Hana Maalej

Ssistant Rofessor

S Sawsan

Doctor Sawsan ffes, Doctor Hana Maalej, Ssistant Rofessor

Suming Li

Doctor Rim Nasri, ssistant rofessor Rania Bdelhedi

Moncef Nasri

Sawsan Affes

Rania Abdelhedi

Rim Nasri

Chitosan Chito-oligosaccharides ilms Maillard reaction conditions hysicochemical characteri ation antioxidant otential

Keywords: Chitosan, Chito-oligosaccharides, Films, Maillard reaction conditions, Physicochemical characterization, antioxidant potential

n this study, the effects of different tem eratures, incubation times and ty es of reducing sugars, including glucose and different low molecular weight (Mw chitooligosaccharides (COS with varying acetylation degree ( D , on the extent of Maillard reaction (MR on chitosan-based films were studied. nterestingly, an im rovement of structural and functional ro erties of all MR-crosslinked films was noted, which is more ronounced by heating at higher tem erature and ex osure time. These findings were roved through T R and -ray diffraction analyses. n addition, color change and ltraviolet s ectra demonstrate that glucose addition rovides the high extent of MR, followed by COS1 and COS . These results were confirmed by enhanced water resistance and thermal ro erties. Moreover, MR-chitosan/COS films showed the highest mechanical ro erties, whereas, glucose-loaded films were brittle, as demonstrated by scanning electron microsco y microgra hs. urthermore, MRchitosan/COS1 films exhibited the better antioxidant behavior followed by Effect of glucose substitution by low-molecular weight chitosan-derivatives on functional, structural and antioxidant properties of Maillard reaction-crosslinked

Introduction

Nowadays, many research has been made to develop natural, biodegradable and non-toxic bio-based packaging to enhance the shelf-life of fresh or packaged commercialized food as alternative to synthetic packaging, which are the main cause of environmental pollution [START_REF] Galiano | Advances in biopolymer-based membrane preparation and applications[END_REF][START_REF] Liu | Current advances and future perspectives of 3D printing natural-derived biopolymers[END_REF]. Indeed, coating material should protect food from all pathogenic contamination, oxidation and drying and perfectly prevent the Ultraviolet (UV) radiation. For this reason, several polymers such as polysaccharides and proteins were widely used for biofilm preparation (Affes et al., 2020;[START_REF] Etxabide | Effects of cross-linking in nanostructure and physicochemical properties of fish gelatins for bio-applications[END_REF][START_REF] Gennadios | Physical properties of egg white-dialdehyde starch films[END_REF][START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF][START_REF] Kchaou | Influence of Maillard reaction and temperature on functional, structure and bioactive properties of fish gelatin films[END_REF]. Thanks to its film-forming properties, antioxidant and antimicrobial activities, chitosan-based film has received growing interest to be applied in different fields including food, biotechnology, cosmetics and biomedical domains [START_REF] Aranaz | Cosmetics and Cosmeceutical Applications of Chitin, Chitosan and Their Derivatives[END_REF][START_REF] Chang | Preparation of chitosan films by neutralization for improving their preservation effects on chilled meat[END_REF][START_REF] Hajji | Optimization of the formulation of chitosan edible coatings supplemented with carotenoproteins and their use for extending strawberries postharvest life[END_REF][START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF].

The physicochemical properties of chitosan films were depended on chitosan characteristics mainly its molecular weight (Mw), acetylation degree (AD) and depolymerization degree [START_REF] Affes | Chitosan derivatives-based films as pH-sensitive drug delivery systems with enhanced antioxidant and antibacterial properties[END_REF][START_REF] Hamdi | Acetylation degree, a key parameter modulating chitosan rheological, thermal and film-forming properties[END_REF]Leceta, Guerrero, & de la Caba, 2013a;[START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. All these parameters should be taken into account to choose the better film formulation that gives good barrier properties against light and water, mechanical resistance and biological activities. Further, in order to enhance chitosan film characteristics several chemical, physical and enzymatic modifications methods were envisaged to enlarge their domain of applications including Maillard reaction (MR), heat treatment and the combination of two polymers (Affes et al., 2020c;[START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF][START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF][START_REF] Hajji | Optimization of the formulation of chitosan edible coatings supplemented with carotenoproteins and their use for extending strawberries postharvest life[END_REF]Leceta, Guerrero, Ibarburu, Dueñas, & de la Caba, 2013b). Given the good antioxidant and antibacterial activities of MR products (MRP) in food industry, their use as preservatives is attracting increasing interest [START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF][START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF].

Maillard reaction is a non-enzymatic crosslinking complex biochemical process, which is done by heat treatment of amino acids in the presence of reducing sugars, and that produces fluorescent, brown final MRP named melanoidins with high biological properties (Affes et al., 2020c;[START_REF] Etxabide | Effect of pH and lactose on cross-linking extension and structure of fish gelatin films[END_REF][START_REF] Sun | Antibacterial activities and preservative effect of chitosan oligosaccharide Maillard reaction products on Penaeus vannamei[END_REF][START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. Three stages in the MR are usually considered. The initial products are called Schiff bases. They form Amadori products via rearrangement, which undergo further reactions to form irreversible advanced glycation end products [START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF]. Having amino groups makes chitosan a candidate to react with the carbonyl group of reducing sugars, and allows it to participate in the MR [START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF][START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. The rate of this chemical reaction is influenced by many factors, including the type, the concentration and the ratio of carbonyl/amino groups, pH, temperature, time of exposure and water activity [START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF][START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF][START_REF] Kchaou | Influence of Maillard reaction and temperature on functional, structure and bioactive properties of fish gelatin films[END_REF][START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF][START_REF] Xu | Comparative study on the Maillard reaction of chitosan oligosaccharide and glucose with soybean protein isolate[END_REF].

In a previous study (Affes et al., 2020c), the factor amino-containing compound was investigated using chitosan and high Mw-chitosan-depolymerization-products (CDP) (1244.7, 482.03 and 163.56 kDa), while maintaining constant the same added-carbonyl-containing compound, which is glucose at a concentration of 0.5 mg/ml, in all films' formulations. Changes undergone by crosslinking and MR development, after heat-treatment during 24 h at 90 °C, demonstrated better functional, thermal and mechanical properties in higher Mw chitosan-based films. Although many studies, dealing with MR crosslinked-chitosan-based films, have been achieved and the factors affecting the reaction, including characteristics (Mw and AD of chitosan) and concentration of amino groups donors, pH and water activity, have been evaluated, there are no many previous reports on the effect of the others factors, particularly the nature of reducing sugars (Fernández- [START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF][START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF][START_REF] Kosaraju | Chitosan-glucose conjugates: Influence of extent of Maillard reaction on antioxidant properties[END_REF][START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. Thus, in the present work, chitosan was used as constant aminocontaining compound and other parameters affecting MR development were studied.

The present research aims to evaluate the effects of different heating conditions, including temperature and incubation time, and types of reducing sugars (glucose and different low molecular weight (Mw) chito-oligosaccharides (COS) with varying acetylation degree (AD)), at a concentration of 5 mg/ml, on the extent of MR on chitosan-based films. To this end, chitosan, chitosan/glucose and chitosan/COS-based films were thermally treated at 90 and 110 °C and for 6 and 24 h and their functional, structural, thermal, mechanical and antioxidant properties were compared.

To the best of our knowledge, this is the first study dealing with the use of low-Mw COS, having different acetylation degree, as carbonyl-containing compounds, to develop MR on chitosan-based films, to optimize films crosslinking conditions, as well as to correlate MR rate with resulted films physico-chemical and antioxidant properties.

