Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin

Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin

Accepted Manuscript Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin Eliana Matta, María José Tavera-Quiroz, N...

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Accepted Manuscript Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin

Eliana Matta, María José Tavera-Quiroz, Nora Bertola PII: DOI: Reference:

S0141-8130(18)34603-8 https://doi.org/10.1016/j.ijbiomac.2018.12.114 BIOMAC 11273

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

30 August 2018 5 December 2018 13 December 2018

Please cite this article as: Eliana Matta, María José Tavera-Quiroz, Nora Bertola , Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.114

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ACCEPTED MANUSCRIPT ACTIVE EDIBLE FILMS OF METHYLCELLULOSE WITH EXTRACTS OF GREEN APPLE (GRANNY SMITH) SKIN

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Eliana Matta 1, María José Tavera-Quiroz 2, Nora Bertola 1

Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA)- CONICET CIC, Facultad de Ciencias Exactas - UNLP, 47 y 116, La Plata (1900), Argentina.

Desarrollo e Innovación de Procesos Alimentarios (DESINPA), Facultad de Ingeniería.

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Departamento de Ingeniería Agroindustrial. Universidad de Sucre. Carrera 28 Nº 5-267 Barrio

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Puerta Roja, Sincelejo (Sucre), Colombia

Corresponding author: Nora Bertola, [email protected]

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telephone numbers: +54 221 4254853

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ACCEPTED MANUSCRIPT ABSTRACT The aim a present study was developed methylcellulose (MC) active edible films with extracts of green apple skin, as model systems of edible coating. Active edible films were developed by incorporation of ethanolic extract of freeze-dried apple skin (EEFD) and aqueous extract of apple skin (AES) at 10, 20 and 25% (v/v) concentrations. Analysis of thermal, mechanical and

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functional properties was carried out. Results showed that incorporation of green apple skin extracts into MC films contribute to total phenolic content and antioxidant properties. Addition of green apple skin extracts generated shifts towards lower glass transition temperature values regarding MC films without extracts. A lower tensile strength and

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increased elongation at break in MC-AES films were observed. Mechanical properties of MCEEFD films were less affected by the increase in extract concentration due to absence of the

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plasticizing effect of sugars present in AES. The methylcellulose films are important for

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actives edibles coatings with applications in the food industry.

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Keywords: Active film; Green apple skin; Methylcellulose; Antioxidants; Polyphenols.

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ACCEPTED MANUSCRIPT 1.

Introduction

Active packaging technology is designed to extend the shelf life of product, maintaining nutritional, sensory quality and microbiological safety [1]. One cause of quality loss in foods is oxidation. Therefore, an active package may contain antioxidants to delay harmful effects. Currently, many researchers have focused on food packaging with antioxidants from natural

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sources. Apple is an excellent source of vitamins and essential ingredients for human health. Some research has indicated that apples contain high levels of biologically active compounds that can help provide protection against cancer [2], [3]. Food industry, especially production of apple juice and dried apple industry generates lots of waste from apples such’s as skin,

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core, seed, calyx, stem, and soft tissue [4]. Granny Smith apples have less susceptibility to browning and higher values of firmness and juiciness compared to other varieties of apples,

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thus, this variety would be the most suitable for industrial processing [5]. Wolfe et al. [6] found that antioxidant and antiproliferative activities from unpeeled apples were higher than

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those from peeled ones. This is due to the concentration of polyphenols, vitamin C and mineral content in skin. [7]. Therefore apple skin, labeled as an agroindustrial waste, is

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potentially useful for making extracts rich in polyphenols to be used as source of functional

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ingredients.

Edible coatings / films technology emerges as a promising alternative for improving quality

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and preservation of food during processing and/or storage. Polysaccharides, proteins, lipids and combinations of these have been used to produce edible coatings [8]. Films and coatings usually can be obtained from the same formulation. Coatings are applied in liquid form before forming the coating meanwhile films are obtained as solid sheets and then applied to food products. Films and coatings act as a barrier and their properties depending on the specific requirements of food preservation. Because the properties of a coating on the surface of a

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ACCEPTED MANUSCRIPT biological matrix cannot be characterized, isolated films have been reported as an alternative for predicting coating properties [9], [10], [11], [8]. Cellulose constitutes the most abundant renewable polymer resource available in nature and has been widely reported as a raw material for biodegradable films mainly because of renewability, low cost, non-toxicity, biocompatibility, biodegradability and chemical stability

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[12], [13]. The simplest cellulose derivative is methylcellulose, in which the hydroxyl residues of cellulose are replaced by methyl groups. Methylcellulose has excellent film making properties, with high solubility, low oxygen and lipid permeability; and it is a promising biopolymer for active food packaging [10], [14].

