 14

Films were obtained by casting and conditioned at different relative humidities 15

(RH) prior to immersion in liquid carvacrol. Incorporation of cyclodextrins in the 16

chitosan matrix slightly increased sorption of carvacrol and it was necessary to 17

use glycerol and water as coadjuvants to control loading of the films with the 18

volatile. Good agreement was found between carvacrol retention and 19

plasticization of the film by glycerol and water. The kinetics of carvacrol sorption 20

by the films was evaluated at 25 °C. Diffusion coefficients of sorption varied 21

from 0.011 x 10-14 m2/s for films incorporating 35% glycerol and conditioned at 22

0% RH to 1.9 x 10-14 m2/s for films incorporating 35% glycerol and conditioned 23

at 75% RH. Release of carvacrol from the films was greatly affected by RH. The 24

films showed antimicrobial activity against S. aureus and E. coli after 20 days of 25

storage at 25 °C and 43% environmental RH. These films could be useful in the 26

design of systems for delivering active volatiles. 27

KEYWORDS 28

Chitosan, hydroxypropyl-β-cyclodextrins, carvacrol, loading and release,

antimicrobial films

29

1. INTRODUCTION 30

Delivery systems based on polymers capable of carrying and delivering a 31

continuous supply of biologically active molecules into a specific environment 32

have become of increasing interest in recent years. These systems are able to 33

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reduce the amount of active agent required for treatment by maintaining an 34

effective concentration in the system applied over a certain period of time 35

(Ouattara, Simard, Piette, Begin, & Holley, 2000). There is a great need for 36

these devices in diverse technological applications encompassing 37

multidisciplinary areas such as biomedicine, pharmacology, agriculture, 38

packaging, food technology, textiles and the cosmetic industry for the 39

entrapment and delivery of drugs, enzymes, nutraceuticals, agrochemicals, 40

flavours and fragrances, biocides, etc. Currently, most of the recently developed 41

delivery systems consist of natural and synthetic polymers, polymer blends, and 42

composites of organic and inorganic materials that form membranes, capsules 43

or micelles, depending on the application required. Issues concerning 44

biodegradability, biocompatibility and non-toxicity of the materials used for the 45

development of carrier systems need to be considered. Renewable polymers 46

are being widely investigated as delivery vehicles because most of them fulfil 47

the aforementioned requirements. 48

Chitosan polymer (poly β-(1, 4)N-acetyl-D-glucosamine) has been intensively 49

studied during recent decades (Dutta, Tripathi, & Dutta, 2012; Lopez-Carballo, 50

Higueras, Gavara, & Hernandez-Munoz, 2013; Valencia-Chamorro, Palou, del 51

Rio, & Perez-Gago, 2011). It is a natural cationic linear aminopolysaccharide 52

obtained from partial N-deacetylation of chitin. Chitosan is receiving a great deal 53

of attention in biomedicine and pharmacology for the delivery of drugs (Ramya, 54

Venkatesan, Kim, & Sudha, 2012). Chitosan can also act as a carrier for 55

sustained release and delivery of compounds other than drugs which are of 56

interest in foods, personal care, agriculture, etc. (Kumar, Muzzarelli, Muzzarelli, 57

Sashiwa, & Domb, 2004; Prabaharan & Mano, 2006; Zhang, et al., 2009). 58

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Antimicrobial carriers are of great interest in the area of food packaging, and 59

great efforts are being made to develop effective antimicrobial food packaging 60

systems (Appendini & Hotchkiss, 2002; Suppakul, Miltz, Sonneveld, & Bigger, 61

2003). Antimicrobial food packaging technologies which are based on the 62

incorporation of active volatiles in polymer matrices do not require the film be in 63

contact with the food product to be active. In this case, the volatile is released to 64

the headspace of the package and exerts its activity when contact with the food 65

surface. 66

There is a wide range of volatile compounds derived from plants or forming part 67

of the aroma profile of fruits presenting biocide properties which could be 68

applied in the design of antimicrobial carriers since most of them are generally 69

recognized as safe (GRAS) and are used as food flavouring or seasoning 70

agents. However, volatiles can be lost to some extent during entrapment or 71

encapsulation in the polymer matrix, which consequently decreases the 72

retention process. Therefore, it would be of great interest to develop suitable 73

carriers with a high entrapment capacity and sustained release properties for 74

volatile compounds. In addition, the release of the volatile from the polymer 75

matrix can be triggered by different stimuli such as the moisture present in the 76

headspace of the package; in this respect, the hydrophilic nature of the polymer 77

and the relative humidity of the headspace are major factors controlling the 78

release kinetics of the agent. 79

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of a three-80

dimensional structure forming a truncated cone with a hydrophobic cavity and a 81

hydrophilic outer surface. CDs are widely used as excipients in pharmacy to 82

solubilise lipophilic molecules by means of inclusion complexes. However, non-83

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inclusion aspects of CDs are being studied, such as solubilisation by formation 84

of self-assembled aggregates or surfactant-like effects. In recent years, CDs 85

and their derivatives have been used as building blocks for the development of 86

a wide variety of polymeric networks and assemblies with a higher drug loading 87

capacity. They have been blended with polymers in the design of 88

nano/microparticles, and micelles, for the sustained release and targeted 89

delivery of bioactive substances (van de Manakker, Vermonden, van Nostrum, 90

& Hennink, 2009). 91

The aim of this work was to develop novel films by blending chitosan with 92

hydroxypropyl-β-cyclodextrins (a water soluble cyclodextrin derivative) in order 93

to improve the capacity of chitosan film to be loaded with carvacrol, a naturally 94

occurring phenolic volatile with antimicrobial properties which is a major 95

component of thyme and oregano essential oils. The loading capacity and 96

sorption kinetics of carvacrol were studied as a function of film formulation, and 97

the release of carvacrol was evaluated at different relative humidities. The 98

antimicrobial activity of the films was tested in vitro in vapour phase against 99

pathogen bacteria S. aureus and E. coli. Finally, the antimicrobial activity of the 100

films was monitored over time. 101

2. MATERIALS AND METHODS 102

2.1. Materials 103

Carvacrol (kosher > 98%), dimethyl sulfoxide (99.9% ACS Reagent grade), 104

phosphorus pentoxide, magnesium nitrate 6-hydrate, glycerol, acetic acid and 105

low molecular weight chitosan (CS) were supplied by Sigma (Barcelona, Spain). 106

Sodium chloride, potassium carbonate and barium chloride 2-hydrate, were 107

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supplied by Fluka (Madrid, Spain). Hydroxypropyl-β-cyclodextrins (HP-β-CDs, 108

CAVASOL® W7-HP) were provided by Wacker Fine Chemicals, S.L. 109

(Barcelona, Spain). Maltodextrins (MDs) from maize starch, Biochemika, 10, 110

was supplied by Sigma-Aldrich (Madrid, Spain). 111

2.2. Film preparation 112

A flowchart with the preparation of chitosan/hydroxypropyl-β-cyclodextrin 113

(CS:CD) films is shown in Fig. 1. Chitosan (1.5% w/w) was solubilised in 0.5% 114