Materials and methods

Materials

Shrimp chitosan (Ch) (Molecular weight (Mw), 1244.70 kDa; Acetylation degree (AD), 7.60 ± 0.54%) was prepared as described in our previous study [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF] and employed as filmogenic biopolymer for films preparation. Two different chito-oligosaccharides (COS) were prepared by chitosan hydrolysis during 24 h using the chitosanolytic crude extract from Portunus segnis blue crab viscera (pH 4.0, 40 °C and E/S ratio = 100 U/g chitosan) [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF] and the chitosanolytic preparation from Bacillus licheniformis strain (pH 5.0, 50 °C and E/S ratio = 70 U/g chitosan) [START_REF] Affes | Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation[END_REF], respectively. The mixtures chitosan/chitosanases were incubated at appropriate conditions and the obtained soluble parts were freeze-dried, characterized by using steric exclusion chromatography (SEC-HPLC) and the first derivative Ultraviolet (UV) spectrophotometric method and referred as COS1 (Mw,< 4.4 kDa;AD,18.20 ± 1.19%) and COS2 (Mw, < 4.4 kDa; AD, 10.63 ± 0.17%) using the digestive and bacterial chitosanases, respectively. COS1, COS2 and D (+) anhydrous-glucose (Glu) (C6H12O6; 180 g mol -1 ) were used as reducing sugars to initiate the Maillard Reaction (MR) in chitosan-based films. Anydrous glycerol was purchased from Fluka (98% purity, Fluka Chemical, Germany) and used as plasticizer for the films. All other reagents were of analytic grade.

Films preparation

Films forming solutions (FFS) were prepared by dissolving chitosan (10 mg/ml) in acetic acid (1%, v/v). Then, three categories of films were prepared by the addition of three different reducing sugars to promote MR development; glucose, COS1 and COS2, at a concentration of 5 mg/ml. Glycerol was added as a plasticizer to the solutions at a concentration of 15% (w/w dry chitosan matter) and the FFS were maintained under stirring for 30 min. Subsequently, a volume of 34.0 ml of each FFS was cast in Polystyrene Petri dishes (13.5 x 13.5 cm diameter) and left to dry for 48 h at 25 °C. Dried films were manually peeled off from the surface and non-heated films were considered as blank and referred as F1 (Ch), F2 (Ch-Glu), F3 (Ch-COS1) and F4 (Ch-COS2).

All prepared films were then conditioned at 25 °C and 50% relative humidity (RH) before analyses, except for Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analyses (TGA), films were equilibrated at 0% RH.

Heating treatment of films

Then, to induce MR, the films (with or without reducing sugar) were heated in an oven at two different temperatures (90 ± 2 and 110 ± 2 °C) and for different incubation times (6 and 6 h, F2-90-24 h, F3-90-6 h, F3-90-24 h, F4-90-6 h and F4-90-24h, respectively for F1, F2, F3 and F4. While, the films heated at 110 °C were named F1-110-6 h, F1-110-24 h, F2-110-6 h, F2-110-24 h, F3-110-6 h, F3-110-24 h, F4-110-6 h and F4-110-24h for F1, F2, F3 and F4 respectively. Eq. ( 2)

UV-visible light spectroscopy

The films were cut into rectangles (1.0 x 3.0 cm) and placed in the test cell of an UVvisible (UV-vis) spectrophotometer (Shimadzu UV-2401PC). Light transmission of the films was determined in the wavelength range from 200 to 800 nm. An empty test cell was used as a reference.

FTIR-ATR analysis

FTIR spectra of the studied films were assessed using a spectrometer (Agilent Technologies, Cary 630 series) equipped with an attenuated reflection accessory (ATR) containing a diamond/ZnSe crystal, at 25 °C. Data analysis and treatment was carried out by using the OMNIC spectra software (Thermo Fisher Scientific).

Water content and solubility

The water content (WC) of the films (g moisture/100 g film), was determined by measuring the weight loss of each film sample (100 mg) after drying in an oven at 105 °C until constant weight was reached according to the following equation:

WC % = m i -m f m i x 100 Eq. ( 3
)
where mi is the initial film weight (g) and mf is the final is the final film dry weight (g). Three samples of each formulation were analyzed.

The solubility of the films in water (WS) was studied according to the Gennadios et al.

(1998) method. WS was calculated as follows:

WS % = m i × -M -m f (m i × -M ) × 100 Eq. ( 4
)
where mi is the initial film weight (g), mf is the final dry film weight (g) and WC is the water content of each film sample (%). All tests were carried out in triplicate.

Water contact angle

The contact angle measurements were measured using the sessile drop method on a GBX equipment (Digidrop, Romans, France), equipped with an image analysis software (Visiodrop).

Six measurements per films were carried out. All the tests were conducted in an environmental chamber at 25 (± 2) °C and with a relative humidity of 50 (± 1) %.

Film thickness

Film thickness was measured using a micrometer (Digimatic IP65, Mitutoyo, France).

Six measurements at different positions were done for each film sample. The mean value was taken into account for mechanical properties parameters calculation.

Mechanical properties

Tensile strength (TS, MPa) and elongation at break (EAB, %) of the films were determined using a rheometer apparatus (Physica MCR, Anton Paar, GmbH, France) equipped with a mechanical properties measuring geometry. Rheoplus software was used for the estimation of TS and EAB, corresponding respectively to the maximum load and the final extension at break from the tensile stress vs. strain curves. Six measurements for each films formulation were carried out at 25 °C.

Thermal stability analysis

The thermal stability of the film was studied, at a temperature range from 30 to 600 °C, using a thermogravimetric analysis (TGA, Q500 High Resolution, TA Istruments). The temperature of maximum degradation (Tmax, °C), the weight loss (Δw, %) and final residue at 600 °C (%) values were determined using TA Universal Analysis 2000 software (Version 4.5

A, TA instruments).

X-ray diffraction analysis

The crystallinity of the selected films was performed by x-ray diffraction analysis, at 2θ from 7 to 40 °, using a Philips diffractometer equipped with monochromatic Cu Kα radiation source.

Films microstructure

The microstructure of the films was assessed by surface and cross-section observations using a scanning electron microscopy (SEM, Hitachi S4800), at different magnifications.

Antioxidant activity

The capacity of film samples (10 mg) to scavenge 2,2′-Azino-bis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was studied according to the methods of [START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF] and [START_REF] Bersuder | Antioxidants from a heated histidine-glucose model system. I: Investigation of the antioxidant role of histidine and isolation of antioxidants by high-performance liquid chromatography[END_REF], respectively. The absorbances of the final solutions were mesured at 734 and 517 nm, after 10 min and 24 h of incubation in the dark, respectively for DPPH and ABTS scavenging activity.

The ability of films (10 mg) to reduce iron (III) was evaluated according to the method described by Yidirim, Mavi, & Kara (2001) with slight modification of the incubation time at 50 °C during 3 h. The absorbance of the final solutions was measured at 700 nm.

