Comprehensive characterization of active chitosan-gelatin blend films enriched with different essential oils

Abstract Natural extracts and plant essential oils (EOs) have long been recognized as valid alternatives to synthetic food additives owing to their proved wide-spectrum antimicrobial capacity. The main aim of this study was to characterize the physical, mechanical, water barrier, microstructural and antimicrobial properties of chitosan-gelatin blend films enriched with cinnamon, citronella, pink clove, nutmeg and thyme EOs. The film microstructure determined by scanning electron microscopy, showed that all active films had heterogeneous surface: in particular, films including cinnamon, nutmeg and thyme EOs showed remarkable pores on the surface. The possible interaction of chitosan-gelatin blend film with incorporated EOs was investigated using Fourier-transform infrared (FT-IR) spectroscopy. Presence of new bands and changes in the FT-IR spectra confirmed intermolecular interactions between the chitosan-gelatin matrix and the EOs. The antimicrobial activity of films was determined using the disk diffusion assay. Active films inhibited the growth of four major food bacterial pathogens including Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella typhimurium and, among the tested EOs, thyme was the most effective (p


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Introduction 35
Environmental concerns as well as consumer demand for natural, minimally processed, 36 preservative-free and high-quality food, have raised the attention of food packaging 37 industries on the development of bio-based films enriched with natural compounds. Bio-38 based films have been considered as attractive alternatives to plastic packaging due to their 39 excellent biodegradability, moreover, they can be blended with active compounds such as 40 antimicrobial agents to protect food against microbial deterioration and to extend the shelf life 41 of food products (De Leo et al., 2018;Shen & Kamdem, 2015). 42 Among biopolymers, chitosan (CS) and gelatin (GL) have shown outstanding film forming 43 property, non-toxicity, biocompatibility, biodegradability, stability and commercial availability. 44 The CS is a linear polysaccharide, commercially obtainable from deacetylation of chitin. This 45 polycationic biopolymer is soluble in solutions with pH below 6.5 due to the protonation of the 46 amino group (Bonilla, Poloni, Lourenço, & Sobral, 2018). The positively charged amino group 47 M A N U S C R I P T

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Preparation of films was adapted from Bonilla & Sobral (2016) with slight modifications. In 113 this study, five different types of films based on a CS-GL blend enriched with EOs 114 (cinnamon, citronella, pink clove, nutmeg and thyme) were analyzed. A film without EO was 115 used as a control. All film forming solutions (FFS) with and without EOs were prepared 116 separately. CS FFS (2%, w/v) was prepared by dissolving CS in an acetic acid solution (1%, 117 v/v) under continuous stirring at 55°C for 30 min. GL FFS (2 %, w/v) was prepared by 118 dissolving GL in distilled water, first being allowed to swell at 7°C for 15 min and then stirred 119 at 55 °C for 30 min. Glycerol (25% w/w of CS or GL) was then added as a plasticizer into 120 both FFS, followed by additional stirring for 30 min. CS-GL blend solution was prepared by 121 mixing CS and GL FFS at 1:1 ratio. Moreover, different types of EOs (1%, v/v) together with 122 Tween 80 (0.2%, v/v EO) were added to FFS, followed by stirring at 55 °C for additional 30 123 min. All FFS were degasified with a vacuum pump (70 kPa) for 15 min to remove bubbles 124 from the FFS. Films were obtained by casting 20 mL of the FFS into Petri dishes (14.4 cm in 125 diameter) and drying at 25±2 °C overnight in the chemical hood at ambient relative humidity 126 (RH) of 45%. 127

Gas Chromatography-Mass Spectrometer (GC-MS) analysis of essential oils 128 volatile profiles 129
The volatile profiling of the EOs used for incorporation in CS-GL films was carried out by

Scanning electron microscopy 141
Scanning electron microscopy (SEM) of the surface and cross-section of the films were 142 obtained with the use of a scanning electron microscope (FEI,Quanta 200,Oregon,USA). 143 Film samples were fixed on a stainless-steel support with a double side conductive adhesive. 144 The analysis was conducted in low vacuum (0.6 Torr) at an acceleration voltage of 20 kV. 145

Attenuated Total Reflection (ATR) / Fourier-Transform Infrared (FT-IR) 146
Spectroscopy 147 The infrared spectra of different films were obtained using an ATR/FT-IR spectrometer (type 148 Alpha, Bruker Optik GmbH, Ettlingen, Germany). Spectra were collected from two different 149 locations from the top and bottom of the same samples in the 4000-400 cm −1 wavenumber 150 range by accumulating 64 scans with a spectral resolution of 4 cm -1 . 151