(w/w) acetic acid solution and filtrated to eliminate impurities. Films were 115

prepared by casting, pouring a suitable amount of the film-forming solution into 116

a flat polystyrene tray and allowing it to dry under controlled environmental 117

conditions (36 h, 40.0 ± 1.5 °C and 20 ± 9% RH). CS :CD films were obtained by 118

adding HP-β-CDs to the chitosan solution in 1:1 weight ratio of HP-β-CDs to 119

CS, and glycerol at 0% (CS:CD-0G), 20% (CS:CD-20G) and 35% (CS:CD-35G) 120

(g glycerol/100 g dry matter) was added to the film-forming solution while 121

stirring at 1500 rpm and 37 °C until complete disso lution. Films were obtained 122

by casting as described above. After peeling the films from the tray, they were 123

plasticized with different amounts of water, for which purpose samples 550 mm 124

in diameter and 55 ± 5 µm in thickness were stored in glass desiccators with 125

phosphorus pentoxide to achieve humidities close to 0%, to dry the films, or at 126

humidities of 52.9 ± 0.2 and 75.3 ± 0.1 RH, using saturated salt solutions 127

(ASTM, 2007) in a temperature-controlled room at 25 ± 1 °C until moisture 128

equilibrium was reached. Films were named as (CS:CD-xxG-xxRH), depending 129

on the amount of glycerol and the relative humidity at which they were 130

conditioned prior to being loaded with carvacrol. 131

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To study how the amount of HP-β-CDs blended with chitosan affects sorption of 132

carvacrol by the resulting films, specimens were prepared at 1:2, 1:0.5 and 133

1:0.25 weight ratio and compared with 1:1 CS:CD proportion; film samples of 134

550 mm in diameter and 55 ± 5 µm in thickness were plasticized with 35% 135

glycerol and conditioned at 75% RH at room temperature as described above 136

prior to immersion in carvacrol. 137

Chitosan film samples of similar size and thickness as mentioned above and 138

plasticized with 35% glycerol were also mixed with MDs in a 1:1 (w/w) 139

proportion and conditioned at three different humidities as described above, and 140

carvacrol sorption properties studied. 141

2.3. Optical properties of the films before and after immersion in carvacrol 142

The colour of the films before and after immersion in carvacrol was measured 143

with a CR-300 Minolta Chroma meter® (Minolta Camera Co., Ltd., Osaka, 144

Japan). The film samples were placed on a white standard plate; the results 145

were expressed in accordance with the CIELAB system with reference to 146

illuminant D65 and a visual angle of 10°. The measu rements were performed 147

through a 6.4-mm-diameter diaphragm containing an optical glass, monitoring 148

L*, a*, b*, chroma (C*ab = (a*2+b*2)1/2) and hue (hab = arctan (b*/a*)). The 149

samples were measured in triplicate by eight measurements in different 150

locations for each film sample. 151

2.4. Loading and release of carvacrol 152

After being conditioned at different relative humidities to achieved the desired 153

water content, film samples with various matrix compositions as described in 154

“Film preparation” section were immersed in liquid carvacrol at 25 °C and the 155

amount of the compound sorbed in the film was measured over time until 156

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sorption equilibrium was reached. For that, after a determined period of time 157

depending of the film composition, a piece of the film was cleaned with a paper 158

tissue to remove any excess of carvacrol on the film surface and then inserted 159

into an empty desorption tube (11.5 x 0.39 cm I.D.) for thermal desorption. The 160

release of carvacrol from the films was evaluated as a function of time at 25 °C 161

and at three relative humidities: 43.2 ± 0.4, 52.9 ± 0.2, and 90 ± 2%. For this 162

purpose, a flow of air of 200 mL/min was bubbled in a saturated salt solution to 163

provide the desired relative humidity (ASTM, 2007), and passed through a 164

hermetically closed 500-mL container where the film sample was placed. The 165

release of carvacrol from the films was calculated by analysing the amount of 166

carvacrol remaining in the film by thermal desorption. 167

2.5. Analysis of carvacrol in a film 168

The amount of carvacrol in a film was determined by thermal desorption 169

coupled to gas chromatography using a Dynatherm Thermal Desorber Model 170

890/891 (Supelco, Teknokroma, Barcelona, Spain) connected in series to the 171

column of an HP5890 gas chromatograph Series II Plus (Agilent Technologies, 172

Barcelona, Spain) via a heated transfer line. The desorption tube containing the 173

film sample was placed in the desorber chamber, which was immediately 174

sealed. Conditions for desorption were as follows: desorption temperature, 210 175

°C; transfer line, 230 °C; desorption time, 7 min; He desorption flow, 8.15 176

mL/min. The GC was equipped with a TRB5 (30 m, 0.32 mm, 0.25 µm) column 177

(Teknokroma, Barcelona, Spain) and a flame ionization detector. The 178

chromatographic conditions were: 260 °C detector te mperature, 7 min at 45 °C, 179

heating ramp to 220 °C at 18 °C/min, and 1 min more at 220 °C. After the 180

analysis, the film sample was recovered from the desorption tube and weighed 181

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on an analytical balance (Voyager V11140 model, Ohaus Europe, Greifensee, 182

Switzerland). 183

2.6. Antimicrobial assays 184

2.6.1. Bacterial strains and growth conditions 185

Two model microbial strains were obtained from the Spanish Type Culture 186

Collection (CECT, Valencia, Spain): the Gram-positive bacterium 187

Staphylococcus aureus CECT 86 and the Gram-negative bacterium Escherichia 188

coli CECT 434. The strains were stored in Mueller Hinton Broth (MHB, 189

Scharlab, Barcelona, Spain) with 20% glycerol at −80 °C until needed. For 190

experimental use, the stock cultures were maintained by regular subculture on 191

Tryptone Soy Agar (TSA, Scharlab, Barcelona, Spain) slants at 4 °C and 192

transferred monthly. In the first step, a loopful of each strain was transferred to 193

10 mL of Tryptone Soy Broth (TSB, Scharlab, Barcelona, Spain) and incubated 194

at 37 °C overnight to obtain early stationary phase cells. 195

2.6.2. Determination of minimal inhibitory concentration of carvacrol in vapour 196

phase 197

The microatmosphere method was selected to carry out an antimicrobial test in 198

which no direct contact between the device containing the volatile and the agar 199

medium is necessary for the former to exert its activity. In this method, the 200

volatile compound migrates from the carrier (filter paper, film) into the 201

headspace of the system, thus becoming available to contact the growth 202

medium and the microorganism. 100 µL of a bacterium suspension containing 203

approximately 107 CFU/mL was spread over the surface of 90-mm-diameter 204

Petri dishes containing approximately 15 mL of solid culture TSA medium. 205

Decreasing quantities of carvacrol were dissolved in dimethyl sulfoxide 206

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(DMSO), and a final volume of 10 µL of the carvacrol solution was added to 25-207

mm-diameter sterilized filter paper. Blanks prepared by adding 10 µL of DMSO 208

to 25-mm-diameter sterile filter disks showed that the DMSO did not have any 209

effect against any of the microorganisms tested. Once the Petri dishes and lids 210

had been assembled, the units were sealed with Parafilm™ to reduce leakage 211

of the volatile agent and incubated at 37 °C for 24 h. At the end of the 212

incubation period, the antimicrobial activity was determined by measuring the 213

diameter in millimetres of the zone below the filter papers where there was no 214

microbial growth. The minimum inhibitory concentration (MIC) is defined as the 215

lowest concentration of active compound that yields inhibition of microorganism 216

