Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts

Author’s Accepted Manuscript Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts Jeannine Bonilla, Paulo J.A. Sobral www.elsevier.com/locate/sdj

PII: DOI: Reference:

S2212-4292(16)30049-9 http://dx.doi.org/10.1016/j.fbio.2016.07.003 FBIO165

To appear in: Food Bioscience Received date: 14 January 2016 Revised date: 12 July 2016 Accepted date: 12 July 2016 Cite this article as: Jeannine Bonilla and Paulo J.A. Sobral, Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts, Food Bioscience, http://dx.doi.org/10.1016/j.fbio.2016.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts Jeannine Bonilla1*, Paulo J.A. Sobral School of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225; 13635-900 Pirassununga (SP) Brazil. *[email protected] Abstract Gelatin and chitosan are edible polymers, which may be used in combination with antimicrobial/antioxidant extracts as thin coatings to extend shelf life of foods. The effect of cinnamon, guarana, rosemary and boldo-do-chile ethanolic extracts and different ratios of gelatin:chitosan on the optical, microstructural, mechanical and barrier properties of the films was investigated, as well as the antimicrobial and antioxidant activity. Both polymers were blended homogeneously in the film matrix as confirmed by the microstructural and FTIR studies. Increments in chitosan proportion increased the elasticity of the films and provided a reduction in the water vapor permeability, which was not significantly reduced with the addition of the extracts. The blends films presented good antioxidants properties in TEAC test and an excellent growth inhibition against E. coli and S. aureus, suggesting that these films based on blends of gelatin and chitosan and additivated with ethanolic extracts could provide an alternative as active packaging material for food applications. Keywords: gelatin, chitosan, plant extracts, antioxidant film, antimicrobial film. 1. Introduction Edible coatings have recently gained more interest for food preservation due to the promising results obtained, mainly improving the quality of food products through 

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Gelatin-chitosan edible film additivated with ethanolic extracts. Telephone number: +55193565-4000 ext. 654186

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their applications (Sánchez-González et al., 2011). Edible films and coatings should be chosen based on their properties. According to McHugh (1996), the macromolecules for edible films production are mainly polysaccharides and proteins. The strengths and/or weaknesses of each (i.e. mechanical or barrier properties) is the main reason of interest to the development of composite or blended edible coatings and films that include the virtues of each class of components, that may be produced by single mixture of two biopolymers, such as gelatin and chitosan. Gelatin is a protein of animal origin widely utilized in the food industry since it is a protein produced in abundance, with relatively low costs and with interesting functional properties. It has been extensively studied for its film forming capacity and applicability as an outer covering to protect food against drying, light, and oxygen, although it does not present good barrier properties to water vapor (Sobral and Habitante, 2001). Chitosan is a cationic polysaccharide obtained from chitin by deacetylation in presence of alkali (Kanatt et al., 2012). Several studies have demonstrated that chitosan has a great potential to be used as film material due to its non-toxicity, low permeability to oxygen, biocompatibility and excellent film forming ability under acidic conditions (Bonilla et al., 2014a), in addition to its inherent antimicrobial and antifungal properties against various groups of pathogenic and spoilage microorganisms (Tan et al., 2015). Antioxidant and antibacterial packaging are main categories of active packaging and very promising for extending food product shelf life (Jridi et al., 2014), where the use of natural antioxidants and antimicrobial ingredients is an alternative to chemical products in the conservation of food. Edible plants, especially those ones rich in secondary metabolites (eg. essential oils, polyphenols) have an increasing interest due to their high concentrations of bioactive ingredients with antioxidant and/or antimicrobial

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activity (Sánchez-González et al., 2011). According to Bitencourt et al., (2014) and Giménez et al., (2013), the incorporation of natural extracts into films based on gelatin can improve the physical and functional properties of films. Among the spices more studied as raw material for bioactive compounds extraction are rosemary and cinnamon (Bubonja-Sonje et al., (2011); Gibis and Weiss (2012); Mathew and Abraham (2006)). Rosemary plant is considered one of the most important sources of both volatile and non-volatile bioactive compounds (Ojeda-Sana et al., 2013). Its antioxidant activity has been attributed mainly to phenolic compounds such as diterpenes, carnosol and carnosic acid. Furthermore, rosemary extract has shown the capacity to inhibit the growth of different foodborne pathogens and spoilage moulds (Bubonja-Sonje et al., 2011). According to Mathew and Abraham (2006), cinnamon is considered a common spice with strong antimicrobial and antioxidant activity. It belongs to the family Lauraceae and possesses significant biological activities (antitumor, antifungal, cytotoxic and antimutagenic) attributed to the cinnamaldehyde. The ability of this extract to retard lipid oxidation is attributed to the ability of its phenolic constituents to quench reactive oxygen species. Less studied components in the active films technology include those from boldo-do-chile and guarana. Boldo-do-chile (Peumus boldus Molina) is an endemic tree from Chile. In particular, boldo-do-chile leaves have been shown to possess more than 30 compounds (quercetin glycosides, phenolic acids, proanthocyanidins, etc), with fungistatic and antioxidant properties. Boldine, the major alkaloid present in the leaves and bark of the boldo, is well known for its antioxidant properties (Soto et al., 2013). On another hand, guarana (Paullinia cupana) is a climbing plant that is native of the Central Amazon basin and cultivated exclusively in Brazil. Guarana is a nutritional supplement that has been studied for its different properties, including antimicrobial and