Thickness and mechanical properties 152
Film thickness was measured with a digital micrometer (SAMA Tools measuring Instruments 153 & NTD equipment, Viareggio, Italia) at five different random positions (one at the center and 154 four at the edges). The means of these five separate measurements were recorded. 155 The tensile stress (TS), elongation at break (EAB) and elastic modulus (EM) were 156 determined using a dynamometer (Z1.0, ZwickRoell, Italy) according to ASTM standard 157 method D882 (ASTM, 2001a). The films with known thickness were cut into rectangular 158 strips (9 x 1.5 cm 2 ). Initial grip separation and cross-head speed were set at 70 mm and 10 159 mm/s, respectively. Measurements were repeated 10 times. The software TestXpert® II 160 (V3.31) (ZwickRoell, Ulm, Germany) was used to record the TS curves. TS was calculated by 161 dividing the maximum load to break the film by the cross-sectional area (thickness) of the film 162 and expressed in MPa. EAB was calculated by dividing film elongation at rupture by the initial 163 grip separation expressed in percentage (%). EM was calculated from the initial slope of the 164 stress-strain curve and expressed in MPa. TS and EAB were evaluated for ten samples from 165 each type of film. 166

UV barrier, light transmittance, opacity value and color 167
The barrier properties of films against UV and visible light were determined at the UV (200,168 280 and 350 nm) and visible (400, 500, 600, 700 and 800 nm) wavelengths onto square film 169 samples (2 × 2 cm 2 ) using a Jasco V -550 UV/Vis spectrophotometer (Jasco Corporation,170 Tokyo, Japan) as described by Bellelli, Licciardello, Pulvirenti & Fava (2018). The opacity of 171 the films was calculated by Eq. (1): 172 where T 600 is the fractional transmittance at 600 nm and x is the film thickness (mm). The 174 greater opacity value represents the lower transparency of the film. For each film, four 175 readings were taken at different points and average values were determined. 176 The color of films was measured with a CR-400 Minolta colorimeter (Minolta Camera, Co., 177 Ltd., Osaka, Japan) at room temperature, with D65 illuminant and 10° observer angle. The 178 instrument was calibrated with a white standard (L* = 99.36, a* = -0.12, b* = -0.07) before 179 measurements. Results were expressed as L* (luminosity), a* (red/green) and b* 180 (yellow/blue) parameters. The total color difference (∆E * ) was calculated using the following 181 Eq. (2): 182 ∆E * = [(∆L * ) 2 +(∆a * ) 2 +(∆b * ) 2 ] (2) 183 where ∆L * , ∆a * and ∆b * are the differences between the corresponding color parameter of the 184 samples and that of a standard white plate used as the film background. For each film, five 185 readings were taken at different points and the average values were determined from the top 186 and bottom sides. 187

Moisture content and water solubility 188
Moisture content (MC) of the films was determined by measuring weight loss upon drying to 189 constant weight in an oven at 105 ± 2 °C according to the following Eq. Where, Mw and Md are the initial weight and dry weight of the film, respectively. 192 The initial dry matter content of each film was determined by drying to constant weight in an 193 oven at 105± 2 °C (Wi) and then each film was immersed in 50 mL distilled water at 25 °C.
After 24 h, the film samples (2 × 2 cm 2 ) were dripped and dried to constant weight at 105± 2 195 °C (Wf) to determine the weight of dry matter which was not solubilized in water. The 196 measurement of water solubility (WS) was determined according to the following Eq. (4): 197 where, Wi and Wf are initial and final weight of the film, respectively. 199

Water vapor transmission rate and water vapor permeability 200
Water vapor transmission rate (WVTR) of the films was determined gravimetrically in 201 triplicate according to the ASTM E96 method (ASTM, 2001b) with some modifications. Films 202 were sealed on top of glass test cups with an internal diameter of 10 mm and a depth of 55 203 mm filled with 2 g anhydrous CaCl 2 (0% RH). The cups were placed in desiccators containing 204 BaCl 2 (75% RH), which were maintained in incubators at 45 °C. WVTR was determined 205 using the weight gain of the cups and was recorded and plotted as a function of time. Cups 206 were weighted daily for 7 days to guarantee the steady state permeation. The slope of the 207 mass gain versus time was obtained by linear regression (r 2 ≥ 0.99). WVTR (g /day m 2 ) and 208 WVP (g mm/kPa day m 2 ) were calculated according to the following Eqs. where ∆W/∆t is the weight gain as a function of time (g/day), A is the area of the exposed 212 film surface (m 2 ), L is the mean film thickness (mm) and ∆P is the difference of vapor 213 pressure across the film (kPa). 214