(Delaquis, Stanich, Girard, & Mazza, 2002; Hammer, Carson, & Riley, 1999). 217

Each assay was performed in triplicate. 218

2.6.3. Antimicrobial activity of the films 219

The procedure to determine the antimicrobial activity of the films was similar to 220

that described above. In this case, films of the same size as the filter papers 221

and loaded with carvacrol were placed in the lid of the inoculated Petri dishes. 222

Each assay was performed in triplicate. 223

2.7. Data analysis 224

Statistical analysis of the results was performed with SPSS commercial 225

software (SPSS Inc., Chicago, Illinois, USA). A two-way analysis was applied to 226

compare the effect of different amounts of glycerol in the same CS or CS:CD 227

matrix. Additionally, one-way analysis of variance was carried out for the other 228

data. Differences between means were assessed on the basis of confidence 229

intervals, using the Tukey-b test at a significance level of P ≤ 0.05. The data are 230

represented as average ± standard deviation. The data were analysed and 231

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plotted using the SigmaPlot 10.0 software (Systat Software Inc., Richmond, 232

California, USA). 233

3. RESULTS AND DISCUSSION 234

CS:CD films prepared at a weight ratio of 1:1 were homogeneous, with no 235

phase separation visible to the naked eye, easy to handle and highly 236

transparent. After immersion in carvacrol, the films mantained their integrity and 237

did not break when handled. 238

3.1. Colour Properties 239

Table 1 shows colour parameters of films incorporating 0, 20 and 35% glycerol 240

and conditioned at different RH before and after being immersed in carvacrol. 241

Before immersion in carvacrol the incorporation of glycerol at 20 or 35% did not 242

significantly (P > 0.05) modify the colour parameters of the films when 243

compared with those prepared without glycerol. Moreover, there were no 244

significant differences in colour parameters (P > 0.05) of glycerol plasticized 245

films conditioned at different relative humidities (data not shown). After 246

immersion in carvacrol, films plasticized with glycerol at 20 or 35% and 247

conditioned at 75% RH, and films plasticized with 35% glycerol and conditioned 248

at 53% RH acquired a vivid yellow-green colour, increasing their chroma and 249

hue, and slightly decreasing their lightness. These changes are related to the 250

amount of carvacrol that the films are capable of retaining. Fig. 2 shows the 251

sorption equilibrium of carvacrol in films incorporating different percentages of 252

glycerol and conditioned at various relative humidities. According to the results 253

obtained, changes in colour parameters were not significant for films retaining 254

less than 6% of carvacrol. 255

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3.2. Sorption properties 256

3.2.1. Sorption equilibrium of carvacrol in films 257

As Fig. 2 shows, the amount of carvacrol retained in the films depended on the 258

level of plasticization by glycerol and moisture. Carvacrol retention in films with 259

a fixed amount of glycerol increased as the relative humidity at which they were 260

conditioned increased; water acts as a plasticizer for CS:CD films, enhancing 261

their carvacrol loading capacity. It can also be observed that at a fixed relative 262

humidity carvacrol sorption increased with the glycerol content in the film. The 263

highest carvacrol loading capacity was achieved by films plasticized with 35% 264

glycerol and conditioned at 75% RH, followed by the same films conditioned at 265

53% RH, and films incorporating 20% glycerol and conditioned at 75% RH; the 266

sorption of carvacrol in these films was 216.3 ± 22.1, 133.3 ± 16.9 and 56.8 ± 267

3.5% (g carvacrol/g dry film), respectively. In spite of having lower water content 268

(Fig. 2), films incorporating 35% glycerol and conditioned at 53% RH absorbed 269

a greater amount of carvacrol than films incorporating 20% glycerol and 270

conditioned at 75% RH. When glycerol was not incorporated in the CS:CD 271

matrix, sorption of carvacrol did not exceed 0.45% (g carvacrol/g dry film), 272

regardless of the relative humidity at which they were conditioned; whereas the 273

maximum amount of carvacrol sorbed by films stored under dry conditions was 274

4.5%, corresponding to films plasticized with 35% glycerol. These observations 275

highlight the fact that glycerol plays a crucial role in the sorption of carvacrol. 276

Thus, the use of HP-β-CD together with glycerol and moisture is required to 277

control the loading of carvacrol in a chitosan matrix. It is worth pointing out that 278

previous studies (Higueras, López-Carballo, Cerisuelo, Gavara & Hernández-279

Muñoz, 2013) showed that chitosan films without HP-β-CD did not retain more 280

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than 1% of carvacrol, whatever the amount of glycerol and water incorporated in 281

the matrix. Incorporation of HP-β-CD in the chitosan matrix slightly increased 282

the sorption of carvacrol compared with plain chitosan films, and the use of 283

water and glycerol as coadjuvants was necessary to control the loading of the 284

films with carvacrol. Kurek et al. (2014) studied the effect of various additives on 285

the retention of carvacrol during the processing of chitosan films by casting and 286

found that carvacrol retention was directly correlated with the incorporation of 287

glycerol and nanoclays into the matrix; moreover, retention of carvacrol was 288

also favoured by blending chitosan with gum arabic because of the formation of 289

coacervates which encapsulate carvacrol and prevent its evaporation from the 290

matrix during the drying of the films . 291

CDs are commonly used to solubilise lipophilic molecules, β-CDsare capable of 292

forming 1:1 and 1:2 host/guest complexes with carvacrol (Locci, Lai, Piras, 293

Marongiu & Lai, 2004; Ravi & Divakar, 2001). In this work, the maximum 294

percentage of carvacrol that could be held in chitosan films incorporating HP-β-295

CD was <10% (g carvacrol/100 g dry matter). When water and glycerol were 296

present, the amount of carvacrol retained in the films exceeded this percentage, 297

so mechanisms other than the formation of inclusion complexes participate in 298

the sorption of carvacrol. In the last few years, a growing body of research has 299

shown that CDs can act as building units able to self-assemble into aggregates 300

driven by CD-CD H-bonds, and these aggregates can act as solubilizers. The 301

size of these aggregates tends to grow with increasing concentration of CDs, 302

and aggregates up to several micrometres in diameter have been reported 303

(Messner, Kurkov, Jansook, & Loftsson, 2010). Water-soluble polymers 304

contribute to the stabilization of these aggregates through formation of CD-305

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polymer hydrogen bonds (Ribeiro, Veiga, & Ferreira, 2003). Formation of CD 306

aggregates could enhance the loading of amphiphilic molecules such as 307

carvacrol by plasticized chitosan films that otherwise present a very low sorption 308

potential. Thus, formation of CD aggregates could explain the high carvacrol 309

sorption values found for some of the films developed in the present work. 310

Moreover, the effect of HP-β-CDs concentration in a chitosan film on carvacrol 311

sorption capacity was also studied. Fig. 3 shows the sorption equilibrium of 312

carvacrol in chitosan films plasticized with 35% glycerol and conditioned at 75% 313