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antioxidant activities. The seeds contain 3.2–7.0% caffeine, containing theobromine and theophylline, in smaller amounts (Dalonso and Petkowicz, 2012). The objective of the present investigation was to study the effect of incorporated rosemary, cinnamon, boldo-do-chile and guarana ethanolic extracts at 1% into edible films prepared with blends of gelatin and chitosan. Furthermore, the most relevant films properties, such as optical, mechanical, microstructural and water vapor permeability properties, and antioxidant and antimicrobial activity were studied. 2. Materials and methods 2.1. Raw materials Guarana seeds (Paullinia cupana), leaves of boldo-do-chile (Peumus boldus Molina), cinnamon barks (Cinnamomum sp.) and leaves of rosemary (Rosmarinus officinalis) were purchased from Florien, Insumos Farmacêuticos (Piracicaba, SP, Brazil). A pigskin gelatin (GEL) (bloom 260; molecular weight ~5.2x104 Da; moisture content = 9.98%) was supplied by GELNEX (Itá, SC, Brazil) and medium molecular weight chitosan (CH) (practical grade, batch STBF3507V, degree of deacetylation: 7585%, viscosity: 200-800 cps) was supplied by Sigma-Aldrich Química (São Paulo, SP, Brazil). Glycerol (PubChem CID: 753) was supplied by Labsynth® (São Paulo, SP, Brazil). 2.2. Preparation of the ethanolic extracts Plant extracts were prepared according to the method of Hoque et al., (2011), with some modifications. Dried seeds, barks and leaves were ground using a mortar and pestle. To prepare extracts, these powders (25 g) were then mixed with absolute ethanol using a powder:solvent ratio of 1:8 (w/v). The mixtures were then stirred continuously at room temperature for 4 hours and subsequently centrifuged at 1792 g-force for 10 min at 20ºC, using a centrifuge (Thermo Scientific IEC™ Centra-GP8, Waltham, MA,

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USA), to remove undissolved debris. The supernatant was collected and filtered through a filter paper. These ethanolic extracts, referred as cinnamon extract (C), guarana extract (G), rosemary extract (R) and boldo-do-chile (B) extracts, were placed in amber bottles, closed tightly and stored in refrigeration temperature (4ºC). 2.3 Preparation of films The edible films were prepared by casting technique. The pigskin gelatin (GEL) and chitosan (CH) solutions were prepared separately and then conveniently mixed to obtain the following formulations: films of pure biopolymers (GEL100 or CH100) and films based on blends of gelatin and chitosan with 25% (GEL75:CH25) or 50% (GEL50:CH50) of CH in blends. The different ethanolic extracts were incorporated at 1%. Gelatin solution was prepared according to Sobral et al., (2001b), where gelatin (4g GEL/100ml of distilled water) was hydrated at room temperature for 30min, and solubilized later in a thermostatic bath (Marconi® MA-184, Piracicaba, SP, Brazil) at 55ºC (±0.5ºC). After this, glycerol (0.2%) was added. In parallel, chitosan solution was prepared according to Bonilla et al., (2013). Chitosan (1% w/w) was dispersed in an aqueous solution of glacial acetic acid (1.0% v/w) for 12 h using a magnetic stirrer (Gehaka AA-2050, São Paulo, SP, Brazil), at 40ºC. The film-forming solutions (FFS) were obtained by mixing GEL and CH solutions in different ratios using a rotor–stator homogenizer (T25 digital ULTRATURRAX, IKA®, Campinas, SP, Brazil) at 10,000 rpm for 5 min at room temperature. The FFS were cast in petri plates (14 cm diameter) and dried at 30ºC and 60% relative humidity (RH) for about 24 h, in a conventional oven (Marconi® MA035/5, Piracicaba, SP, Brazil). The films were peeled off from the casting plates and conditioned for 7

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days at 25ºC-53% RH in chambers containing saturated solutions of Mg(NO3)2 prior to all analyses. 2.4. Characterization of films 2.4.1 Physical properties 2.4.1.1 Color, opacity and gloss Color measurements of film samples were taken by using a colorimeter MiniScan MSEZ 1049 (HunterlLab, Reston, VA, USA) with a spectral range from 400 to 700 nm, D65 (day light) lamp, angle of 10º and a measuring cell with an opening of 30 cm. The coordinates CIE L*a*b* (L* for luminosity and a* and b* for the coloropponent dimensions) were obtained from the reflection spectra of the samples placed on the surface of a standard white plate, allowing the calculation of the total color difference. All the film samples for each formulation were measured in triplicate. Moreover, film opacity was evaluated following the Hunterlab method in the reflectance mode, using the same equipment for the color analysis. The gloss of the films was measured at 60º incidence angle, according to the ASTM standard D523 (ASTM, 1999), using a glossmeter Novo-gloss LiteTM (Rhopoint Statistical Ltd, St Leonards, East Sussex, UK). Ten replicates were taken per each formulation. 2.4.1.2 UV-Vis light barrier The UV-Vis light barrier properties of films were determined in transmittance mode in the wavelength range of 200-900 nm, using a UV-Vis spectrophotometer LAMBDA 35 (Perkin Elmer, Waltham, MA, USA). Film samples were fixed in the cuvette so the light beam could pass over the film surfaces. The analyses were performed in triplicate.