The total antioxidant ability of the films (10 mg) to reduce Mo (VI) to Mo (V) and to form a green phosphate/Mo (V) complex at acidic pH was studied as reported by [START_REF] Prieto | Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of Vitamin E[END_REF]. The absorbance was measured at 695 nm. All the experiments were carried out in triplicate. Results illustrated in Table 1 showed that L* values of the control unheated films F1, F3

and F4 are similar (p > 0.05). However, it decreased significantly in the film F2 containing glucose (Glu). Further, after thermal treatment of the films, L* values decreased significantly indicating that these films turn darker. For all the formulations, this decrease was more noticeable after heating at 110 °C, as compared to 90 °C and after incubation time during 24 h in comparison with those incubated for 6 h. Furthermore, the decrease of L* values was more pronounced in the films containing glucose followed by the films containing COS1 and COS2

as compared to the films of chitosan. After heating at 90 °C, there are no significant decrease in L* values of chitosan films without reducing sugar, however, negligible change of L* values was observed after heating at 110 °C. Darker color of the films indicate the development of MR, due to the interaction between chitosan and reducing sugars (Glu or COS), allowing to the production of dark MRP. These brown products produced during the final stage of MR are melanoidins (Affes et al., 2020c).

Interestingly, thermal treatment of all the films decreased their a* values and increased their b* values, as compared to the control unheated films, turning to green and yellow color region, respectively (p < 0.05). These variations were more significantly pronounced as temperature and heating time increased and in the films containing glucose followed by the films containing COS1 and COS2 as compared to chitosan films.

Moreover, ΔE* values, obtained by comparing the color of the films with the film F1, showed a slight difference between the control films more pronounced in the film F2 (3.6 ± 0.90). However, a high significant change of color was obtained in the MR-crosslinked films, which is correlated with the increase of the heating time and temperature and differed toward the used reducing sugar. Indeed, this effect was highest at 110 °C and after 24 h of treatment and when glucose was added as reducing sugar (18.80 ± 0.22), followed by COS1 (11.30 ± 0.34) and COS2 (8.34 ± 0.36) comparing to chitosan film (4.20 ± 0.34).

Besides, the BI, which give an overall evaluation of the progress of the MR according to the development of colored compounds, was measured and results are illustrated in Table 1. In all the heated films, the BI values were higher as temperature and incubation time were higher.

Further, low BI values were obtained in the treated free-reducing sugar chitosan-based films (2.30 ± 0.39 for F1-110-24 h). However, similarly to the trend of ΔE, the BI of heated films conjugated with Glu (120.85 ± 5.27 for F2-110-24 h) was higher than that of chitosan/COS1 (33.79 ± 0.91 for F3-110-24 h) and chitosan/COS2 films (18.01 ± 1.87 for F4-110-24 h).

All of these results indicates that MR was more pronounced for chitosan/Glu films followed by chitosan/COS1 and chitosan/COS2 films, comparing with chitosan film, as evidenced by the intensity of the change of color to dark-yellowish. This fact could indicate that severer reactions occurred in the chitosan films conjugated with Glu than in the films containing COS, allowing to the generation of higher amount of melanoidins. Similarly, Xu et al. (2019) illustrated that milder color change was obtained for soybean protein isolate (SPI) films conjugated with COS as compared to Glu, implying that COS was less susceptible to the MR with SPI than Glu. Moreover, it appeared that color change was higher in the films containing COS1 than those containing COS2, this fact can be attributed to the difference in depolymerisation degree (DP) allowed by the different method of chitosan depolymerization despite the higher AD of COS1 (18.20%) than COS2 (10.63%).

Furthermore, results demonstrate that the development of MR was more significant when temperature and heating time increased. In this context, Leceta et al. (2013a) reported that color parameters changed notably for chitosan-based films after being heat-treated at 105 °C for 24 h, which could mean that the film structure changed. [START_REF] Kchaou | Influence of Maillard reaction and temperature on functional, structure and bioactive properties of fish gelatin films[END_REF] reported that the color change of heated gelatin-glucose films increased as a function of heating temperature indicating better extent of MR of these films at higher temperature. Further, [START_REF] Etxabide | Effects of cross-linking in nanostructure and physicochemical properties of fish gelatins for bio-applications[END_REF] and [START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF] proved that color change of gelatin-lactose and gelatin-glucose films, respectively, was more pronounced as heating time increased, turning toward dark yellow at longer times, due to the production of higher amount of brown melanoidins (Affes et al., 2020c). Darker films, generated after MR development, have stronger barrier ability in the visible region and are advantageous to prevent oxidative deterioration by coating sensitive to light foods [START_REF] Yang | Effect of hsian-tsao gum (HG) content upon rheological properties of filmforming solutions (FFS) and physical properties of soy protein/hsian-tsao gum films[END_REF].

Ultraviolet-visible (UV-vis) spectroscopy

UV-vis spectroscopy was carried out in the range 200-800 nm in order to analyze the effect of crosslinking as a function of heating time and temperature and using different reducing sugars. Spectra, presented in Fig. 1A, 1B, 1C, 1D, showed that the control unheated films had poor barrier properties to light in the UV region, being the preventive effect slightly better for films conjugated with reducing sugars, particularly with glucose.

Further, as can be seen, the absorbance of all the heated films highly increased as function of temperature and heating time. Interestingly, in all cases, the absorbance was higher when the films were heated at 110 °C than 90 °C and after 24 h of reaction time than 6 h. Indeed, the conditions 110 °C -24 h were more efficient than 110 °C -6 h followed by 90 °C -24 h and 90 °C -6 h to develop high UV absorbance compounds. Furthermore, when chitosan films without reducing sugar were heated a slight increase was obtained, mainly after heating at 110 °C, as compared to F1. This modification was attributed to the caramelisation reaction due to heating of chitosan [START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. Moreover, there was a sharp increase in absorbance (A) in the range of 200 -500 nm for the heated films F2, F3 and F4, as compared to the control unheated films. This increase was clearly higher in the films F2 conjugated with Glu followed by the films containing COS1 and the films conjugated with COS2. These results are in line with the color change.

The concomitant rise of absorption of the heated films containing reducing sugars indicate the formation of MRP generated by the development of MR, which is a condensation reaction between nitrogen-containing compounds of chitosan and carbonyl group of reducing sugars [START_REF] Li | Effect of chitosan molecular weight on the functional properties of chitosan-maltose Maillard reaction products and their application to freshcut Typha latifolia L[END_REF]. The interactions between functional amino and carbonyl groups, present in chitosan and glucose or COS, respectively, are associated to the development of intermediate colorless compounds (Amadori) absorbing at 294 nm, prior to the generation of brown pigments (melanoidins), absorbing at 420 nm, as final MRP [START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF][START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF].

Results showed that in all cases, the values of absorbance at 294 nm (A294 nm) were significantly higher than those obtained at 420 nm (A420 nm). Similarly, [START_REF] Kosaraju | Chitosan-glucose conjugates: Influence of extent of Maillard reaction on antioxidant properties[END_REF] proved that the thermal treatment of chitosan with the addition of glucose found higher rates of intermediate browning products than final browning products. In addition, they were greater when temperature and heating time were higher. Moreover, the absorbance at 280 and 420 nm of the heated films conjugated with glucose were higher than those of the heated films 1550, and 1450 cm -1 , respectively. These three latter absorption bands showed a shift to a lower wavenumber as compared to chitosan powder, which were 1650, 1562 and 1420 cm -1 , respectively as described in our previous study [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF]. This shift was due to the relaxation of the chains additionally to the effect of glycerol addition and its interaction with chitosan (Affes et al., 2020a ;[START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF]. Furthermore, for the control films, the intensity of the N-H band obtained at 1550 cm -1 was higher than the intensity of the band of the carbonyl groups C=O at 1630 cm -1 . The higher intensity of the amine groups is a result of the presence of activated amine groups (-NH3 + ) generated during the films evaporation (Fernández- [START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF].