RH and incorporating HP-β-CDs in CS:CD weight ratios of 1:0, 1:0.25, 1:0.5, 314

1:1 and 1:2. The sorption of carvacrol greatly depended on the HP-β-CDs 315

content in the film. The highest sorption value was obtained for the 1:2 316

formulation. However, although 1:2 films were plasticized with glycerol and 317

water, they were very brittle and difficult to handle because of the high CD 318

content incorporated into the chitosan matrix, which exerts an anti-plasticizing 319

effect. 320

Maltodextrins (MDs)are non-cyclic oligosaccharides consisting of linear and 321

branched amylose and amylopectin degradation products. These starch 322

derivatives can form complexes with hydrophobic molecules, host-guest 323

complexation being the most common. MDs conformation goes from flexible coil 324

to helix as the dextrose equivalent (DE) number decreases. The inside of the 325

helical structure is hydrophobic, as in CDs, but more flexible than the cavity of 326

them, which means less steric hindrance. As in the case of CDs, in addition to 327

hydrophobic interactions MDs participate in hydrogen-bonding with guest 328

molecules (Garnero, Aloisio, & Longhi, 2013). In an attempt to establish the 329

effect of the molecular shape of oligosaccharides on the carvacrol sorption 330

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capacity of chitosan films, non-cyclic oligosaccharide maltodextrins were 331

incorporated in the chitosan matrix as an alternative to HP-β-CDs and carvacrol 332

sorption properties were studied. Fig. 4 shows the sorption equilibrium of 333

carvacrol in films formulated with chitosan and maltodextrins at a ratio of 1:1 by 334

weight, plasticized with 35% glycerol, and conditioned at 0, 53 and 75% RH 335

prior to immersion in carvacrol. In no case did sorption of carvacrol exceed 336

1.5%. These results show the effect of the molecular geometry of low molecular 337

weight starch derivatives incorporated in chitosan films on the carvacrol loading 338

capacity. 339

3.2.2. Kinetics of sorption of carvacrol in CS:CD films 340

Fig. 5 shows the kinetics of sorption of carvacrol in 1:1 CS:CD films plasticized 341

with 35% glycerol and conditioned at 0, 53 or 75% RH, and films plasticized 342

with 20% glycerol and conditioned at 75% RH. The plots represent the sorption 343

of liquid carvacrol into the films versus time. It can be observed that the 344

equilibrium times varied among films, depending on their glycerol and water 345

content. Sorption equilibrium was achieved faster for films which presented a 346

greater level of plasticization, i.e. films incorporating 20 or 35% glycerol and 347

conditioned at 75% RH, whereas it took longer to reach sorption equilibrium for 348

films conditioned under dry conditions or at 53% RH before being immersed in 349

carvacrol. 350

According to Alfrey et al.(1966), the diffusion of a sorbate in a polymer sheet 351

can be classified as Fickian (Case I) or non-Fickian (anomalous, Case II and 352

Super Case II) depending on the solvent diffusion and polymer relaxation rates. 353

Diffusion categories can be distinguished by the shape of the sorption uptake 354

curve of a polymer-penetrant system: 355

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)1(nt ktM

M =∞

where Mt is the mass of solute uptake at time t, and M∞ is the mass of solute 356

uptake when the polymer has reached equilibrium, k is a constant and n a 357

diffusion exponent which denotes the type of diffusion mechanism. If the 358

exponent n is equal to 0.5 the diffusion is Fickian and the solvent diffusion rate 359

is slower than the polymer relaxation rate; n equal to 1 refers to Case II type 360

diffusion, with the solvent diffusion rate faster than the polymer relaxation 361

process. A value of n between 0.5 and 1 refers to anomalous diffusion, which 362

happens when the diffusion and relaxation rates are comparable. Super Case 363

diffusion occurs for n > 1. Experimental sorption uptake curves plotted in Fig. 5 364

were fitted to the power law described by Equation 1, and the mass transport 365

mechanism was evaluated by calculating parameter n. The values of n were 366

between 0.5 and 0.6 (Table 2). Therefore the mathematical model based on the 367

one-dimensional solution of Fick's second law of diffusion in a plane sheet was 368

applied to the experimental sorption uptake data. This model considers the 369

diffusion coefficient independently of the concentration of the sorbed compound. 370

Assuming the initial/boundary conditions: 371

� = 00 < � < �� = � � > 0� = 0, � = �� = �∞

where� is the initial concentration of sorbate in the polymer (� = 0) and �∞ is 372

the concentration of the sorbate in both surfaces of the plane sheet which is 373

assumed to be constant throughout the experiment, � is the concentration of 374

sorbate as a function of time (�) and position (�) in the film of thickness �. The 375

solution under these conditions is: 376

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� = ∞ �1 − 8���� 1�2� + 1�� ��� �−�� · · �2� + 1�� · ��� !"

∞

#$%�2�

where � is the thickness of the film (m) and the diffusion coefficient (m2/s) 377

(Crank, 1975). Fig. 5 shows that sorption uptake data were well fitted by 378

Equation 2. Table 2 shows values of diffusion coefficients for films plasticized 379

with 35% glycerol and conditioned at 0, 53 and 75% RH prior to immersion in 380

carvacrol, and films plasticized with 20% glycerol and conditioned at 75% RH. 381

The diffusion coefficient of carvacrol in films incorporating 35% glycerol and 382

conditioned at different relative humidities increased as the water content 383

increased. Diffusion coefficients of films conditioned at 0 and 53% RH differed 384

by one order of magnitude, and also of films conditioned at 53 and 75% RH. 385

Water has the ability to plasticize and swell hydrophilic polymers such as 386

chitosan, increasing chain mobility and interchain distance. As the RH at which 387

the films were conditioned increased, they experienced a successive 388

plasticization and swelling by water molecules, giving rise to a looser polymer 389

matrix, which enhanced the diffusion of carvacrol. Films conditioned at 75% RH 390

are expected to be greatly plasticized by water and an increase in the amount of 391

glycerol from 20 to 35% did not greatly affect the diffusion coefficient. 392

3.3. Desorption kinetics of carvacrol 393

The release of carvacrol vapour from 1:1 CS:CD films plasticized with 35% 394

glycerol and conditioned at 75% RH (CS:CD-35G-90RH) prior to immersion in 395

carvacrol, was evaluated at 25 °C and under different relative humidities: 43, 53 396

and 90%. Fig. 6 shows the normalized experimental curves of release of 397

carvacrol vapour from these films. As in the sorption process described in 3.2 398

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section, the one-dimensional solution of Fick's second law of diffusion in a plane 399

sheet for the boundary conditions in a desorption process: 400

� = 00 < � < �� = �∞

� > 0� = 0, � = �� = 0

The solution under these conditions is: 401

� = ∞ � 8���� 1�2� + 1�� ��� �−�� · · �2� + 1�� · ��� !"