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A few studies have been reported on properties of methylcellulose-based films and edible coating added with extracts rich in polyphenols and antioxidants [15], [16]. Thus, the main

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objective as present study was to develop edible films, as model systems of edible coating, by using different extracts of apple skin and analyzing the influence of these extracts on

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physicochemical, microstructural, thermal and mechanical properties of the methylcellulose-

Materials and methods

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2.

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based film.

Methylcellulose (A4M, Methocel) was provided by COLORCON S.A. (Argentina).

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According to specifications of the supplier, the molecular weight was 65.103 Da. Granny Smith apples (Malus domestica), selected by size and appearance, were purchased from the local market.

2.1.

Obtaining extracts of green apple skin

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ACCEPTED MANUSCRIPT In present study were obtained and characterized extracts of green apple skin. Granny Smith apple peel was used, since this variety presents a high phenolics content and long shelf life [17]. The apples were washed, and the skin was removed with a peeler kitchen. Extracts of green apple skin were obtained from two different methods. First, ethanolic extract of freeze-dried

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apple skins were obtained. Skin obtained were placed in plastic trays and frozen at -80°C for 24 hours. After this step, the apple skin was freeze-dried and milled to obtain a powder, which was used for the preparation of the ethanolic extract of freeze-dried apple skin (EEFD). The extract was prepared with 1.5 g powder of freeze-dried apple skin mixed with 25 ml of 70%

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ethanol solution, under constant stirring for 15 min and centrifuged. In the second method, apple skins were processed in a domestic extractor (Black and Decker, Argentina) obtaining

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the juice (AES). The juice was centrifuged for 10 minutes (Rolco CM2036, Argentina) and finally the supernatant was obtained. Both extracts were used as active components for the

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preparation of films.

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2.2. Characterization of extracts obtained

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The polyphenol content was measured by using the Folin–Ciocalteau method [18]. Chlorogenic acid (Fluka, USA) was used as a standard. The results were expressed as mg

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chlorogenic acid/100 g dry sample. The antioxidant properties were obtained by the DPPH method [19]. Results were expressed as radical scavenging activity or inhibition of free radical percentage (I%). The absorbance of the reaction mixture containing both the DPPH free radical and the antioxidant is related to the absorbance of the reaction mixture without any antioxidant an incubation period of 60 min, using:

(1)

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ACCEPTED MANUSCRIPT where A0 is the absorbance of the reaction mixture without any antioxidant and A1 is the absorbance of the reaction mixture with the addition of the antioxidant. For all assays, the absorbance was measured in a spectrophotometer Shimadzu UV-Mini 1240 (Japan). The determination of the content of ascorbic acid (AA) was carried out by high-performance liquid chromatography (Waters, model R-414, USA). The assay was performed by isocratic

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elution procedure with UV-Visible detector at 245 nm. AA (Sigma Aldrich, USA) was used as a standard. The extracts obtained were mixed with 5 ml of an aqueous solution 50 g L-1 of metaphosphoric acid (Sigma Aldrich, USA) for 15 min and centrifuged at 2000 rpm for 10 min (Rolco CM 2036, Argentina). Separations were carried out on a 5 mm RP C18 column of

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150 mm-4.6 mm (WAT 045905, Waters, Ireland). A mixture of 5 g metaphosphoric acidacetonitrile (93:7) was employed as mobile phase [19], [20].

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The content of glucose, fructose, sucrose determination was made using a modified HPLC method of Tavera-Quiroz et al. [19]. The analysis was performed isocratically on a Microsorb

Films preparation with extracts of apple skin

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2.3.

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R0086700, amino column (KNAUER, Germany) attached to a refractive index detector.

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1.5 g MC were dispersed in 50 ml of distilled water at 80°C under constant stirring for one hour. With the aim to obtain solutions with extracts of green apple skin concentrations of 10,

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20 and 25% (v/v) of AES or EEFD were tested. In all cases, a total volume of 100 ml was made up of cold distilled water. About 20 g of film-forming solutions with AES and EEFD were cast onto Petri dishes (9 cm diameter) and dried at 37°C in an oven until reaching a constant weight. The obtained films were removed from the dish and then were stored at 20°C and 65% RH in a controlled room. Film thickness was determined by using a coating thickness gauge microprocessor digital

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ACCEPTED MANUSCRIPT meter CM-8822 (China) for non-conductive materials on non-ferrous substrates. Thickness values of films are important for permeability determinations.