Furthermore, small shifts of the amide I and amide II peaks from 1630 and 1550 cm -1 to around 1615 and 1536 cm -1 , respectively, were observed for all MR-crosslinked films. Indeed, the shift of the absorption peak of the Amide I to 1615 cm -1 suggests the formation of Schiff base (C=N) between the reducing sugar termination and the chitosan amino groups (Affes et al., 2020c ;[START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF]. The shift of the characteristic peak of the Amide II of MRcrosslinked films was ascribed to the chitosan conformational transformations occurring during the crosslinking reaction between chitosan and reducing sugars.

Nevertheless, after thermal treatment of all the films, the intensity of the different bands of the spectra decreased as compared to unheated films. This decrease was more pronounced in the films containing glucose, especially F2-110-24 h, and when the films were heated during higher time (24 h) and at higher temperature (110 °C). These results were in accordance with WS values variation between control and heated films. As well, the difference in the intensity between the 1630 and 1550 cm -1 absorption bands became smaller. Similarly, Gullón et al.

(2016) stated that the intensity of chitosan polymer powder decreased after thermal treatment at 60 °C during 32 h.

All these results proved the crosslinking of the films due to MR development between carbonyl and amine groups thanks to heating treatment. This reaction was more noticeable when reducing sugars were added, especially glucose. These results corroborate with the study of Fernández-de [START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF] for heated chitosan and chitosan/COS films. Leceta et al.

(2013b) demonstrated also the change in the chemical structure of chitosan films after heat treatment by FTIR spectra variation analysis.

Water resistance features of chitosan based films modified by MR

Water sensitivity of the films, which is an important factor in the shelf life of packaging materials, was studied by measurement of moisture content (WC), water solubility (WS) and water contact angle (WCA).

Results illustrated in Table 2 showed that WC values of the control unheated films containing glucose (14.07%), COS1 (13.98%) and COS2 (14.15%) were significantly higher than WC value of chitosan film (12.28%). This is probably due to the humectant character of glucose and to the WC of COS1 and COS2. Additionally, the heat treatment of the different films proved a high decrease of WC values as compared to the control films. This decrease was higher as the temperature and the heating time were higher (p < 0.05). Indeed, heating conditions of 110 °C for 24 h showed the lowest WC values followed by 110 °C-6 h, 90 °C-24 h and 90 °C-6 h, for all the films. Furthermore, the highest WC decrease was observed in the heated films containing glucose, especially F2-110-24 h (from 14.07 to 9.60%). The decrease of WC by thermal treatment is due to the interaction between the amino group of chitosan and the carbonyl group of the different reducing sugars (glucose, COS1 or COS2) through MR.

Similarly, [START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF] reported that WC values of gelatin films increased with the addition of glucose and decreased gradually after heating at 90 °C with heating time and glucose content. [START_REF] Rivero | Heat treatment to modify the structural and physical properties of chitosan-based films[END_REF] stated that heating of chitosan film at 100, 160 and 180 °C decreased its moisture content value which is lower at higher temperature.

In addition to its importance to assess the extension of MR, it is crucial to control film solubility to be applied as food coatings, allowing several potential benefits. To this end, films WS was evaluated. As shown in Table 2, the addition of glucose, COS1 and COS2 to control unheated chitosan film lead to a significant increase of its WS from 12.12% to 16.19, 18.56 and 19.00%, respectively. The higher WS of chitosan/COS films is due to the total water solubility of the added COS as described in our previous studies [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF][START_REF] Affes | Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation[END_REF]. [START_REF] Etxabide | Effects of cross-linking in nanostructure and physicochemical properties of fish gelatins for bio-applications[END_REF] reported that the WS of gelatin films decreased with the addition of lactose and that this reduction of WS is more pronounced at higher concentrations of lactose and after treatment at 105 °C for longer time.

Water contact angle (WCA) values are considered as a good indicators of the degree of hydrophilicity of the film surface, being higher when hydrophilicity is lower. The final state of a water drop informs about the surface wettability (Leceta et al., 2013a). The surface properties of the films were evaluated by WCA measurements. Results depicted in Table 2 showed the variation of WCA of chitosan films as a function of temperature, heating time and type of reducing sugars. WCA values slightly decreased after 20 s (T20s) of depositing the drop as compared to 10 s (T10s), due to water evaporation. Additionally, control chitosan film (F1)

showed the higher WCA values. However, the addition of glucose (85.09 °), COS1 (65.50 °)

and COS2 (59.20 °) to chitosan films decreased significantly the WCA as compared to F1. The hydrophilicity of these latter films was probably attributed to their higher WC that make them more susceptible to absorb water molecules. After thermal treatment, WCA regress significantly for all the films as compared to the unheated ones indicating that heating leads to an increase of films hydrophilicity as a result of Maillard crosslinking reactions. This reduction was more important when temperature and heating time were higher. Further, control and heated chitosan-based films without reducing sugar addition were considered as hydrophobic films as they exhibit WCA values higher than 90 °. Similarly, [START_REF] Kchaou | Enhancement of structural, functional and antioxidant properties of fish gelatin films using Maillard reactions[END_REF] stated that WCA of gelatin-glucose films decreased after thermal treatment at 90 °C as a function of heating time. Furthermore, Leceta et al. (2013b) proved that the heat-treatment of chitosan films containing 15% glycerol for 24 h at 105 °C caused a slight decrease in WCA values as compared to un-heated films.

Thickness and mechanical properties changes of MR treated chitosan-based films

Results from Table 2 show that overall films possess similar thickness of approximately 31.0 µm (p > 0.05). The mechanical properties, including tensile strenght (TS) and elongation at break (EAB), of the different films were measured and displayed in Table 2, except for the heated films containing glucose because they were too brittle to be tested.

Based on the tensile stress vs. strain curves, TS and EAB values of chitosan-based film showed slight non-significant decrease when glucose was added to the control film F2. This decrease was probably attributed to the high concentration of glucose (5 mg/ml) incorporated in the film. Heating of this films generate slightly more tearable and brittle films, with not significant differences. Similarly, [START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF] demonstrated that no significant effect on mechanical properties of control chitosan film was obtained with the addition of glucose, at a concentration of 0.4 mg/ml, and that heating of chitosan/glucose film increase its mechanical behavior.

In addition, a non-significant decrease of TS and EAB was obtained with the addition of COS1 and COS2 for the control films F3 and F4. Further, mechanical parameters values of the film F4 were slightly higher than F3. This variation can be explained by the higher AD of COS1 (18.20%) than COS2 (10.63%). Similarly, [START_REF] Hamdi | Acetylation degree, a key parameter modulating chitosan rheological, thermal and film-forming properties[END_REF] reported that AD influences chitosan film-forming mechanical properties and that lower AD chitosan provides better film mechanical behavior.

Moreover, heat treatment of chitosan, chitosan/COS1 and chitosan/COS2 films slightly improve their mechanical properties. The film F3-110-24 h showed the highest increase of EAB as compared to F3 and the film F1-110-24 h showed the highest TS value (p < 0.05). Further, this increase of mechanical properties values was correlated with the thermal treatment conditions in the following order of reactivity: 110 °C-24 h > 110 °C-6 h > 90 °C-24 h > 90 °C-6h. Thus suggesting that higher temperature and heating time were more efficient to obtain more flexible and stretchable chitosan and chitosan/COS films.