∞

#$%�3�

The theoretical curves modelled with Equation 3 for a desorption process are 402

also shown in Fig. 6. The rate of carvacrol release largely depended on the 403

relative humidity to which the films were exposed. It is known that the diffusion 404

of small molecules in hydrophilic polymers such as chitosan strongly depends 405

on the moisture content of the matrix (Chalier, Ben Arfa, Guillard, & Gontard, 406

2009; Mascheroni, Guillard, Gastaldi, Gontard, & Chalier, 2011); at intermediate 407

and high relative humidities hydrophilic materials absorb moisture, which 408

triggers the release of volatiles entrapped in their structure. Moisture acts as a 409

plasticizer, thereby governing the rate of volatile loss. The release of carvacrol 410

was greatly accelerated when films were exposed to high RH conditions (90%) 411

compared to those exposed to intermediate relative humidities (43 and 53%). 412

All the desorption curves had a two-step pattern. In the first step there was a 413

fast release of carvacrol, which was more acute for films conditioned at 90% 414

RH, followed by a slower release of the remaining volatile. Films conditioned at 415

90% RH released 96% of the carvacrol in the first 34 h, and took approx. 8 days 416

to release 99% of the initial amount. Films conditioned at 53% RH had released 417

94% of the initial amount of carvacrol after 15 days, and 1.5% still remained in 418

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the film after 23 days. Films exposed to 43% RH released 95% of the carvacrol 419

content in 22 days, and after 62 days the residual amount of volatile was 2.6%. 420

Carvacrol diffusion coefficients were calculated for films exposed to different 421

relative humidities by fitting experimental release kinetics data to Equation 2 422

and the values obtained are shown in Table 3. Increasing the RH at which the 423

desorption was carried out led to an increase in diffusivity of carvacrol. Films 424

exposed to 90% RH experienced a rapid loss of carvacrol, having a diffusion 425

coefficient one order of magnitude greater than that obtained for films exposed 426

to intermediate relative humidities. Films conditioned at 43 or 53% RH had 427

diffusion coefficients with the same order of magnitude. Diffusion coefficient 428

values were of the same order of magnitude as those obtained by Mascheroni 429

et al. (2011) at 30 °C in wheat gluten coated paper containing 15% (wt) of 430

carvacrol, where the diffusion coefficients ranged from 0.143 x 10-14 to 2.8 x 10-431

14 m2 s-1 for RH ranging between 60 and 100%. Chalier et al. (2009) studied the 432

diffusivity of carvacrol at 30 °C in soy protein coated paper, where the diffusion 433

coefficients ranged from 0.02 to 1.4 x 10-14 m2 s–1 for RH varying between 60 434

and 100%. Kurek et al. (2014) evaluated the diffusion coefficient of carvacrol in 435

chitosan films measured at RH >96%, finding values of 3.8 x 10–15 at 20 °C and 436

5.5 x 10–13 at 37 °C. 437

3.4. Antimicrobial activity 438

3.4.1. Antimicrobial activity of carvacrol vapour against S. aureus and E. coli 439

The antimicrobial activity of carvacrol was tested against S. aureus and E. coli 440

using the microatmosphere method as described in Materials and Methods, and 441

the MIC (minimal inhibitory concentration) was determined. Table 4 shows that 442

the minimum dose of carvacrol found to produce inhibition zones on agar was 1 443

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mg. The inhibition zone diameters produced by disks with carvacrol were 15 444

and 13 mm for S. aureus and E. coli, respectively. The inhibition zones 445

increased as the amount of carvacrol added to the filter paper disk increased; 446

thus, 5 mg of carvacrol produced an inhibition zone of 44 mm against S. aureus 447

and E. coli. 448

Several studies regarding the antimicrobial activity of essential oils against food 449

spoilage microorganisms and foodborne pathogens agree on their slightly 450

greater activity against Gram-positive bacteria compared to Gram-negative 451

bacteria (Burt, 2004). This has been attributed to differences in the cell wall of 452

the two types of microorganism, since the outer membrane of Gram-negative 453

bacteria restricts diffusion of hydrophobic compounds through its 454

lipopolysaccharide covering (Vaara, 1992). On the other hand, some studies 455

suggest that Gram-positive bacteria are more resistant than Gram-negative 456

bacteria to the antibacterial properties of essential oils (Zaika, 1988). However, 457

other authors have not obtained evidence for a general greater effectiveness of 458

essential oils against Gram-positive or Gram-negative bacteria, and the results 459

found depend on the essential oil tested (Dorman & Deans, 2000). With regard 460

to the antimicrobial activity of carvacrol, the major component of oregano and 461

thyme oil, a similar antibacterial activity against E. coli and S. aureus was found 462

in the present work; these results are in agreement with other studies (Ben Arfa, 463

Combes, Preziosi Belloy, Gontard, & Chalier, 2006; Griffin, Wyllie, Markham, & 464

Leach, 1999). 465

3.4.2. Study of the antimicrobial activity of films loaded with carvacrol 466

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The antimicrobial properties of carvacrol loaded 1:1 CS:CD films plasticized 467

with 20 or 35% glycerol and conditioned at different RH values were tested 468

against S. aureus and E. coli. 469

Table 5 shows the carvacrol content of the films and the diameter of the 470

inhibition zone of S. aureus and E. coli. It is worth mentioning that the 471

antimicrobial activity of carvacrol loaded chitosan films having different amounts 472

of glycerol and water incorporated into the matrix but without the incorporation 473

of HP-β-CDs was also evaluated. The retention capacity of these films was less 474

than 1% carvacrol and none of them exerted antimicrobial activity in vapour 475

phase. The addition of HP-β-CDs to the chitosan matrix increased sorption of 476

carvacrol and therefore changed the antimicrobial capacity. No inhibition zone 477

was observed for carvacrol loaded 1:1 CS:CD films without glycerol and 478

conditioned at 0, 53 and 75% RH prior to immersion in carvacrol, since the 479

amount of carvacrol in the film did not reach the minimum inhibitory dose of 1 480

mg. However, there was observed a reduction in growth density across the Petri 481

dish for films without glycerol and conditioned at 53 and 75% RH, the amount of 482

carvacrol retained by these films was next to 0.6 mg. Dry CS:CD films 483

plasticized with 20% glycerol retained less than 0.4 mg of carvacrol, thus they 484

did not also experience any antimicrobial activity. CS:CD films plasticized with 485

35% glycerol and conditioned at 75% and 53% RH, and CS:CD films plasticized 486

with 20% glycerol and conditioned at 75% RH possessed the greatest content 487

in carvacrol ( > 60 mg) and produced complete inhibition of bacterial growth in 488

the Petri dish (85 mm diameter), whereas the inhibition halo was reduced to 79 489

mm and 76 mm against S. aureus and E.coli respectively for dry films 490

plasticized with 35% glycerol and around 19 mg carvacrol content. An inhibition 491