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2.4.1.3 Fourier transformed infrared spectroscopy Fourier transformed infrared (FTIR) spectroscopy, based on the identification of absorption bands concerned with the vibrations of functional groups present in GEL and CH macromolecules, was used to analyze the chain interactions in blend films. A Spectrum One FTIR Spectrometer (Perkin Elmer) was used to record the FTIR spectra between wave numbers 650 and 4000 cm-1. The transmission mode was used, and the resolution was 1 cm-1. All tests were performed in triplicate at room temperature. 2.4.1.4 Microstructure Micrographs of the cross-sections of the films were obtained by Scanning Electron Microscopy (SEM), using a HITACHI Tabletop Microscope TM3000 (Hitachi Ltd, Tokyo, Japan), with wide magnification range of 15 to 30,000x. The film samples were equilibrated in P2O5 to eliminate water, and then mounted on copper stubs perpendicularly to their surface. The images were captured using an accelerating voltage of 15 kV. Three samples per film formulation were analyzed. 2.4.1.5 Mechanical properties According to ASTM standard method D882 (ASTM, 2001), a texturometer TA.XTplus model (Stable Micro Systems, Haslemere, Surrey, UK) was used to obtain the true stress–Hencky strain curves. From these curves, elastic modulus (EM), tensile strength (TS) and elongation at break (EB) of the films were calculated. Rectangular samples (15 x 90 mm) were cut and stored and equilibrated film samples were mounted in the film-extension grips and stretched at 50 mm*min-1 until breakage. Fifteen replicates of each formulation were measured. 2.4.1.6 Water vapor permeability Water vapor permeability (WVP) was determined at 25ºC and at 53–100% RH gradient using a modification of the ASTM E96–95 gravimetric method (1995) for

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flexible films. Films were selected based on their lack of bubbles or holes. Payne permeability cups of 8 cm in diameter were filled with 5 ml distilled water (RH 100%). Once the films were secured, each cup was placed in a pre-equilibrated cabinet, where the shiny side of the films was exposed to the atmosphere at the lowest RH. The cups were weighed periodically (±0.00001 g) after the steady state was reached. The slope of the weight loss vs. time plot was divided by the exposed film area to calculate the water vapor transmission rate (WVTR). The vapor pressure on the film’s inner surface was obtained by means of the method subsequently proposed by Mc Hugh et al. (1996) to correct the effect of concentration gradients established in the stagnant air gap inside the cup. Three replicates per formulation were analyzed. 2.4.2 Antioxidant properties 2.4.2.1 Chemical materials The reactants for the antioxidant capacity assay - Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) (PubChem CID: 40634), ABTS (2,2-azino-bis[3-etilbenzotiazol-6-sulfonic acid]) (PubChem CID: 6871216) were supplied by Sigma– Aldrich (São Paulo, SP, Brazil). Potassium persulfate (K2S2O8) (PubChem CID: 54602120) was provided by Merck (Darmstadt, Germany). Absolute ethanol (PubChem CID: 702) was supplied by Labsynth® (São Paulo, SP, Brazil) and filter papers (Whatman 1) were supplied by GE Healthcare (Buckinghamshire, UK). 2.4.2.2 TEAC (Trolox-equivalent-antioxidant-capacity) Assay The antioxidant capacity of the films was determined by means of a spectrophotometric method described by Re et al., (1998). Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) was used as standard antioxidant. ABTS (2,20azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) was dissolved in water at 7 mM and allowed to react with a 2.45 mM potassium persulfate solution (final

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concentrations) in the dark, for 16 h. An aliquot of the stock solution was diluted with distilled water in order to prepare the working solution of ABTS•+ radical with an absorbance of 0.70 ± 0.03 at 734 nm. For the analysis of the films, the method described by Bonilla et al., (2013b) was followed, where 1 g of each film sample, cut into small pieces, was previously immersed in 5 mL of a hydroalcoholic mixture to favor the extraction of the antioxidants. The samples were maintained under stirring overnight and then 10 µl aliquot was added to 990 µl of ABTS + dilution to produce a decrease in absorbance of 20–80% within 6 min. Absorbance at 734 nm was registered every minute during the test using a UV-Vis spectrophotometer LAMBDA 35 (PerkinElmer). The TEAC of the films extracts was determined by comparing the corresponding percentage of absorbance reduction to the Trolox concentration–response curve and expressed as the mass of the extract which produces the same percentage of absorbance reduction as 1 mM Trolox solution. All the analyses were carried out in triplicate. 2.4.3 Antimicrobial activity 2.4.3.1 Microbiological materials and Microbial strains The materials for antimicrobial capacity assay – brain heart infusion (BHI) broth and Müller-Hinton agar were supplied by Acumedia (Lansing, MI, USA) and chloramphenicol