The slight enhancement of mechanical properties of chitosan films by heating treatment, especially for F1-110-24 h, is an accordance with results obtained by Leceta et al. (2013b) who demonstrated that heating of chitosan film at 105 °C for 24 h increased its TS and EAB values.

Moreover, these results were in agreement on with those of [START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF] who stated that heat treatment at 90 °C for 24 h did not affect significantly the mechanical parameters of chitosan blank film. The better flexibility and stretchability of heated films as compared to control films was attributed to the formation of more compact network through MR crosslinking [START_REF] Park | Mechanical and barrier properties of chitosan-based biopolymer film[END_REF].

Thermal properties of MR crosslinked chitosan-based films

The thermal stability was assessed by means of thermogravimetric analysis (TGA), in a range of temperature between 30 and 600 °C, in order to study the changes occurred by the effect of different heating conditions on chitosan-based films with and without reducing sugars addition. The thermal decomposition data in terms of corresponding weight loss (∆w), temperature of maximium degradation (Tmax) and final residual mass (R) of the films, measured from the curve of the derivate weight loss as fonction of temperature as shown in Fig. 1E, 1F, 1G, 1H, are detailed in Table S1.

On the TGA derivate weight curves of the control chitosan-based films F1, F2, F3 and F4 three transformation steps corresponding to the main stages of weight loss were distinguished.

The first stage of transformation (∆w 1) observed below 100 °C is related to the loss of free and bound water. ∆w 1 values increased for the chitosan-based films conjugated with reducing sugars F2, F3 and F4 as compared to F1, which is in line with the increase of WC values of these films. Whereas, ∆w 1 values of overall heated films decreased comparing to control unheated films. This decrease, that was further correlated with the decrease of WC of heated films, was higher as temperature and heating time were higher.

A second narrow weight loss (∆w 2) appeared at approximatly 140 -230 °C and was probably attributed to the entrapped water through hydrogen bonds and the elimination reaction of NH3 as suggested by [START_REF] Martins | Influence of α-tocopherol on physicochemical properties of chitosan-based films[END_REF] or due to the evaporation of glycerol as considered by Leceta et al. (2013b). ∆w 2 values decreased for all the heated films and disappeared for chitosan films containing glucose heated at 110 °C (F2-110-6 h and F2-110-24 h).

The third major stage of weight loss (∆w 3), corresponding to ∆w around 44.94 and 57.14%, displayed the degradation or the decomposition of chitosan and COS chains and were higher for heated films comparing to control films [START_REF] Martins | Influence of α-tocopherol on physicochemical properties of chitosan-based films[END_REF].

Regarding Tmax values, no change was noted for the film F2 incorporated with glucose (300 °C) as compared to F1 (297 °C). However, the addition of COS to control chitosan-based films decreased their Tmax values, that were 277.3 and 267 °C for F3 and F4, respectively. After thermal treatment, Tmax values of all heated films increased as compared to unheated films. This increase was more pronounced when the films were heated at 110 °C comparing to 90 °C and during 24 h as compared to 6 h. When comparing to the chitosan heated films, glucose incorporation increased Tmax values of heated films, however, Tmax values of chitosan films conjugated with COS remains lower despite having increased. Similarly, the residual weight at 600 °C (R) was higher for all the heated films as compared to unheated films.

These variations of thermal stability behavior were attributed to the different extent of MR crosslinking, through creation of new bonds in film matrix, between the different films formulations and the different heating conditions. The increase of thermal stability of heated chitosan films containing reducing sugars is explained by the development MRP which could interact with chitosan chains to stabilize the film network leading to more thermally stable films with enhanced functional properties [START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF][START_REF] Kchaou | Influence of Maillard reaction and temperature on functional, structure and bioactive properties of fish gelatin films[END_REF]Leceta et al., 2013b). An increase of thermal stability was further stated for heated free-reducing sugars chitosan films by thermal treatment, but in a less extend than for MR.

3.5. Effect of MR crosslinking on the structural properties of chitosan-based films 3.5.1. X-ray diffraction (XRD) analysis XRD analysis is a proven tool to study the changes of the molecular conformation of chitosan-based films acquired by reducing sugar addition and thermal treatment. The XRD patterns of the different films formulations before and after treatment at 90 and 110 °C for 24 h, were illustrated in Fig. 2E, 2F, 2G, 2H.

As shrimp chitosan is a semi-crystalline polysaccharide due to its regular chain it present two peaks at 2 θ = 10 and 20 °associated with its hydrated conformation [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF].

Similarly, XRD chromatogram of chitosan film showed that it was in a partially crystalline state and has two main characteristic peaks with a slight shift, at 2 θ around 12 and 20 °. Comparably XRD chromatograms were obtained by [START_REF] Chang | Preparation of chitosan films by neutralization for improving their preservation effects on chilled meat[END_REF], Leceta et al. (2013) and [START_REF] Rivero | Heat treatment to modify the structural and physical properties of chitosan-based films[END_REF] for chitosan-based films. When glucose was added to chitosan film (F2), the same peaks were observed but with slightly lower intensity as compared to F1. Whereas, it is clearly shown that the addition of COS reduced the crystallization of chitosan films F3 and F4, where the peak at 12 ° disappeared and the intensity of the peak at 20 ° decreased comparing to F1.

This change in the crystallinity open the incorporation of COS1 and COS2 was ascribed to the non-crystalline structure of these two chitosan derivatives [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF][START_REF] Affes | Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation[END_REF].

After thermal treatment of the different films, the crystallinity decreases remarkably as a function of heating temperature as evidenced in the decrease in the intensity of the peak at 20 °. Indeed, films heating at 110 °C provides less crystalline structure than 90 °C. Further, the intensity of this peak (20 °) was lower in the films containing COS followed by glucose containing films, as compared to F1. As well, the peak at 12 ° disappeared in all the heated films. Similarly, diffractograms of chitosan films cured at different temperatures showed the disappearance of the peak located at 2 θ = 15 ° and demonstrated that films heating at higher temperature, 160 °C, leads to lower crystallinity than 100 °C [START_REF] Rivero | Heat treatment to modify the structural and physical properties of chitosan-based films[END_REF]. Furthermore, Leceta et al. (2013b) stated that chitosan film structure was influenced by the effect of temperature. These results were in contradiction with those of Fernández-de [START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF] who stated that chitosan and chitosan/COS (1:1) films heat treated and non-treated showed the same XRD patterns with amorphous behaviour and a single wide diffraction peak at 20.2 °.

Films microstructure

In order to investigate the microstructural modifications in the elaborated chitosan-based films through MR, the final films microstructure was qualitatively visualized by surface electron microscopy (SEM) analysis. Crosslinked films through MR at 110 °C for 24 h were selected for the SEM analysis, and compared with control unheated films, except for heated film containing glucose where the film F1-90-6 h was tested because F1-110-24 h was too brittle and it is difficult to be fixed on SEM support.

Characteristic SEM micrographs of surface and cross-sections of films were shown in Fig. 3. The surface of all the films was flat, smooth and homogenous without apparent porosity.

However, the films F2 and F2-90-6 h showed the appearance of some cracks more pronounced in the heated film F2-90-6 h, due to the addition of glucose (5 mg/ml).

Moreover, cross-sectional images of the control films showed compact and homogenous structure without any evidence of irregularities. Further, crosslinking provides and increases of films cross-sections homogeneity and order, except of the film F2-90-6 h were some cracks were visualized. Results were in accordance with mechanical properties values that increased for heated chitosan and chitosan/COS films and decreased for heated chitosan/glucose films.