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halo of 67 mm was observed for CS:CD films plasticized with 20% glycerol, 492

conditioned at 53% RH and having around 11 mg of carvacrol. In conclusion, 493

the major antimicrobial activity was exerted by CS:CD films possessing 494

adequate combination of glycerol content and relative humidity conditioning, 495

which allowed to be loaded with a higher content of carvacrol (≥60 mg) and 496

produce bactericide effect against Gram positive and Gram negative bacteria. 497

498

3.4.3. Antimicrobial activity of films during long-term storage 499

CS:CD-35G-75RH films, which showed the greatest capacity for sorption of 500

carvacrol, were chosen to follow their antimicrobial capacity over time. For this 501

purpose, the antimicrobial activity in vapour phase of films submitted to 502

desorption at 25 °C and 43% RH was monitored for 20 days (every 2 days for 2 503

weeks and then after 8 days). Fig. 7 shows the effect of time on the amount of 504

carvacrol remaining in the film and the inhibition zone created against E. coli 505

and S. aureus. As expected, the inhibition halo experienced a reduction as the 506

carvacrol content in the film decreased over time. The initial amount of carvacrol 507

in the film was 146 mg, producing total inhibition of bacterial growth. A rapid 508

reduction of carvacrol in the films was observed during the first week of storage, 509

giving rise to almost total growth inhibition on the agar plate. Thus, the amount 510

of carvacrol in the film was reduced by 93% after 8 days of storage, and being 511

the inhibition zone reduced to 64 and 66 mm in diameter for S. aureus and E. 512

coli respectively. After that, film experienced a slow release of carvacrol; at the 513

middle of the storage period, the carvacrol content in the film was 6.5 mg having 514

inhibition zones of around 60 mm. After 12 days of storage, the carvacrol 515

content retained in the film was 4.5 mg, and the inhibition zones produced were 516

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57 and 56 mm in diameter for E. coli and S. aureus, respectively. After 20 days 517

of storage, the amount of carvacrol remaining in the film did not change greatly, 518

this was found to be 4 mg and the inhibition zone against the bacteria tested 519

was similar to the 12th day. 520

It is worth pointing out that the inhibition zone created by the film disk containing 521

4.5 mg of carvacrol was slightly greater that that produced by a filter paper of 522

similar dimensions and incorporating 5 mg of carvacrol when the two samples 523

(film and filter paper) were submitted to the same microatmosphere test 524

conditions (37 °C for 24 h and >99% RH). Because of the high relative humidity 525

reached in the Petri dish and the temperature used in the antimicrobial 526

experiment, it was expected that the small amount of carvacrol incorporated in 527

the paper (5 mg) and remaining in the film (4.5 mg) would be released in 24 h. 528

Slight differences in inhibition could be related to carvacrol release behaviour 529

from these two materials during the antimicrobial test, which could affect 530

microbial growth. 531

532

4. CONCLUSIONS 533

This work shows that it is possible to control the loading of hydrophobic 534

antimicrobial carvacrol in hydrophilic chitosan. For this purpose, the polymer 535

was blended with HP-β-CDs and glycerol and water were incorporated as 536

coadjuvants. Depending on the amount of these compounds in the chitosan 537

matrix, the films can be loaded with different input amounts of carvacrol. 538

Moreover, the release rate of carvacrol from the films depends greatly on the 539

environmental relative humidity. The films developed have antimicrobial 540

properties and can be active for an extended period of time. They could be 541

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applied in the design of active packages to inhibit microbial growth on the 542

surface of solid foods. Because of the volatile properties of carvacrol, direct 543

contact of the film with the food would not be required, since the moisture 544

present in the package triggers and controls release of the compound 545

entrapped in the film. 546

ACKNOWLEDGEMENTS 547

The authors would like to thank the Spanish Ministry of Science and Innovation 548

for financial support (AGL2012-39920-C03-01), and CSIC/European Social 549

Fund (JAE-Predoc, L.H. fellowship). 550

REFERENCES 551

Alfrey, T., Gurnee, E. F., & Lloyd, W. G. (1966). Diffusion in glassy polymer. 552

Journal of Polymer Science Part C: Polymer Symposia, 12, 249-261. 553

Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food 554

packaging. Innovative Food Science & Emerging Technologies, 3(2), 555

113-126. 556

ASTM. (2007). Standard practice for maintaining constant relative humidity by 557

means of aqueous solutions. In (Vol. ASTM E104 - 02). West 558

Conshohocken, PA: ASTM International. 559

Ben Arfa, A., Combes, S., Preziosi Belloy, L., Gontard, N., & Chalier, P. (2006). 560

Antimicrobial activity of carvacrol related to its chemical structure. Letters 561

in Applied Microbiology, 43(2), 149-154. 562

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

25

Burt, S. (2004). Essential oils: their antibacterial properties and potential 563

applications in foods - a review. International Journal of Food 564

Microbiology, 94(3), 223-253. 565

Crank, J. (1975). The mathematics of diffusion (2nd ed.): Oxford Series 566

Publications. 567

Chalier, P., Ben Arfa, A., Guillard, V., & Gontard, N. (2009). Moisture and 568

temperature triggered release of a volatile active agent from soy protein 569

coated paper: effect of glass transition phenomena on carvacrol diffusion 570

coefficient. Journal of Agricultural and Food Chemistry, 57(2), 658-665. 571

Delaquis, P. J., Stanich, K., Girard, B., & Mazza, G. (2002). Antimicrobial 572

activity of individual and mixed fractions of dill, cilantro, coriander and 573

eucalyptus essential oils. International Journal of Food Microbiology, 574

74(1/2), 101-109. 575

Dorman, H. J., & Deans, S. G. (2000). Antimicrobial agents from plants: 576

antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 577

88(2), 308-316. 578

Dutta, J., Tripathi, S., & Dutta, P. K. (2012). Progress in antimicrobial activities 579

of chitin, chitosan and its oligosaccharides: a systematic study needs for 580

food applications. Food Science and Technology International, 18(1), 3-581

34. 582

Garnero, C., Aloisio, C., & Longhi, M. (2013). Ibuprofen-maltodextrin interaction: 583

study of enantiomeric recognition and complex characterization. 584

Pharmacology & Pharmacy, 4, 18-30. 585

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

26

Griffin, S., Wyllie, S. G., Markham, J., & Leach, D. (1999). The role of structure 586

and molecular properties of terpenoids in determining their antimicrobial 587

activity. Flavour and Fragrance Journal, 14(5), 322-332. 588

Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of 589

essential oils and other plant extracts. Journal of Applied Microbiology, 590

86(6), 985-990. 591

Higueras, L., López-Carballo, G., Cerisuelo, J. P., Gavara, R. & Hernández-592

Muñoz, P. (2013). Preparation and characterization of chitosan / HP-β-593

cyclodextrins composites with high sorption capacity for carvacrol. 594

Carbohydrate Polymers 97 (2), 262-268. 595

Kumar, M. N., Muzzarelli, R. A., Muzzarelli, C., Sashiwa, H., & Domb, A. J. 596

(2004). Chitosan chemistry and pharmaceutical perspectives. Chemical 597

Reviews, 104(12), 6017-6084. 598

Kurek, M., Guinault, A., Voilley, A., Galic, K., & Debeaufort, F. (2014). Effect of 599

relative humidity on carvacrol release and permeation properties of 600

chitosan based films and coatings. Food Chemistry, 144, 9-17. 601

Locci, E., Lai, S. M., Piras, A., Marongiu, B., & Lai, A. (2004). C-13-CPMAS and 602