(2,2-dichloro-N-[1,3-dihydroxy-1-(4-nitrophenyl)propan-2-

yl]acetamide) was purchased in Sigma-Aldrich Ltda (São Paulo, SP, Brazil). All chemicals were of analytical grade. Cultures were obtained from André Tosello Foundation-Collection of Tropical Crops (Campinas, SP, Brazil). One gram-negative bacteria, Escherichia coli (ATCC 25922), and one gram-positive bacteria, Staphylococcus aureus (ATCC 29213), were kept frozen (-25 °C) in Tryptone Soy Broth supplemented with 30% glycerol. The cultures were regenerated by transferring a 100 μl aliquot of each bacterium into 10 mL

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of BHI broth and incubated at 37°C for 24 h until the end of the exponential phase of growth. After incubation, the bacterial suspensions were standardized by adjusting on the scale of Mac Farland 0.5 Mac, which corresponds to 0.08 to 0.10 absorbance (1x108 CFU/ml), using a spectrophotometer at 625 nm. 2.4.3.2 Quantitative analyses The disk diffusion method was used to determine the antimicrobial activity of the films. E. coli and S. aureus were selected for this study. The plates containing Müller-Hinton agar were inoculated after preparation of the bacterial suspension, and then a sterile swab was used for dipping the bacterial suspension in all the plate avoiding leaving uncovered areas. Filter paper disks (18 mm diameter) were soaked with 15 ml of absolute ethanol and chloramphenicol (2.5%) used as negative and positive controls, respectively. Then films samples (18 mm diameter) and controls were laid onto the inoculated plate’s surface. These plates were incubated for 24 h at 37ºC. At the end, the halos formed by inhibition zones surrounding clear areas were considered as a measurement of the antimicrobial activity. All analyses were performed in triplicate. 2.5 Statistical Analyses The results were analyzed by means of a multifactor analysis of variance with a 95% significance level using Statgraphics® Plus 5.1 (Manugistics Corp., Rockville, MD, USA). Multiple comparisons were done using 95% least significant difference (LSD) intervals.

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3. Results and discussion 3.1 Characterization of films 3.1.1 Colour, opacity and gloss In spite of some significant difference observed in the L* of films (GEL100 = 91.7 ± 0.0240 and CH100 = 90.3 ± 0.0240), this value varied around 91 (Table 1), which means that these films were almost clear. This behavior was similar to that observed by Pereda et al., (2011) and Rivero et al., (2009), both working with films of bovine-hide gelatin type B and CH. Nevertheless, Jridi et al. (2014) prepared films of cuttlefish skin gelatin and chitosan by casting in different concentrations and reported that the lightness of composite films was reduced as a function of the increasing chitosan proportion in the formulation, where pure gelatin film appeared slightly clearer (L*= 93) as compared to pure chitosan film (L*= 79). The parameter a*, which corresponds to the redness of films, stayed around -1, which means that films were not truly red. The films of pure chitosan (CH100) presented lower value (-1.14 ± 0.009) (p≤0.05) than pure gelatin films (GEL100) (0.740 ± 0.009) (Table 1). The presence of ethanolic extracts produced a* values varying between -0.680 ± 0.009 and -0.977 ± 0.009, increasing these values when the proportion of CH increased into films (p≤0.05). Notwithstanding, in practical terms, that variation can be neglected. Nonetheless, a more visible effect was observed in the parameter b*, whose values increased (p≤0.05) as a function of the CH proportion, and, in overall, the values varied between 3 and 6 (Table 1). The b* values showed an increment as the proportion of chitosan increased in the film as a consequence of the incorporation of extracts (p≤0.05), where boldo-do-chile (6.17 ± 0.05) and rosemary (5.13 ± 0.05) extracts provoked the highest values as compared to cinnamon and guarana extracts (p≤0.05), meaning that the yellowness effect became stronger in the first two films.

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GEL100 and CH100 films presented b*= 2.56 ± 0.05 and 3.42 ± 0.05, respectively (p≤0.05). This tendency of a* and b* parameters were previously reported by other authors (Jridi et al., (2014); Pereda et al., (2011); Rivero et al., (2009)). Regarding opacity, all films were almost transparent (opacity  0). The films of pure chitosan (0.500 ± 0.033) presented values similar to pure gelatin films (0.300 ± 0.033) (p>0.05). The incorporation of different extracts did not affect the opacity of the blended films, where an increment of chitosan proportion in the films resulted in a slightly increment of this parameter (p≤0.05), where boldo-do-chile extracts showed the highest value (1.23 ± 0.033). The gloss of pure gelatin and pure chitosan films showed no significant difference, with values of 90 ± 3 and 93 ± 3, respectively (p>0.05). The incorporation of extracts increased significantly these values (p≤0.05), with no significant differences by type of extract added (p>0.05). The GEL75:CH25 films with guarana (144 ± 3) and boldo-dochile (139 ± 3) extracts showed the highest values as compared with GEL50:CH50 films. These latter showed a reduced gloss with increasing chitosan proportion. 3.1.2 UV-Vis light barrier It can be observed in Figure 1 that the GEL100 film presented higher barrier to UV (<250 nm) than the CH100 film, but this behavior was inversed for visible light (>300 nm), where the CH100 films presented higher barrier than GEL100 films. The incorporation of all extracts in the films provoked transmittance values in the ultraviolet (UV) and visible light region (250-300 nm) similar to pure gelatin film (GEL100), showing a significant increment of these values for visible light above of 300 nm, where GEL100 films with guarana (G) and cinnamon (C) extracts presented higher transmittance values than pure gelatin films.