In vitro antimicrobial activity 215
Antibacterial activity test on films was assessed against four typical food bacterial pathogens 216 including Listeria monocytogenes (UNIMORE 19115), Escherichia coli (UNIMORE 40522), 217 Salmonella typhimurium (UNIMORE 14028) and Campylobacter jejuni (UNIMORE 33250) 218 using the disk diffusion assay according to (Haghighi et al., 2019). Films (sterilized with UV 219 light) were cut into a disc shape of 22 mm diameter and placed on the surface of BHIA agar 220 plates, which had been previously streaked with 0.1 mL of inocula containing 10 6 CFU/mL of M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT tested bacteria. The plates were then incubated at 30 °C for 24 h (C. jejuni plates were 222 incubated at 37 °C). The diameter of the inhibition zones was measured with a caliper and 223 recorded in millimeters (mm). All tests were performed in triplicates. 224

Statistical analysis 225
The statistical analysis of the data was performed through analysis of variance (ANOVA) 226 using SPSS statistical program (SPSS 20 for Windows, SPSS INC., IBM, New York). The 227 differences between means were evaluated by Tukey's multiple range test (p<0.05). The 228 data were expressed as the mean ± SD (standard deviation). 229

3.
Results and discussion 230

Composition of the essential oils 231
The volatile profiles of the tested EOs are shown in Tab. 1, which reports the major 232 compounds with their relative abundance (%). Typical chromatograms for each EO are 233 available in the supplementary material (Appendix A). As it can be inferred, eugenol alone 234 accounted for more than 51% of the total peak area of cinnamon EO, while 14 other 235 components contributed from 1 to 6.7% to the total peak area, with β-caryophyllene and 236 benzyl benzoate prevailing, followed by acetyleugenol and linalool, among the most 237 represented. Some differences between our results and other studies were observed, as 238 reported by Wang et al. (2018), cinnamaldehyde was the most representative components of 239 cinnamon EO. The other main constituents were eugenol (19.188%), linalool (4.563%), and 240 beta-caryophyllene (4.551%). In fact, the chemical compositions of the EOs may be varied 241 depending on geographical and climate conditions, herbal species, age, ecotypes, 242 geographical origins and method of drying and isolation of the EOs (Khezrian & Shahbazi, 243

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Pink clove EO was the simplest among the studied substances, since it was mainly 250 composed of eugenol (96.5% of total peak area), with minor contributions of carvacrol, β-251 caryophyllene and vanillin. 252 Nutmeg EO was composed by about 22.7% sabinene, 14.9 and 10.3% αand β-pinene, 253 respectively, and many other terpenic compounds, 7 representing 3-7% and 7 more ranging 254 from 1 to 3% of total peak area. Our results on chemical profiling of the nutmeg EO was in 255 accordance with Morsy (2016). 256 Thyme EO was characterized by p-cymene, thymol and carvacrol, which, together, 257 represented almost 80% of the total chromatographic area. Linalool, α-pinene and borneol 258 contributed for another 13%, while β-myrcene, limonene, β-caryophyllene, camphene and  reported that CS-GL blend film containing eugenol and ginger EOs had uncompact texture 283 with sponge-like structure due to the uneven dispersion of EOs with hydrophobic nature from 284 the aqueous phase during the film drying process. 285

Attenuated Total Reflection (ATR) / Fourier-Transform Infrared (FT-IR) 286
Spectroscopy 287 ATR/FT-IR spectroscopy was performed to characterize the structural and spectroscopic cm -1 can be assigned to antisymmetric and symmetric ν as (CH 3 /CH 2 )/ν s (CH 3 /CH 2 ) stretching 300 vibrations of CH 3 and CH 2 functionalities. The peaks at 849, 898, 995, 1030, 1150 cm -1 can 301 be assigned to saccharide structures of the CS biopolymer in the CS-GL blend film network 302 (Shen & Kamdem, 2015). 303 The ATR/FT-IR spectra of the active films showed partly characteristic additional bands of 304 the incorporated EOs. It has to be mentioned, however, that due to the low amounts of admixed EOs, only the most intense absorptions of specific functionalities are observable in 306 the spectra. In Fig. 3 the spectra have been arranged (from top to bottom) in the order of 307 increasing ν(C=O) bands in the wavenumber range 1720-1740 cm -1 that can be assigned to 308 ester, aldehyde or ketone functionalities of the EO admixtures. Thus, pink clove and thyme 309 do not show these bands. However, while the spectrum of thyme is -with the exception of 310 weak additional bands in the 2800-3000 cm -1 range due to aliphatic functionalities -very 311 similar to the control spectrum, the spectrum of pink clove shows a very characteristic  This might be due to an increase in homogeneity and to the creation of a well-organized and 341 dense network upon addition of citral EO, but also to the extended drying time required. 342