H-1-NMR study of the inclusion complexes of beta-cyclodextrin with 603

carvacrol, thymol, and eugenol prepared in supercritical carbon dioxide. 604

Chemistry & Biodiversity, 1(9), 1354-1366. 605

Lopez-Carballo, G., Higueras, L., Gavara, R., & Hernandez-Munoz, P. (2013). 606

Silver ions release from antibacterial chitosan films containing in situ 607

generated silver nanoparticles. Journal of Agricultural and Food 608

Chemistry, 61(1), 260-267. 609

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

27

Mascheroni, E., Guillard, V., Gastaldi, E., Gontard, N., & Chalier, P. (2011). 610

Anti-microbial effectiveness of relative humidity-controlled carvacrol 611

release from wheat gluten/montmorillonite coated papers. Food Control, 612

22(10), 1582-1591. 613

Messner, M., Kurkov, S., Jansook, P., & Loftsson, T. (2010). Self-assembled 614

cyclodextrin aggregates and nanoparticles. International Journal of 615

Pharmaceutics, 387(1-2), 199-208. 616

Ouattara, B., Simard, R. E., Piette, G., Begin, A., & Holley, R. A. (2000). 617

Diffusion of acetic and propionic acids from chitosan-based antimicrobial 618

packaging films. Journal of Food Science, 65(5), 768-773. 619

Prabaharan, M., & Mano, J. F. (2006). Chitosan derivatives bearing cyclodextrin 620

cavities as novel adsorbent matrices. Carbohydrate Polymers, 63(2), 621

153-166. 622

Ramya, R., Venkatesan, J., Kim, S. K., & Sudha, P. N. (2012). Biomedical 623

applications of chitosan: an overview. Journal of Biomaterials and Tissue 624

Engineering, 2(2), 100-111. 625

Ravi, P., & Divakar, S. (2001). Stereoselective hydrogenation of thymol over 626

Rh/alumina in the presence of beta-cyclodextrin and its derivatives. 627

Journal of Inclusion Phenomena and Macrocyclic Chemistry, 39(1-2), 27-628

33. 629

Ribeiro, L. S. S., Veiga, F. J. B., & Ferreira, D. (2003). Physicochemical 630

investigation of the effects of water-soluble polymers on vinpocetine 631

complexation with beta-cyclodextrin and its sulfobutyl ether derivative in 632

solution and solid state. European Journal of Pharmaceutical Sciences, 633

20(3), 253-266. 634

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

28

Suppakul, P., Miltz, J., Sonneveld, K., & Bigger, S. W. (2003). Antimicrobial 635

properties of basil and its possible application in food packaging. Journal 636

of Agricultural and Food Chemistry, 51(11), 3197-3207. 637

Vaara, M. (1992). Agents that increase the permeability of the outer membrane. 638

Microbiological Reviews, 56(3), 395-411. 639

Valencia-Chamorro, S. A., Palou, L., del Rio, M. A., & Perez-Gago, M. B. 640

(2011). Antimicrobial edible films and coatings for fresh and minimally 641

processed fruits and vegetables: a review. Critical Reviews in Food 642

Science and Nutrition, 51(9), 872-900. 643

van de Manakker, F., Vermonden, T., van Nostrum, C., & Hennink, W. (2009). 644

Cyclodextrin-based polymeric materials: synthesis, properties, and 645

pharmaceutical/biomedical applications. Biomacromolecules, 10(12), 646

3157-3175. 647

Zaika, L. L. (1988). Spices and herbs - their antimicrobial activity and its 648

determination Journal of Food Safety, 9(2), 97-118. 649

Zhang, X. G., Wu, Z. M., Gao, X. J., Shu, S. J., Zhang, H. J., Wang, Z., & Li, C. 650

X. (2009). Chitosan bearing pendant cyclodextrin as a carrier for 651

controlled protein release. Carbohydrate Polymers, 77(2), 394-401. 652

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Fig. 1. Flowchart of film preparation

Fig. 2. Sorption equilibrium of carvacrol in CS:CD films with a 1:1 (w/w) blend

ratio, incorporating several percentages of glycerol and conditioned at different

relative humidities prior to immersion in the solvate and measured at 25 ºC.

Figure also shows the equilibrium moisture content of the films at 25ºC before

being immersed in carvacrol.

Fig. 3. Effect of hydroxypropyl-β-cyclodextrin content in a CS:CD-35G-75RH

film on the sorption capacity of carvacrol at 25 ºC.

Fig. 4. Carvacrol sorption capacity by films of chitosan incorporating

maltodextrins (1:1 weight ratio). Films were plasticized with 35% glycerol and

conditioned at 0, 53 and 75% RH prior to immersion in carvacrol.

Fig. 5. Comparison between experimental sorption curves (symbols) of liquid

carvacrol in CS:CD films with a 1:1 (w/w) blend ratio at 25 ºC and modelled

curves (continuous line) obtained from Equation 2. Inset graph: Experimental

data and predicted values (line) for CS:CD-35G-0RH films.

Fig. 6. Experimental (symbols) and theoretical (continuous line) normalized

time-desorption curves of carvacrol from CS:CD-35G-75RH film measured at 25

ºC as a function of environmental relative humidity.

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Fig. 7. Carvacrol remaining in CS:CD-35G-75RH film at 25 ºC and 43% RH and

inhibition halo produced against S. aureus and E. coli over time.

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Table 1

Colour parameters of CS:CD (1:1 weight ratio) films.