The more compact structure of the heated chitosan and COS films, as compared to control films, was attributed to the crosslinking through MR thanks to the action of temperature leading to high interaction between chitosan and COS. Likewise, Fernández-de [START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF] reported that chitosan and chitosan/oligosaccharides based films had homogenous microstructure with relatively roughness and that no irregularities, caused by crosslinking through heat treatment at 105 °C overnight, were observed in the heat treated films.

However, the less compact structure of heated chitosan/glucose films despite the high extent of MR in this films, was probably attributed to the very high concentration of glucose. [START_REF] Hamdi | Conception of novel blue crab chitosan films crosslinked with diff erent saccharides via the Maillard reaction with improved functional and biological properties[END_REF] reported that the incorporation of glucose at concentrations from 0.5 to 2%

(w/w chitosan) to chitosan film improves its microstructure.

Antioxidant property of activated films by Maillard reaction

To evaluate the antioxidant activity of blank and MR-crosslinked chitosan-based films and to correlate antioxidant potential to films structural characteristics, differents in vitro antioxidant tests were assayed including DPPH and ABTS + radicals scavenging activities, reducing power test and the total antioxidant activity.

As shown in Fig. 4, for the four tested mechanisms, chitosan-based film F1 exhibited the lowest antioxidant behavior. The addition of glucose increased slightly the antioxidant activities values of the film F2 (p < 0.05). Further, when COS were incorporated in the control films, a significant increase of the antioxidant potential, via the different tests, was noted which is more pronounced for the film F3 than F4. The higher antioxidant activity of the film F3 than F4 was attributed to the better antioxidant activity of COS1 (Mw, < 4.4 kDa; AD, 18.20 ± 1.19%) than COS2 (Mw, < 4.4 kDa; 10.63 ± 0.17%) as shown in our previous studies [START_REF] Affes | Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan[END_REF][START_REF] Affes | Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation[END_REF]. Thus can be a result of the difference in the degree of polymerization, ion composition, and the presence of high units of D-glucosamine in the structure of COS1 despite its high AD than COS2 [START_REF] Anraku | Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies[END_REF][START_REF] Li | Preparation, characterization and antioxidant activity of two partially N-acetylated chitotrioses[END_REF]. Therefore, for the control films, F3 showed the better antioxidant behavior, followed by F4 and F2 as compared to F1.

Regarding thermally treated films, values were significantly higher in heated films than in the blank films, even by heating of the film F1, due to the development of active MRP. This increase more noticeable after heating at 110 °C for 24 h followed by 110 °C-6h, 90 °C-24 h and 90 °C-6 h allowing to conclude that heating at higher temperature (110 °C) and incubation times (24 h) generates better antioxidant films. Further, the heated film F1 showed the lowest increase of antioxidant values which is negligible as compared to heated reducing sugar containing films. However, heated glucose containing films, especially F2-110-24 h showed the better increase of antioxidant potential comparing to the blank film F2. In addition, the heated films containing COS1 (F3 group) exhibited the better antioxidant activities, for all the tests, followed by F2 and F4. Where, the film F3-110-24 h reached 100% for ABTS and DPPH radical scavenging activities, 2.44 ± 0.07 for reducing power test and 179,325 ± 1.65 αtocopherol (µmol/ml) for the total antioxidant activity.

The antioxidant activity was enhanced subsequently to the development of MR. This increase was directly correlated to the incorporated reducing sugars in the film matrix, the different mechanisms, of all the crosslinked films as compared to blank films thanks to the formation of highly active MR products. Among all treated films, the most actives were chitosan/COS1 crosslinked films, followed by chitosan/glucose and chitosan/COS2 films.

These films possess interesting functional and antioxidant properties that were suitable for food packaging and biomedical applications.

2. 4 .

 4 Physicochemical characterization of the films 2.4.1. Color properties Color development was studied using a CIE colorimeter (CR-5, Konica Minolta, Sensing Europe B.V). Color of the films was expressed as L* (lightness/brightness), a* (greenness/ redness) and b* (blueness/yellowness). The total color change (ΔE*) of the films was determined as follows: a0*, b0* are the colorimetric parameters of the film F1, as standard, and L*, a*, b* are the values of the modified films. The obtained CIE Lab values were then used to calculate the browning index (BI) as mentionned in equation 2:

2. 6 .

 6 Statistical analysis Statistical analyses were performed with IBM SPSS Statistics v.20. Normal distribution and equality of variance were tested with the Shapiro-Wilk and Levene's tests respectively. Statistically significant differences were then calculated using the Kruskal-Wallis test for nonnormal distributed data with the dunn's post hoc test. Differences were considered significant at p value < 0.05. Standard deviation errors were used to compare all experiments. 3. Results and discussion 3.1. Effect of Maillard reaction on films optical and spectral properties 3.1.1. Films color change To evaluate the effect of MR on chitosan-based films, different reducing sugars including glucose and chito-oligosaccharides (COS1 and COS2) were added to the FFS. Films were incubated at 90 and 110 °C for 0, 6 and 24 h to perform MR. During reaction, reducing sugars reacted with amino acid residues of chitosan to form the final MR products (MRP). The final stage of MR was evaluated by color change measurement. Films color was measured using CIELab scale and L* (darkness/whiteness), a* (greenness/redness) and b* (blueness/yellowness) parameters were used to calculate difference in color (ΔE*) and browning index (BI).

  containing COS1 and COS2 comparing to heated chitosan films. This means much more intermediate compounds of the MR and darker color. These results indicated that temperature, incubation time and the type of reducing sugar affected crosslinking by MR extension. In the same context,[START_REF] Gullón | Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction[END_REF] reported that reaction conditions, especially temperature and reaction time, affect the formation of chitosan/glucose derivatives by MR.3.1.3. Fourier-transform infrared spectroscopy (FTIR)Chemical changes of chitosan film structure, due to reducing sugars addition and MR development at different heating times and temperatures, were studied using FTIR spectroscopy. FTIR spectra presented in Fig.2A, 2B, 2C, 2D showed that similar characteristic peaks were obtained for all the films. All films spectra revealed the presence of the stretching vibrations of hydroxyl group (O-H), CH2 and CH3 groups, amide I (C=O in the NH-COCH3 group), amide II (N-H) and C-H bending vibration of CH2 groups obtained at 3370, 2875, 1630,

  Contrarily, when the films were heat-treated, WS values decreased significantly, as compared to unheated films, and proportionally to the increase of temperature and heating time indicating a change in the chemical structure of the films by thermal treatment. The high decrease of WS values in the films containing reducing sugars, especially in the films containing glucose, is due to the development of interactions between amino and carbonyl groups of chitosan and reducing sugars, respectively, induced by temperature, allowing to the crosslink of the films through MR. The decrease of free amino groups in film matrix lead to obtain more compact structure less sensitive to water with WS values[START_REF] Umemura | Modification of chitosan by the Maillard reaction using cellulose model compounds[END_REF]. Likewise, Fernández-de[START_REF] Castro | Films of chitosan and chitosan-oligosaccharide neutralized and thermally treated: Effects on its antibacterial and other activities[END_REF] proved that chitosan/oligosaccharide film showed higher WS value than chitosan film and that thermal treatment at 105 °C decreased significantly WS by the development of brown compounds through MR. In addition,[START_REF] Umemura | Modification of chitosan by the Maillard reaction using cellulose model compounds[END_REF] stated that the solubility in acetic acid of chitosan films conjugated with glucose and cellobiose decreased after thermal treatment at 50 °C for 15 h. In this context,