2.4. Determination of moisture content and solubility of films Moisture content of films was determined by measuring their weight loss upon drying in an

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oven at 105°C until constant weight. Moisture results were expressed as g of water per 100 g of sample.

MC-based films were cut into 4 cm2 pieces to determine film solubility. Samples were weighed, placed into a metallic mesh and immersed into test beakers with 50 ml distilled

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water. The samples were maintained under constant agitation for 1 h at room temperature (approximately 25 ºC). After soaking, remaining pieces of the films were dried again in an

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oven at 105°C until reaching a constant weight. Film solubility (% soluble matter) was calculated as it was described by Rivero et al. [21].

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Average values of two measurements of moisture and solubility, were calculated for each

Thermal properties

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2.5

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sample.

2.5.1 Differential scanning calorimeter (DSC)

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Thermal properties of films were determined by using a differential scanning calorimeter DSC-Q100 controlled by a TA 5000 module (TA Instruments, USA). Film samples of 6–7 mg were weighed in aluminum pans and were hermetically sealed; an empty pan was used as reference. Indium was used to calibrate temperature and heat flux. The assay was performed as described in Tavera-Quiroz et al. [10]. The first scan was performed in the -70ºC to a 200ºC range at 10º/min. After the first scan was completed, the samples were cooled until 70°C and then, a second scan was recorded between -70 and 250ºC at 10º/min. The glass

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ACCEPTED MANUSCRIPT transition temperatures (Tg) were obtained from thermograms by using the Universal Analysis V1.7F software (TA Instruments, USA).

2.5.2 Dynamic mechanic analysis Thermal properties of films were determined by using a dynamic-mechanical thermal

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equipment DMA Q800 (TA Instruments, USA) using a tension clamp with a liquid N2 cooling system. Film strips (6 mm x 30 mm) were assayed. Amplitude sweep from 1 to 50 μm at a fixed frequency (5 Hz) was performed. At fixed amplitude, multi-frequency sweeps (1, 5, 10 and 15 Hz) from −100 to 200°C at 5°C/min were carried out, with an isotherm of 10 min at

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−100°C. Storage (E′), loss (E′′) modulus and tan δ (E′′/E′) curves as a function of temperature were recorded and analyzed using the software Universal Analysis 2000. Temperatures of the

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relaxation processes associated with glass transition temperatures were determined through the inflection point of the maximum peak in the tan δ curves [14]. Tg of the samples was

Mechanical properties

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2.6.

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analyzed in duplicate.

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Stress-strain assays were conducted in dynamic-mechanical thermal equipment (DMA). Film strips (6 mm x 30 mm) were assayed using a ramp force constant strain rate of 0.2 N/min to a

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static force of 18 N or even rupture of the samples. Elongation percentage at break (%E) was obtained from stress-strain curves. A mathematical model was applied to describe the stressstrain curve [22] using: 𝜎𝑇 (𝜀𝑇 ) = 𝐸𝐶 𝜀𝑇 exp(𝜀𝑇 𝐾)

(2)

Where: εT and σT (MPa) are the true strain and the true stress, respectively, EC (MPa) is the elastic modulus and K is a fitting parameter. The relationship between stress and strain was here affected by an exponential term acting as a damping factor [22].

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ACCEPTED MANUSCRIPT The model allowed to estimate EC, and with this, it was possible to characterize the elastic region of the curves. For that, the points immediately before the rupture of the film were not considered to obtain a better fitting of the experimental points at initial and intermediate deformations. For each batch, 3 samples were analyzed separately, obtaining the corresponding

2.7.

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Force deformation curves.

Water vapor permeability

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Water vapor permeability (WVP) tests were performed by using a modified ASTM method E96 [14]. Each film sample was sealed over a permeation cell that was stored at 20ºC in a

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desiccator. A driving force of 1753.55 Pa, corresponding to a 75% RH gradient across the film was used. To maintain the driving force corresponding to a 75% RH gradient across the

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film, anhydrous calcium chloride (0% RH) was placed inside the permeability cell and a sodium chloride saturated solution was used in the desiccator. After steady-state conditions

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were reached, the permeation cells were weighed at a 1 h interval for about 7 h. The water

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vapor transmission rate (WVTR) was calculated from the slope of the straight line (g/s) divided by the cell area. WVP was calculated as WVP = [WVTR /S (R1 − R2)] d, where S is

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the saturation vapor pressure of water (Pa) at the test temperature, R1, the RH in the desiccator, R2, the RH in the permeation cell and d is the film thickness. Triplicates samples were tested and mean values of WVP were reported.