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Moreover, a reduction of gelatin content, substituted by up to 50% chitosan (GEL50:CH50) with added extracts, showed higher transmittance values in visible light (250-300 nm) than pure gelatin films, noting that GEL50:CH50 films with guarana and cinnamon extracts in visible light above 300 nm presented transmittance values similar to pure chitosan film (CH100) and lower than pure gelatin films (GEL100). According to Sobral and Habitante, (2001a), gelatin has excellent UV barrier properties due to the presence of amino acids residues containing aromatic rings. Similar values have been reported by Hoque et al., (2011), who reported the transmission of UV and visible light (200-800 nm) through films from cuttlefish ventral skin gelatin with 1% ethanolic extracts of cinnamon, clove and star anise. Likewise, Gómez-Estaca et al., (2009) concluded that bovine-hide gelatin films with added oregano or rosemary extracts maintain the transparency of the pure films and showed similar values of transmittance to the control film in the range below 270 nm, increasing these transmittance values (80–90%) at 400 nm. Jridi et al. (2014) studied gelatin– chitosan composite films and concluded that the transmission in visible range (350–800 nm) of gelatin films was from 38.7 to 90.9%. At 280 nm, chitosan film had higher absorbance compared with pure gelatin and composite films. 3.1.3 Fourier transformed Infrared spectroscopy Fourier transformed infrared spectroscopy (FTIR) spectra of GEL100 and CH100 films and GEL:CH films (GEL75:CH25 and GEL50:CH50) with extract are shown in Fig. 2. In this figure only the result of the blend films (GEL50:CH50 and GEL75:CH25) are presented with rosemary (R) extracts, since the other extracts (B, C and G) showed the same tendency to be incorporated into these formulations. For pure gelatin films (GEL100), four characteristic peaks were identified. The band at about 1237 cm-1 was assigned to Amide III, previously reported by Bergo et al.,

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(2013) and Liu et al., (2012), who characterized pigskin gelatin films plasticized with glycerol and fish gelatin films, respectively. The band at about 1450 cm-1 was assigned to vibrations of the -OH group of the primary alcoholic group from gelatin (Bergo et al., 2013), 1545 cm-1 band was assigned to Amide II (N-H) and C-N vibrations (Bergo et al., (2013); Liu et al., (2012)) and 1630 band cm-1 was assigned to Amide I (C=O stretch) (Bergo et al., (2013). For pure chitosan films (CH100), the main characteristic bands were assigned to saccharide structures at 894, 1019 and 1155 cm-1 (Bonilla et al., 2014a). Different authors (Bonilla et al., 2014a; Sionkowska et al., 2004; Liu et al., 2012) reported strong characteristic bands at around 1406, 1543 and 1643 cm-1, which were assigned to vibrations of -OH group of the primary alcoholic group, amide I and amide II bands, respectively. The gelatin-based films containing different chitosan proportions (GEL75:CH25 and GEL50:CH50) with added rosemary extract showed a slight decreasing in the intensity of the band corresponding to Amide I (1643 cm-1) as compared with pure chitosan film. According to other authors (Sionkowska et al., 2004; Liu et al., 2012), these modifications could result from molecular interactions between the two polymers, where a decrease in the vibrational wavelength and a broadening of the OH and NH vibration bands are indicative of a hydrogen bonding interaction between polymer molecules in the film. 3.1.4. Microstructure The microstructure of GEL:CH films was accessed by scanning electronic microscopy (Fig. 3). A continuous and homogeneous phase in blended films (GEL75:CH25 and GEL50:CH50) can be observed, without any evidence of phase separation, as a result from the well interactions between the polymers in the blend.

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This indicates the high compatibility of the two biopolymers, and the perfect incorporation of rosemary (R), guarana (G), cinnamon (C) and boldo-do-chile (B) extracts in the films. Similar results were reported by other authors, such as Rivero et al., (2009), which reported that the composite films of gelatin and chitosan were characterized by a compact, uniform, dense structure and homogenous appearance; and Wu et al., (2013), who studied silver carp skin gelatin films with green tea extract incorporated. On the other hand, Bitencourt et al., (2014) studied pigskin gelatin type A films with 0, 5, 50, 100, 150 and 200 g of curcuma extract/100 g of gelatin and observed less homogeneous and compact structures than the control film, more pronouncedly for higher curcuma ethanol extract concentrations. And, Bodini et al., (2013) studied films of gelatin type A with ethanol-propolis extract (EPE) and observed that the increasing of propolis extract concentrations apparently produced an increase in the porosity of the matrix. 3.1.5. Mechanical properties Elastic modulus (EM), tensile strength (TS) and elongation at break (EB) values of all films were shown in Table 1. For films based on pure biopolymers, the mechanical properties for GEL100 were TS= 2.4 ± 1.8 MPa, EM= 95 ± 2 MPa, and EB= 4.4 ± 0.14 %. The CH100 films showed to be less resistant (TS= 3.4 ± 1.8 MPa) and rigid (EM= 61 ± 2 MPa), but significantly more stretchable (EB= 28 ± 0.1 %) than GEL100 films (p≤0.05). The incorporation of different extracts in gelatin films (GEL100), when compared with pure gelatin films without extracts, showed a reduction of up to 70% in EM (values between 20 ± 2 and 28 ± 2 MPa for films with cinnamon and guarana extracts, respectively). In addition, 30% in TS (values between 3.2 ± 1.8 and 3.8 ± 1.8