Figures captionsFigure 1 :

 1 Figures captions

Figure 2 :

 2 Figure 2: FTIR spectra of chitosan (A) chitosan-glucose (B) chitosan-COS1 (C) and

Figure 3 :

 3 Figure 3: Section and surface SEM micrographs of control films and heated films at 110 °C

Figure 4 :

 4 Figure 4: Antioxidant activities of chitosan-based films conjugated or not with different

  Fig.1

Table 1 :

 1 Color parameters (L*, a* and b*), total color change (ΔE*) and browning index (BI) of the different films.

	Films	L*	a*	b*	ΔE*	BI
	F1	28.36 ± 1.09 AB	-0.45 ± 0.05 A	-0.46 ± 0.07 M -	-
	F1-90-6 h	27.54 ± 0.43 ABCD -0.55 ± 0.02 AB	0.52 ± 0.01 L	1.32 ± 0.26 H	0.40 ± 0.03 H
	F1-90-24 h	27.30 ± 0.85 ABCD -0.60 ± 0.03 AB	0.64 ± 0.04 L	1.68 ± 0.51 H	0.70 ± 0.11 H
	F1-110-6 h	26.40 ± 0.82 CDE	-0.77 ± 0.05 BC	0.71 ± 0.02 L	2.35 ± 0.70 H	0.67 ± 0.23 H
	F1-110-24 h 24.54 ± 0.43 GH	-0.88 ± 0.02 CD	1.22 ± 0.12 K	4.20 ± 0.34 G	2.30 ± 0.39 H
	F2	25.97 ± 1.33 DEF	-0.64 ± 0.19 B	2.03 ± 0.01 J	3.60 ± 0.90 G	-
	F2-90-6 h	16.93 ± 0.60 J	-1.11 ± 0.05 EF	6.20 ± 0.14 D	13.25 ± 0.58 C 38.65 ± 2.65 C
	F2-90-24 h	14.88 ± 0.40 K	-1.29 ± 0.08 FGH 9.20 ± 0.08 B	16.61 ± 0.38 B 81.60 ± 4.03 B
	F2-110-6 h	14.68 ± 0.21 K	-1.41 ± 0.07 GHI 9.36 ± 0.14 B	16.87 ± 0.25 B 84.94 ± 3.53 B
	F2-110-24 h 12.96 ± 0.16 L	-1.68 ± 0.08 J	10.26 ± 0.14 A 18.80 ± 0.22 A 120.85 ± 5.27 A
	F3	27.83 ± 0.13 ABC	-0.95 ± 0.02 DE	1.41 ± 0.02 K	2.00 ± 0.09 H	-
	F3-90-6 h	24.78 ± 0.65 EFG	-1.07 ± 0.05 E	3.90 ± 0.05 G	5.68 ± 0.66 F	13.35 ± 1.70 EF
	F3-90-24 h	22.26 ± 0.20 H	-1.26 ± 0.04 FG	5.46 ± 0.04 E	8.54 ± 0.14 E	22.77 ± 0.03 D
	F3-110-6 h	22.30 ± 0.07 H	-1.26 ± 0.01 FG	5.48 ± 0.01 E	8.53 ± 0.08 E	22.85 ± 0.90 D
	F3-110-24 h 19.69 ± 0.47 I	-1.54 ± 0.03 IJ	6.70 ± 0.03 C	11.30 ± 0.34 D 33.79 ± 0.91 C
	F4	28.68 ± 0.23 A	-1.35 ± 0.01 GHI 1.39 ± 0.29 K	2.10 ± 0.22 H	-
	F4-90-6 h	26.57 ± 0.04 BCD	-1.39 ± 0.02 GHI 2.58 ± 0.02 I	3.65 ± 0.03 G	5.99 ± 0.15 H
	F4-90-24 h	24.47 ± 0.02 GH	-1.47 ± 0.01 HI	3.38 ± 0.36 H	5.56 ± 0.26 F	9.84 ± 1.58 FG
	F4-110-6 h	24.16 ± 0.06 G	-1.47 ± 0.02 HI	3.42 ± 0.13 H	5.81 ± 0.13 F	10.18 ± 0.56 FG
	F4-110-24 h 21.89 ± 0.22 H	-1.51 ± 0.01 IJ	4.69 ± 0.31 F	8.34 ± 0.36 E	18.01 ± 1.87

DE

Values are means ± standard deviation (n = 3). Means with different letters (A-M) and within a column indicate significant difference (p < 0.05). ΔE* was the change of color measured as compared to F1.

a (

Table 2 :

 2 Water content (WC, %), water solubility (WS, %), water contact angle (WCA, °), thickness (µm) and mechanical properties (TS, % and EAB, MPa) of the different films.

			Water resistance properties			Mechanical properties
	Films	WC (%)	WS (%)	WCA (°)	Thickness (µm)	TS (MPa)	EAB (%)
				T10 s	T20 s
	F1	12.28 ± 0.16 B	12.12 ± 1.01 CDE	108.60 ± 2.24 A	106.93 ± 2.26 A	29.4 ± 1.5 A	17.99 ± 0.25 A	15.26 ± 0.74 A
	F1-90-6 h	10.93 ± 0.23 E	10.71 ± 0.04 HI	96.54 ± 1.78 B	94.18 ± 1.73 B	28.00 ± 2.0 A	18.29 ± 0.01 A	15.35 ± 0.26 A
	F1-90-24 h	10.33 ± 0.33 FG	9.33 ± 0.50 JK	95.70 ± 0.62 B	95.10 ± 0.94 B	30.0 ± 4.0 A	18.44 ± 0.73 A	15.50 ± 0.85 A
	F1-110-6 h	10.13 ± 0.38 G	8.85 ± 0.27 KL	95.30 ± 1.98 B	94.11 ± 1.77 B	30.0 ± 2.0 A	18.49 ± 0.23 A	15.71 ± 0.12 A
	F1-110-24 h	9.31 ± 0.19 H	7.92 ± 0.07 L	94.64 ± 0.67 B	92.93 ± 1.06 B	32.0 ± 4.0 A	18.60 ± 0.30 A	15.93 ± 0.10 A
	F2	14.07 ± 0.04 A	16.19 ± 0.32 B	85.09 ± 3.06 C	82.38 ± 3.16 C	30.0 ± 1.5 A	14.05 ± 0.66 A	12.80 ± 0.44 A
	F2-90-6 h	11.45 ± 0.16 CD	11.83 ± 0.31 DEFG	71.13 ± 2.07 D	69.19 ± 2.33 D	30.0 ± 3.0 A	-	-
	F2-90-24 h	11.06 ± 0.06 DE	11.01 ± 0.09 FGHI	70.49 ± 0.91 D	68.44 ± 0.78 D	31.0 ± 1.2 A	-	-
	F2-110-6 h	10.15 ± 0.05 G	10.81 ± 0.22 GHI	69.33 ± 2.15 D	68.24 ± 2.43 D	32.0 ± 5.0 A	-	-
	F2-110-24 h	9.60 ± 0.10 H	9.97 ± 0.23 IJ	67.84 ± 0.74 D	66.30 ± 0.80 D	33.0 ± 2.0 A	-	-

h). The films heated at 90 °C during 6 and 24 h were named F1-90-6 h, F1-90-24 h, F2-90-

Acknowledgement

The "Ministry of Higher Education and Scientific Research", Tunisia, funded this work.