2.8. Fourier transform infrared spectra of the films The Fourier transform infrared (FT-IR) spectra of the films were recorded in an IR spectrometer (Nicolet, iS10, Thermo Scientific, USA) in the wavenumber range 4000–400

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ACCEPTED MANUSCRIPT cm−1 by an accumulation of 64 scans at 4 cm−1 resolution. The spectral analysis of the data was performed using the software Omnic 8 (Thermo Scientific, USA).

2.9. Determination of total polyphenols content and antioxidant capacity analysis The analysis of total polyphenol content and antioxidant activity was carried out films

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obtained [23], [19]. Each film was weighed and immersed in 50 ml of distilled water, and stirred until complete dissolution. 50 l of each solution were taken and carried out the determination of polyphenols content and antioxidant capacity. The antioxidant capacity and

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of total polyphenols content of samples were analyzed in triplicate

2.10. Determination of ascorbic acid

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The films obtained were mixed with 5 ml of an aqueous solution 50 g L-1 of metaphosphoric acid (Sigma Aldrich, USA) for 15 min and centrifuged at 2000 rpm for 10 min (Rolco CM

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2036, Argentina). The content of ascorbic acid was determined, in duplicate, with the same

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procedure used for its determination in the extracts [19]

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2.11. Statistical Analysis

All experiments were performed on triplicate samples. Analysis of variance (ANOVA) was

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used to analyze the results obtained using the statistical program Infostat v2009 software (Argentina). Comparison of means by Fisher LSD mean was tested, and if P0.05 the difference was considered statistically significant.

3.

Results and discussion

3.1.

Characterization of obtained extracts

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ACCEPTED MANUSCRIPT Total polyphenol content (TPC) in EEFD was 850 ± 41.4 mg AES showed values of 131.3 ± 8.8 mg

chlorogenic acid/100

chlorogenic acid

gds (Table 1).

/100gds, while the

EEFD had higher

polyphenol content compared to AES. Huber and Rupasinghe [24] informed values of TPC between 150 to 700 mg/100gds, depending on variety and cultivar. Bustos-Hipólito et al. [25] observed a similar behavior by testing different solvents for the extraction of antioxidant

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compounds of the Red Delicious apple skin. They concluded that the most efficient extraction of these compounds is achieved when using boiling water followed by methanol and ethanol. With ethanol, the highest levels of anthocyanins were obtained.

Results of inhibition

percentage were 78% ± 0.85 inhibition DPPH radicals for AES, and 83% ± 2.09 for EEFD,

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indicating that EEFD showed a higher inhibition percentage compared with AES. These results may be related to polyphenol content obtained from analyzed extracts. Although the

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content of polyphenols is much higher in EEDF than in AES, it is not reflected in %I since in AES the content of AA is higher and produces an increase in % I. Several authors reported

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that apple skins are found to have a high antioxidant capacity [26], [27]. Some authors assert that there is a relationship between polyphenols content and antioxidant activity [24]; [27].

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Regarding AA content, results indicate that fresh apple skin aqueous extract contains higher

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AA concentrations compared to freeze-dried ethanolic extract. AA content in AES was 30.3 ± 3.75 mgAA/mlextract, while in EEFD, it was 20,2 ± 0.09 mgAA/mlextract. This would indicate that

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AA was degraded during the freeze-drying process. Ascorbic acid is one of the organics acids found in green apple skin and one of the components responsible for antioxidant action [28]. This bioactive compound is an abundant vitamin in apples. Nonetheless, its content is lower regarding polyphenols found in apples [29]. Content of the malic acid was not analyzed in present study, but the peak found in AES before ascorbic acid (retention time 1.3 min) could be attributed to malic acid present in apple skin [30]. For sugars evaluation, high sugar

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ACCEPTED MANUSCRIPT content was observed in the AES, mainly fructose (Table 1). Sugars have a high solubility in water. However, this property decreases in the presence of other solvents including ethanol.

3.2.