% Glycerol L* a* b* C*ab h*ab Prior to immersion in carvacrol 0 95.5 ± 0.2a,wx -0.27 ± 0.06a,w 5.9 ± 0.3a,w 5.9 ± 0.3a,w 92.7 ± 0.5a,w

20 95.5 ± 0.2a,w -0.33 ± 0.05a,w 5.9 ± 0.2a, 5.9 ± 0.3a,x 93.3 ± 0.3a,x

35 95.3 ± 0.3a,w -0.48 ± 0.07b,x 6.2 ± 0.3a,z 6.2 ± 0.3a,z 93.4 ± 0.4a,x

After immersion in carvacrol 0% RH

0 95.6 ± 0.8a,wx - 0.32 ± 0.03a,w 5.9 ± 0.3a,w 5.9 ± 0.3a,w 93.2 ± 0.3a,w

20 95.8 ± 0.7a,w - 0.32 ± 0.03a,w 5.7 ± 0.4a,x 5.7 ± 0.4a,x 93.1 ± 0.4a,x

35 95.6 ± 0.5a,w - 0.33 ± 0.02a,w 5.9 ± 0.5a,y 5.9 ± 0.6a,y 93.1 ± 0.6a,x

53% RH 0 95.5 ± 0.5a,wx - 0.29 ± 0.04a,w 5.8 ± 0.4b,w 5.9 ± 0.4b,w 92.9 ± 0.3b,w

20 95.6 ± 0.9a,w - 0.35 ± 0.06a,w 5.9 ± 0.4b,x 5.9 ± 0.4b,x 93.4 ± 0.4b,x

35 91.9 ± 0.6b,x - 3.76 ± 0.07b,y 24.7 ± 0.9a,x 24.4 ± 1.2a,x 98.7 ± 0.4a,w

75% RH 0 96.8 ± 0.4a,w - 0.23 ± 0.05a,w 5.7 ± 0.5c,w 5.7 ± 0.5c,w 92.5 ± 0.4b,w

20 93.6 ± 0.8b,x - 3.03 ± 0.23b,x 19.6 ± 0.9b,w 19.8 ± 0.6b,w 99.2 ± 0.5a,w

35 91.3 ± 0.8c,x - 4.51 ± 0.14c,z 27.2 ± 0.6a,w 27.5 ± 0.5a,w 99.5 ± 0.5a,w

a-c Different letters in the same column indicated significant differences (P ≤ 0.05) when comparing films incorporating different content of glycerol and conditioned at the same relative humidity.

w-z Different letters in the same column indicated significant differences (P ≤ 0.05) when comparing films conditioned at different relative humidities prior immersion in carvacrol and incorporating the same amount of glycerol.

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Table 2. Power law parameters and diffusion coefficients of carvacrol in CS:CD

films at 25ºC.

Film K (min -n)·102 n R2 D (m2/s) ·1014 R2 CS:CD-35G-0RH 0.26 ± 0.09c 0.52 ± 0.04 0.988 0.0110 ± 0.0005c 0.990 CS:CD-35G-53RH 0.98 ± 0.19b 0.56 ± 0.03 0.987 0.22 ± 0.02b 0.992 CS:CD-35G-75RH 3.37 ± 0.82a 0.55 ± 0.05 0.951 1.90 ± 0.08a 0.988 CS:CD-20G-75RH 2.86 ± 0.85a 0.59 ± 0.06 0.988 2.10 ± 0.13a 0.981

a-c Different letters in the same column indicated significant differences (P ≤ 0.05)

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Table 3. Diffusion coefficient of carvacrol in CS:CD-35G-75R films at different relative humidities and 25 °C.

% RH D (m2/s) ·1015 R2

43 1.21 ± 0.04c 0.998 53 1.45 ± 0.08b 0.988 90 25.3 ± 2.1a 0.968

a-c Different letters in the same column indicated significant differences (P ≤ 0.05)

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Table 4

Antimicrobial activity produced by carvacrol in vapour phase against S. aureus and E. coli.

S. aureus E. coli Carvacrol (mg) Inhibition zone (mm) Inhibition zone (mm)

5.0 44 44 2.0 29 30

1.50 20 21 1.25 18 18 1.00 15 13 0.75 - - 0.50 - -

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Table 5. Antimicrobial activity of CS:CD films loaded with carvacrol against S. aureus and E. coli.

Film S. aureus E. coli

Carvacrol (mg) Inhibition zone (mm) Carvacrol (mg) Inhibition zone (mm) CS:CD-0G-0RH 0.124 - 0.124 -

CS:CD-0G-53RH 0.567 (slight decrease in growth density) 0.581 (slight decrease in growth density) CS:CD-0G-75RH 0.626 (marked decrease in growth density) 0.605 (marked decrease in growth density) CS:CD-20G-0RH 0.315 - 0.340 - CS:CD-20G-53RH 11.6 67 11.1 67 CS:CD-20G-75 RH 61.4 85 61.1 85 CS:CD-35G-0RH 19.3 79 18.4 76 CS:CD-35G-53RH 84.3 85 80.8 85 CS:CD-35G-75RH 153 85 146 85

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Fig. 1

chitosan

1.5% (w/w) chitosan solution

solubilisation in

0.5% (w/w) acetic acid

film-forming solution

HP-β-CD

0, 20 or 35% glycerol

filtration

CS:CD-G films

CS:CD-G-RH films

casting40.0 ± 1.5 °C

20 ± 9% RH

36 h

conditioning0, 53 or 75% RH

25 ± 1 °C

stirring 1500 rpm

37 °C

cut (⌀=550 mm)

thickness selection (55 ± 5 µm )

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Fig. 2.

Film

CS:

CD

-0G

-0R

HC

S:C

D-2

0G-0

RH

CS:

CD

-35G

-0R

HC

S:C

D-0

G-5

3RH

CS:

CD

-20G

-53R

HC

S:C

D-3

5G-5

3RH

CS:

CD

-0G

-75R

HC

S:C

D-2

0G-7

5RH

CS:

CD

-35G

-75R

H

Sor

ptio

n eq

uilib

rium

of c

arva

crol

(%

dry

wei

ght)

0.1

1

10

100

1000

Equ

ilibr

ium

moi

stur

e co

nten

t (%

dry

wei

ght)

0

5

10

15

20

25

30

35

40

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ACCEPTED MANUSCRIPTFig. 3.

CS:CD (weight ratio)1:2 1:1 1:0.5 1:0.25

So

rpti

on

eq

uili

bri

um

of

carv

acro

l (%

dry

wei

gh

t)

0

50

100

150

200

250

300

350

24.33

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% Relative humidity

0 53 75

So

rpti

on

eq

uili

bri

um

of

carv

acro

l (%

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

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Time (minutes)

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

Car

vacr

ol r

atio

0,0

0,2

0,4

0,6

0,8

1,0

1,2

CS:CD-35G-75RHCS:CD-20G-75RHCS:CD-35G-53RH

Time (minutes)

0 5e+4 1e+5 2e+5 2e+5

Car

vacr

ol r

atio

0,0

0,2

0,4

0,6

0,8

1,0

1,2

CS:CD-35G-0RH

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Fig. 6.

Time (minutes)

0 10000 20000 30000 40000 50000

Car

vacr

ol r

atio

0.0

0.2

0.4

0.6

0.8

1.043% RH 53% RH90% RH

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Time (hours)

0 100 200 300 400 500

Inh

ibit

ion

zo

ne

(mm

)

0

20

40

60

80

100R

esid

ual

car

vacr

ol (

mg

)

0

20

40

60

80

100

120

140

160

S. aureus E. coli Carvacrol content in the film

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• HP-β-CD and hydrophilic coadjuvants can tailor carvacrol sorption properties of chitosan films.

• Carvacrol sorption kinetics depends on plasticization degree of chitosan/ HP-β-CD films.

• Release kinetics of carvacrol is controlled by environmental relative humidity.

• Films exhibited long-lasting antimicrobial properties against E. coli and S. aureus.