The authors wish to express their thanks for financial support provided from the framework of PHC-Utique program financed by CMCU project, grant N°: 19G0815. The authors gratefully acknowledge Doctor Arie Van Der Lee and Doctor Thierry Thami, from European Institute of Membranes, University of Montpellier, France, for x-ray diffraction analyses and water contact angles measurements, respectively. The authors would like to thank also Professor Ahmed Rebaї and Doctor Rania Abdelhedi from Laboratory of Molecular and Cellular Screening Processes, Centre of Biotechnology of Sfax, University of Sfax, Tunisia, for there helpful aid regarding the statistical analysis.

Conflict of interest

The authors declare that there are no conflicts of interest.


Renaud Hedouin

Olivier Commowick

Gabriel Robert

Julie Coloigner

Imaging biomarker discovery in major depressive disorder with diffusion MRI multi-compartment models

Introduction

• Major depressive disorder (MDD) is a disease widespread all over the world associated with a large and increasing economic, societal, and personal burden [Ferrari 2013]

• Despite a large number of studies, the pathophysiology underlying MDD is not well understood

Results

• We developed a pipeline to perform tractography analysis using multi-compartments models

• We tested this pipeline on the Actidep database

• In the future we will extend this study to explore different database with additional MRI sequences, such as relaxometry, to derive new metrics and develop a multi-modal approach

Conclusion & perspectives

Multi-compartments models

Input:

• DWI with several b-values to estimate complex models

••

  T1-weighted image for registration purpose • Estimate MCM [Stamm 2016] that decomposes the water diffusion as a weighted sum of anisotropic and isotropic compartments Tractography For each subject: • TractSeg performs a tractography for all registered DWIs based on ODF resulting in 72 identified fiber bundles [Wasserthal 2018] • For each bundle a centroid is estimated and values (FA, MD, …) are provided [Chandio 2020] Statistics • Performs group to group comparison along tracts • Covariable (age, sexe …) can be removed • Rigid registration of preprocessed DWIs and scalar maps on MNI152 Atlas • Extract scalar maps (FA, MD, FW …) from MCMs MCM FA is the average of each anisotropic compartment FA DTI FA Data • Actidep: Major depressive disorder in the elderly population 40 patients and 28 controls • Clinical interest: Depressive symptoms in late life are associated with an increased risk of developing dementia [Barnes 2012] • MRI acquisitions • Cusp: DWI with 1.5 mm isotropic resolution, 60 gradient orientations and 7 b0 [Scherrer 2012] • MPRAGE: T1-weighted images 1 mm isotropic • Resting state fMRI Statistic along fiber tracts • Pipeline tested on the Actidep database • Group controls (26 subjects) vs group patients (39 subjects) for different MCM metrics • Corrected for age, sex and duration of depression Expected highlight within the corpus callosum

  Robert 1,2 , J. Coloigner 1 1 INRIA Rennes -Bretagne Atlantique, VISAGES Research Team 2 Adult University Psychiatry Department, Guillaume Régnier Hospital, Rennes, France

	Imaging biomarker discovery in major
	depressive disorder with diffusion MRI multi-
	compartment models
	diffusion
	under the voxel resolution	SCD versus CTL	Silent strokes

• We propose a tract-based pipeline analysis using multicompartment models (MCM) that allows to model water R. Hedouin 1 , O. Commowick 1 , G.


domain: shs sdv

text:
E Delanoue

R Duclos

L Journaux

D Guéméné

M Sourdioux

A C Dockès

R Baumont

J P Bidanel

Perceptions of genome editing in farm animals by livestock stakeholders

 Unstabilized knowledge about a scientific or technical object  Goes beyond the scientific field  Broader uncertainties (ethical, social, legal, etc.)  GE : a topic that straddles two controversies

Material & methods

 Qualitative survey among different livestock stakeholders

• 48 semi-structured interviews (1h25 on average) conducted by Raphaëlle Duclos

• Open-ended questions, complemented by cards presenting typical cases of GE applications (for people who did not know applications of GE)

• The interviews were registered and analysed a posteriori used a common framework

Activity sector # interviews

Research 12

Public authorities 4

Breeding sector 8

Agriculture (except breeding) 7

Retailing sector 3

Civil society (NGO…) 14 • What are the uncertainties put forward by the interviewees

• An attempt at a stakeholder typology "As long as it is a matter that is validated by the society as a whole, whether from a legal, consumer or NGO perspective, we have no issue with it" "A necessary tool to feed the world in the future"

•

Typology of stakeholders

ENTHUSIASTS CAUTIOUS SCEPTICS E1- 2 E 2 E7 E21 E40 E16 E10 E2 7 E9 E5 E8-1 E3 E11 E15 E17- 1 E22 E1-1 E12 E17-2 E17-3 E34-1 E34- 2 E38 E39 E23 E4 E26-2 E29 E26-1 Specialists Profanes Favourable Unfavourable E33-1 E33-2 E20-1 E20-2 E37 E8- 2 E3 0 E28
We have exactly the same claims and promises with GE as with GMOs 30 years ago"

p. 9

Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

-An additional tool in the geneticist toolbox -"Could be used for traits that are difficult to improve with other techniques" -Could be useful to break down unfavourable associations between antagonistic traits" -Particularly promising to improve animal health related traits or animal welfare when an animal suffers, we are ready to do anything, or at least to increase our range of action, because we have the feeling that morally we are doing the right thing.

-But there is no consensus even for these traits -"animal welfare washing" -"should not be a solution for maintaining untenable livestock practices"

Additional arguments

p. 10

Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

• Even if it is limited to France, this study has made it possible to identify the main uncertainties related to the potential use of GE in farm animals and the range of positions and arguments regarding these technologies The controversy on GE is still confined to specialised spheres and current positions are subject to change • it also highlighted some rather profound discrepancies that reflect divergent visions on future agricultural systems and the on the role on science in society, at least in France and, to some extent, in Europe • The controversy about GE is partly related to the controversy about livestock farming systems. Its use is often associated with an industrial agricultural model criticized or rejected by some actors • Scientific, legal, societal … aspects regarding GE are rapidly changing, so that the controversy about GE might soon leave the sphere of the specialists. Thank you for your attention

Conclusion

  Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.  The application of Genome editing (GE) in agriculture is controversial  Sociotechnical controversy :



  Objective of the study : better understand the position of the actors of the livestock sector regarding the use of GE Objective p. 3 Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

  Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al. Results -What are the uncertainties ? Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al. Results -What are the uncertainties ? Level of expertise of the tool What are collateral effects ? Are the changes irreversible? ETHICAL Status of living organisms FINALITY TECHNICAL Are any alternatives? Is the level of knowledge sufficient to propose interesting applications? In which farming system(s) will these techniques fit? p. 7 Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

  Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

  Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al. • Raphaelle Duclos, which did much of the work • The groupe of people who followed Raphaelle's work • The co-authors of the paper • A larger group of people who defined the strategy of the work : co-authors + Jean-Marc Bèche CNIEL, CNE), Joël Bidanel (IFIP), Isabelle Bouvarel (ITAVI), Corinne Cotinot (INRAE), Stépahen Devillers (Allice), Alain Ducos (ENVT), Spéphane Patin (RDF), Laurent Schibler (Allice) Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.

Table -

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	Distribution by activity sector
	of the interviews
	p. 4

Perceptions of genome editing in farm animals by livestock stakeholders 05/09/2022/ EAAP Meeting, Porto / Delanoue et al.