Solubility and moisture content of films

Solubility and moisture content are important films and coating properties because they affect

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the capacity of water-resistant properties and in the case of edible films solubility in water affects the usage of the film [31]. For example, films on high-moisture foods must be insoluble, while edible coatings must be readily soluble. Results of these parameters of MC films with AES and EEFD are shown in Table 2. Methylcellulose films without any added

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extract exhibited moisture content close to 10.3%. Moisture content of films increased with a higher extracts concentration, and when films were immersed in water at room temperature,

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samples underwent swelling and matrix dissolution. MC films showed a lower water content and high solubility values in present study (93.8 % soluble matter). This was explained by the

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higher hydrophilic character of methylcellulose films at room temperature and it can be associated to the higher water binding capacity of this polymer [14]. Incorporation of extracts

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into the polymeric matrix promoted an increase in the average water content of MC films

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without affecting solubility, which can be explained by interactions between these hydrocolloids with polyphenols. According to Mathew et al. (2006) the high values of

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swelling degree and water solubility of the films added with extracts rich in bioactive compounds may be attributed to the hydrophilic groups of polyphenols that can easily interact with water molecules. The addition of extracts elevated the capacity of the matrix to bind with water and thus improved its hydrophily. A similar effect was observed by Sun et al. [31] in chitosan films with added thinned young apple polyphenols.

3.3 Analysis of thermal and mechanical properties

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ACCEPTED MANUSCRIPT Films with added EEFD and AES extracts showed a shift to lower Tg obtained by DSC when compared with the control. Concentration of extract analyzed showed a significant effect on Tg values (Table 3). In MC-AES films, there was a major decrease of Tg values as the content of extract increased in films tested when it was compared with films without added extract. Glass transition

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indicates the passage of a metastable thermodynamic state, characterized by high molecular order and aggregation, to another thermodynamic state, where there are more molecular mobility and thus lower stability [32]. According to Antoniou et al. [33], an increase in mobility of polymer chains by incorporating plasticizers (water, sugars) presented in extracts

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of apple skin, causes a decrease of intermolecular forces among them and consequently reduces Tg.

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Figure 1 shows Tan δ curves for MC with and without AES films after drying process. Curves of MC-AES films showed two relaxations, β, and α. Relaxation β around -50°C is

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characteristic of the polysaccharides presence. Other authors consider this relaxation β in hydrophilic materials as a typical water relaxation favored by hydroxyl groups presence [34].

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The second peak or the maximum of the Tan δ curve corresponds to relaxation α, which is

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attributed to Tg. The addition of AES in samples had a powerful plasticizing effect. This is observed due to shift of the Tg toward lower values. This also can be related to increased

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moisture content in samples having a higher AES content. As the AES content increases, β relaxation undergoes a shift to higher temperatures and a better definition too. Other authors have found similar behavior in MC films with added sugars as plasticizers [35]. The model (Eq. (2)) used to estimate the elastic modulus from true stress – true strain curves satisfactorily fitted the experimental data (R2 > 0.99) for all films tested. Table 3 exhibits the elastic modulus and the percentage of elongation obtained. It was observed that the value of the elastic modulus decreased with increasing AES concentration, while elongation increased.

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ACCEPTED MANUSCRIPT The addition of AES might facilitate macromolecular mobility, increasing the elongation and the formation of a more effective coating. Concentrations of 10 and 25% show a major elongation when compared with films without extract (MC), However, the higher AES content (25%) in films resulted in samples with a very sticky behavior. For this reason, this treatment was discarded. These findings are consistent with those reported by Tavera-Quiroz

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et al. [14], who observed that 1% addition of plasticizer (sorbitol) increased elongation percentage and diminished elastic modulus values of MC films dramatically. They consider that, in MC films, plasticizers interfere with hydrogen bonds among different hydroxyl groups with increasing sorbitol concentrations, similar to those obtained in films with AES and

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EEFD. This could be indicative of the plasticizer saturation of the network. Table 3 shows

as well as Tg followed a similar trend.

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that mechanical and thermal properties are strongly related, since both elongation percentage

Mechanical properties of films formulated with EEFD were less affected by increase in

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extract concentration due to absence of plasticizing effect of sugars present in AES. According to Sablani et al. [36] values of elastic modulus in films based on starch from apple

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skin decreased as the concentration of glycerol increased, demonstrating a lubricant effect

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from the addition of glycerol in films. However, this effect was not reflected in the films mechanical properties with different concentrations of glycerol. Another authors obtained

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similar results in films derived of cellulose with incorporation of natural compounds like Lippia alba extract, resveratrol or α-tocopherol [37], [15].