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MPa for films with boldo-do-chile and guarana extracts, respectively) (p≤0.05). Moreover, a marked increment of EB (values between 51 ± 0.1 and 65 ± 0.1% for films with cinnamon and rosemary extracts, respectively) was observed (p≤0.05). Similar results were determined by Bitencourt et al., (2014), who showed a reduction in the elastic modulus (EM) and revealed a significantly increased EB with the incorporation of diverse concentrations of ethanolic extract of Curcuma in gelatin based films, possibly due to interactions between phenolic compounds (present in the extract) and gelatin peptides. The increasing of the proportion of CH in the blend GEL:CH resulted in an decrement of the elastic modulus (EM) as compared with pure gelatin films, presenting values between 50 ± 2 and 61 ± 2 MPa for GEL50:CH50 films with guarana and rosemary extracts, respectively. However, no significant differences regarding the type of extracts were observed (p>0.05). Moreover, the elongation at break (EB) of polymers was significant increased with the addition of different extracts (p≤0.05) (values between 65 ± 0.1 and 51 ± 0.1 %, for GEL100:CH0 with rosemary and cinnamon extracts respectively). Bitencourt et al., (2014) reported an increment in EB value with the incorporation of curcuma ethanol extracts in GEL films. They concluded that these results may be caused by possible interactions between phenolic compounds (present in the extract) and gelatin peptides (observed in the infrared spectra), which form covalent cross-links that may lead to the formation of more cohesive and flexible matrices. On the other hand, chitosan proportion in the blended films significant reduced (p≤0.05) these values, showing values between 23 ± 0.1 and 24 ± 0.1 % for GEL75:CH25 films with rosemary and cinnamon extracts, and values between 20 ± 0.1 and 21 ± 0.1% for GEL50:CH50 films with cinnamon and guarana extracts, respectively. Similar results of EB were reported by Liu et al., (2012), who concluded that the interactions between

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gelatin and chitosan became stronger, owing to hydrogen bonds and the formation of polyanion-polycation complexes between gelatin and chitosan, suggesting that chitosan may provide more reactive groups within one chain for interaction with gelatin via hydrogen bonds and hydrophobic interactions, leading to film strengthening. 3.1.6 Water vapor permeability The values of water vapor permeability (WVP) of the films are shown in Table 1, where it can be observed that the WVP of pure chitosan films (0.55 ± 0.28 x10-10 g.s1

.m-1.Pa-1) were significantly lower than that of pure gelatin films (2.03 ± 0.28 x10-10

g.s-1.m-1.Pa-1) (p≤0.05). In a general manner, the presence of all extracts, excepted C, slightly increased the WVP of GEL100 films, but without significant effect (p>0.05). Nonetheless, for both studied proportions of CH, the WVP was lowered by CH without a monotonic behavior, with minimum value of 1.17 ± 0.28 x10-10 g.s-1.m-1.Pa-1 for films of GEL50:CH50 with B. In overall, all variation observed (Table 1) can be considered as negligible regarding the WVP of films, or in others words, the films were neither improved nor worsened by extracts and/or CH proportion. Similar results were presented by Li et al., (2014), who studied the WVP of fish skin gelatin-based films incorporated with different natural antioxidants (green tea extract, grape seed extract, grape seed extract, ginger extract and gingko leaf extract). They had no significantly difference compared to pure gelatin films (p>0.05). Hoque et al., (2011) studied the properties of cuttlefish skin gelatin films with cinnamon, clove and star anise extracts added. No differences in WVP were observed among films incorporated with the three non-oxidized herb extracts (p>0.05), showing a higher WVP, compared with their pure gelatin film (p≤0.05). Similar behavior was also observed by Bitencourt et al., (2014), working with gelatin-based films with curcuma ethanol extract (5, 50, 100, 150 and 200 g extract/100 g gelatin), where the

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incorporation of different concentration of extracts resulted in no significant differences of WVP values. 3.2. Antioxidant activity 3.2.1 TEAC (Trolox-Equivalent-Antioxidant-Capacity) Assay The antioxidant capacity determined by the TEAC (Trolox Equivalent Antioxidant Capacity) assays of the pure and blend films at 1, 3 and 6 min are shown in Table 2, where it can be observed that CH100 films presented an antioxidant activity of 0.16 ± 0.13 mg/l and that GEL100 films did not present antioxidant activity in this study. Also, the incorporation of chitosan and extracts in the blend (GEL75:CH25) provoked an increment of film antioxidant activity, where the films with boldo-do-chile (1.5 ± 0.13 mg/l) and guarana (1.5 ± 0.13 mg/l) extracts presented the highest antioxidant activity (p≤0.05). According to García et al. (2015), the use of chitosan as antioxidant additive had been reported in numerous researches, which had demonstrated the capacity of this polymer for interacting with free radicals through ionic interactions with its amino groups. On the other hand, previous studies have been reported the antioxidant activity of rosemary (Ojeda-Sana et al., (2013); Gibis and Weiss (2012)), boldo-do-chile (Valle et al., (2005); Mazutti et al., (2008); Soto et al., (2013)), guarana (Majhenic et al., (2007); Dalonso and Petkowicz, 2012) and cinnamon (Mathew and Abraham, 2006; Przygodzka et al., ( 2013)) extracts, studied by TEAC or DPPH tests. An increment of chitosan proportion (GEL50:CH50) increased the antioxidant activity of films (p≤0.05), being higher for films with rosemary extract (1.9 ± 0.13 mg/l) at 6min (p≤0.05). Sabaghi et al., (2015) reported the antioxidant activity of kefiran films by chitosan addition and observed that an increment in chitosan proportion increased significantly the antioxidant activity of the composite films, possibly due to the increased free amino group of chitosan in combined films.