Mechanical properties 343
The tensile strength (TS), percent elongation at break (EAB%) and elastic modulus (EM) are 344 the most common mechanical parameters for food packaging applications (Acevedo-Fani, that the strong interaction between CS and EO determined a cross-linking effect leading to 361 an increase in TS. The TS of packaging film must be more than 3.5 MPa, according to 362 conventional standards (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015). In this study, 363 the TS value of control and active CS-GL films ranged from 29.54 to 47.72 MPa which is a 364 high value for its application as packaging material. 365 The EAB is related to the film flexibility and stretchability. The EAB values ranged from 366 2.18% to 2.90% indicating that all films were quite brittle. No significant difference was 367 observed in the EAB of control and active films (p>0.05). Souza et al. (2017) also found that 368 the incorporation of different EOs and hydroalcoholic extracts into CS film did not induce 369 significant differences in EAB values. 370 The EM stands for the resistance of the film to elastic deformation and this parameter 371 indicates the rigidity or stiffness of the film. A low EM value corresponds to a flexible film 372 while a larger EM value indicates a more rigid material. The cinnamon-added films showed 373 the lowest EM value (1340 MPa) meaning that the CS-GL film lost its stiffness and became 374 more flexible with the addition of cinnamon EO (p<0.05). However, films containing citronella, 375 pink clove, nutmeg and thyme EOs showed EM values similar to the control film. Overall, it 376 seems that cinnamon EO acts as plasticizer, since it determines a lower TS and a higher 377 EAB (softer and more extensible film). Nutmeg and thyme seem to act as crosslinkers, 378 slightly increasing TS. However, the effects on mechanical properties, are hardly noticeable 379 and may depend on the low relative amounts of EO in the FFSs.

Color 399
The color values (L*, a* and b*) and total color difference (∆E*) of control and active films are 400 shown in Tab which suggest that the compounds present in cinnamon and pink clove EOs absorb in this 420 range, which corresponds to the yellow-red color measured by the a* and b* coordinates. 421 Nevertheless, the color of the developed films can change the overall appearance of the food 422 inside the packaging and affecting customer acceptance (Atarés & Chiralt, 2016). 423

Moisture content, water solubility and water vapor permeability 424
The moisture content (MC), water solubility (WS) and water vapor permeability (WVP) of 425 control and active films are presented in Tab The shelf life of some food products is directly related to the transfer of water between the 450 product and the external environment in which they are introduced. Generally, packaging 451 material should reduce this transfer of water to preserve foods from moisture (de Moraes 452 Crizel et al., 2018;Hosseini, Rezaei, Zandi, & Farahmandghavi, 2016;Kim et al., 2018). determining factor in WVP value. In this study, despite the statistical differences, the WVP 463 varied between 0.8 and 1.2 (g mm/kPa day m 2 ). In practical terms, this means that all films 464 were highly permeable to water vapor. The results showed that the incorporation of different EOs could notably improve the UV 502 barrier properties of CS-GL film, however, light transparency was reduced. The developed 503 films, with special regards for those including thyme EO, possessed noticeable antimicrobial 504 activity against common food pathogens. The moisture content and water vapor permeability 505 of CS-GL film increased by EOs incorporation due to the microstructure change and 506 presence of pores on the surface as confirmed by SEM. The results suggest that the CS-GL 507 films enriched with different EOs could be used as environmentally friendly, active food 508 packaging with antimicrobial properties and potential to extend the shelf life of food products. 509

Declarations of interest 510
None.

723
Different letters in the same column indicate significant differences (p<0.05). Table 4 726

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Color parameters (L*, a* and b*) and total color difference (∆E*) of the films based on chitosan-gelatin 727 blend (CS-GL) as a control and those enriched with EOs (1%, v/v). Table 5  Table 6 739 Inhibition zone diameters of the film disks (22 mm diameter) based chitosan-gelatin blend (CS-GL-

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Control) as a control and those enriched with EOs (1%, v/v).

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Highlights: • Production of films based on chitosan-gelatin enriched with essential oils • Determination of the physical, mechanical and barrier properties • Demonstraion of the interaction between chitosan-gelatin and essential oils • Improving UV barrier of chitosan-gelatin film by addition of essential oils • Effectiveness of active films against common food bacterial pathogens M A N U S C R I P T

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Keywords: Bio-Based Active Packaging; Chitosan-Gelatin Blend; Essential Oil; Scanning Electron Microscopy (SEM); Fourier-Transform Infrared Spectroscopy (FT-IR)