18

3.3.

Antimicrobial activity

The results of antimicrobial activity tests for pure biopolymers films (GEL100 and CH100) and GEL50:CH50 blend films with different extracts, against S. aureus and E. coli, are presented in Fig. 4. As expected, the chloramphenicol, used as antibiotic control, presented the highest (sensitive) inhibitory effect in comparison to all tested films. The diameter of the inhibition zone of pure chitosan films (CH100) indicated inhibition on growth against S. aureus and E. coli strains tested, but, contrarily, the pure gelatin films did not show any antimicrobial activity. Different authors have demonstrated the antimicrobial activity of chitosan (Bonilla et al., (2014b); SánchezGonzález et al., (2011), and explained this behavior for its positively charged amino group which interacts with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Dutta et al., (2009). An increase of chitosan proportion and the presence of rosemary, cinnamon, boldodo-chile and guarana extracts in films (GEL50:CH50) allowed to observe an increased diameter of the inhibition zone during the period analyzed. The films showed their antimicrobial activity with diameters of the inhibition zone ranging from 23 to 26 mm against S. aureus with rosemary and cinnamon extracts added, respectively, and from 21 to 24 mm against E. coli with boldo-do-chile and guarana extracts added, respectively. Bodini et al., (2013) studied gelatin-based films with ethanol-propolis extract added in different concentrations (0, 5, 40 and 200 g of extract/100 g of gelatin), and concluded that only the films with concentrations of 40 and 200 g of extract inhibited the growth of S. aureus, possibly associated with the increase in polyphenol concentrations.

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4. Conclusions This investigation shows how the incorporation of ethanolic extracts and different proportions of gelatin:chitosan can affect the physic-chemical, antimicrobial and antioxidant properties of the blend films obtained. The luminosity of the pure gelatin films decreased with chitosan addition and the gloss was significantly increased (p≤0.05) with the incorporation of extracts. The mechanical properties were significantly improved (p≤0.05) as the chitosan ratio increased in the films, these being more resistant and extensible; at the same time that chitosan reduced the WVP of the pure gelatin films (p≤0.05). The studies of microstructure and FTIR confirmed the good compatibility between the two polymers. Furthermore, the antioxidant activity of the films was significantly enhanced (p≤0.05) with the addition of extracts and chitosan up to 50% (GEL50:CH50), and a notable antimicrobial effect was detected in the blend films when the proportion of chitosan was 50% (GEL50:CH50). The obtained results are important because it suggests the possibility of developing edible antibacterial and antioxidant films with good physicochemical properties by combining gelatin and chitosan. These films could be applied to the life extension of food products. Acknowledgements The authors acknowledge the financial support from the São Paulo Research Foundation (FAPESP) (13/07914-8), Postdoctoral fellowship of Jeannine Bonilla (14/03288-8) and Brazilian National Council for Scientific and Technological Development (CNPq) for the Research fellowship of Paulo J.A. Sobral. Conflict of Interest Statement We declare that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all

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authors and tacitly or explicitly by the responsible authorities where the work was carried out.

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Table 1. Luminosity (L*), a*, b*, opacity, gloss, elastic modulus (EM), tensile strength (TS), elongation at break (EB) and water vapor permeability (WVP) of the all films equilibrated at 58%.

FILM S EXT RAC T L* a* b* OPA CITY GLO SS EM (MPa ) TS (MPa ) EB (%) WVP (g.s1 .m1 .Pa1 ) x1010

GEL 100

CH1 00

GEL GEL10 75: 0:CH0 CH2 5

Without extract 91.7 A,a

0.74 0 C,a 2.56 C,c

B,a

4.4

Boldo 90.6 90.4

91.5 B,c

-1.14

-0.977

1.18 1.43

-0.713

4.36 6.17

2.65

0.46 1.23 A,a 7 B,a 139 126

0.400

AB,bc

3.42 BC,bc

C,b

B,ab

C,b

3.45

B,b

C,a

0.233 C,a

132 A,a 22 C,a

B,a

A,a

64

D,ab

A,b

A,a

B,a

C,a

C,bc

C,a

150 A,a

AB,a

AB,a

55

28 C,a

3.4

3.2 A,a

4.0

3.1

3.8 A,a

24

21

60 A,ab

AB,a

C,ab

B,ab

28

56 A,a

2.03

0.55

2.07

A,a

C,ab

C,b

A,ab

AB,a

B,a

AB,a

B,a

1.58 1.17 B,ab

GEL 75: CH2 5

GEL 50: CH5 0

GEL10 0:CH0

Guarana

91.3

E, d

95 A,a 61 B,a 2.4

GEL10 0:CH0

90.3

0.30 0.50 0 BC,a 0 BC,a 90 93 C,b

GE L50 : CH 50

BC,ab

2.15 A,a

90.9

0.86 7 B,a 3.54

0.95 7 A,a 4.40

0.40 0 B,a 144

0.86 7 A,a 123

B,bc

A,a

61

D,c

A,bc

B,a

91.4 B,bc

-0.680 C,a

2.57 C,bc

0.333 C,a

149 A,a

AB,a

AB,a

50

20 C,a

1.7

3.0

AB,a

24

GEL 50: CH5 0

GEL10 0:CH0

Cinnamon

90.9 C,c

GEL 75: CH2 5

AB,a

90.8

0.86 3 B,a 3.44

0.97 3 A,a 4.50

0.63 3 B,a 139

0.66 7 A,a 117

B,bc

A,a

75

D,bc

A,bc

B,a

91.5 B,bc

-0.827 C,c

2.91 C,ab

0.333 C,a

131 A,a

AB,a

AB,a

59

28 C,a

3.3 A,a

3.6

4.2

AB,a

AB,a

90.6 C,bc

0.99 3 B,c 3.75 B,ab

0.56 7 B,a 134 A,a

60

2.2

2.7 21

AB,a

51 A,a

24

20

65 A,b

23

1.47

1.32

1.99

1.53

1.26

2.68 A,a

1.47

BC,a

A,ab

B,ab

BC,ab

61

3.3 A,a

21

B,a

5.13 0.0 A,ab 5 0.60 0.0 A,a 0 33 131 3 A,a AB,a

B,ab

B,a

90.3 0.0 D,bc 240 0.0 1.18 09 A,c

AB,a

B,ab

B,a

SE M

Rosemary

90.9 C,bc

GEL GEL 75: 50: CH2 CH5 5 0

B,b

B,a

AB,a

B,b

2

1.8 0.1

1.29 0.2 BC,a 8

GEL: Gelatin; CH: Chitosan. Means with the same superscript (ABCDE) were compared horizontally for different GEL:CH ratios, and (abcd) were compared horizontally for different extracts in LSD test. SEM: Standard error of the mean based on Mean Square Error of ANOVA analysis.

25

Table 2. Trolox equivalent antioxidant capacity (TEAC) of the films (mg/l) with or without ethanolic extract at specific time points. Film

Extract

GEL100 CH100 GEL 75:CH25

Without

1 min --

3 min --

6 min --

SEM --

Without

0.15 C,d

0.16 C,d

Boldo

0.94

B,a

1.4

0.16 C,d

0.13

B,a

1.5 B,a

0.13

GEL 50:CH50

1.1 A,a

1.5 A,a

1.8 A,a

0.13

GEL 75:CH25

0.17 B,b

0.21 B,b

0.27 B,b

0.13

1.2 A,b

1.6 A,b

1.9 A,b

0.13

Rosemary

GEL 50:CH50 GEL 75:CH25

Cinnamon

0.17

B,c

0.23

B,c

0.31

B,c

0.13

GEL 50:CH50

0.27 A,c

0.31 A,c

0.32 A,c

0.13

GEL 75:CH25

0.89 B,ab

1.2 B,ab

1.5 B,ab

0.13

Guarana

0.93 A,ab 1.3 A,ab 1.6 A,ab 0.13 GEL 50:CH50 ---- Not showed antioxidant capacity. GEL: Gelatin; CH: Chitosan. Means with the same superscript ( ABCD) were compared vertically for different GEL:CH ratios, and means (abcd) were compared vertically for different extracts in LSD test. SEM: Standard error of the mean based on Mean Square Error of ANOVA analysis.

100

Transmittance (%)

90 80

G C

70

B and G R and B G C

60 50 40

CH100 GEL100

30

GEL100:CH0 with extracts GEL50:CH50 with extracts

20 10 0 200

250

300

350

400

Wavenumber(nm) Figure 1. UV-Vis spectra for pure chitosan films (CH100), pure gelatin films (GEL100) and GEL100:CH0 and GEL50:CH50 blend films with guarana (G), boldo (B), cinnamon (C) and rosemary (R) extracts.

26

Figure 2. FTIR spectra of rosemary (R) extract, pure chitosan films (CH100), pure gelatin films (GEL100) and GEL75:CH25 or GEL50:CH50 blend films with R extract.

27

GEL75:CH25 R

GEL75:CH25 G

GEL50:CH50 R

GEL50:CH50 G

GEL75:CH25 C

GEL50:CH50 C

GEL75:CH25 B

GEL50:CH50 B

Figure 3. Scanning electron microscopy (SEM) images of cross-sections of blend films (Gel75:CH25 or GEL50:CH50) with guarana (G), boldo (B), cinnamon (C) and rosemary (R) extracts.

28

A

B C

+

B

C

R

G

+ B

R

G

Figure 4. Inhibition of Staphylococcus aureus and Escherichia coli following antibiotic disc diffusion assay. GEL: gelatin, CH: chitosan, R: Rosemary, C: cinnamon, B: boldo and G:-guaraná. A: GEL50:CH50 films with extracts added against S. aureus B: GEL50:CH50 films with extracts added against E. coli.

29