3.4. Water vapor permeability The shelf life of multicomponent food systems depends, among other things, on how fast the water transfer between components takes place. To control this migration, several principles

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ACCEPTED MANUSCRIPT can be utilized, including adding edible layers between components [38]. Edible coatings can be used to control moisture migration in foods when these have a low WVP. EEFD addition of in films produces a WVP increase between 10 and 20% when compared with control (Table 3). Whereas, when AES was used in films formulation, permeability values had a more considerable increase (55 and 80 % for AES concentrations of 10 and 20%,

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respectively). Permeability is influenced the hydrophobic or hydrophilic nature of the material, by the presence of voids or cracks, and by stearic hindrance and tortuosity in the structure. Sugar presence in AES increases plasticization effects. This was probably due to increase of the polymer chains mobility and the void spaces between polymer chains. [39].

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These results suggest that high aqueous apple skin extract (AES) content in methylcellulose

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films is not very suitable for applications requiring an excellent barrier to water vapor.

3.5. FTIR spectra

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FTIR spectroscopy technique was used to observe functional groups and interactions among methylcellulose and compounds present in extracts once films were obtained. When two

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components are mixed, chemical interactions are reflected by changes in the bands of the

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characteristic spectra [40].

FTIR spectra of MC and MC-AES presented very similar characteristic peaks due to their

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chemical structures (Figure 2 a, b). Except in the range of 1800-1600 cm-1 for the samples with different extract contents where the appearance of a new band around 1724 cm -1 was observed (Fig. 2 b). This band is characteristic of carbonyl groups such as carboxylic acids and esters, such as citric acid [41]. Xie and Liu [42] suggests that when citric acid was heated, it dehydrated to yield an anhydride, which could react with starch to form a starch-citrate derivative. The MC structure is similar to starch and is made up of linear chains with -(1 → 4) bond with one free –OH and two free –OCH3 groups per unit. The amide-I peak appears at

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ACCEPTED MANUSCRIPT 1630 cm-1 for control films and films with different AES content, corresponding to C–O, stretching from the glucose of the cellulose. In MC-AES films, this band showed lower absorbance values. The region of 1500-1200 cm-1 -CH groups are mostly absorbed, and vibrations of -COC and -OH groups predominate from 1200-900 cm1 (Fig. 2 a). The methylcellulose films strongly absorb in this region due to presence of the glycosidic bond (-

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COC). It was observed that for MC-AES films, the addition of the aqueous apple extract had an important effect. It was possible to observe a new peak at 1120 cm−1 and 1040 cm-1 and between the region of 800- 720 cm-1 (ring stretching). These peaks were attributed to the stretching vibration of C-O in C-O-H groups (1120 cm-1) and the stretching vibration of C-O

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in C-O-C groups (1040 cm-1) [43]. A significant number of bands in films with extracts suggests presence of sugars and bioactive compounds in the samples. The spectra obtained by

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FTIR for films with EEFD show the similar spectra for MC-AES films.

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3.6. Total polyphenols content, antioxidant capacity analysis, and ascorbic acid in films Antioxidant capacity of a bioproduct could be explained by inhibition percentage of the

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DPPH radical for samples with bioactive or polyphenols in its composition. In case of films

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formulated with AES and EEFD, DPPH radical scavenging activity was observed. Inhibition percentage and total polyphenols were similar in the analyzed samples (Table 4). Aqueous

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extract (AES) had a high percentage of inhibition and polyphenols content, however, addition of aqueous extracts in MC films formulations and, the agitation, drying and oxidation processes during formation of the films polymer network generated a decrease in content of total polyphenols and therefore in I %. When films with added EEFD were evaluated, results obtained suggested a dominant I % compared with films with AES (about 20% in all cases). Polyphenols content in EEFD films was about 9.0 mg of chlorogenic acid per film gram (Table 4).

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ACCEPTED MANUSCRIPT Similarly, López De Dicastillo et al. [16] obtained methylcellulose films with high antioxidant capacity by adding ethanolic extracts of murta fruit. Espitia et al. [44] found a relationship between a higher polyphenols content with significant antimicrobial activity in edible films formulated with açaí and apple skin polyphenols. The antioxidant activity observed on apple components is due to a variety of phytochemicals in apples, such as

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quercetin, catechin, phloridzin, and chlorogenic acid, with strong antioxidant and antimicrobial activities [45].

AA content in AES was 30.3 ± 3.75 mgAA/mlextract, while for films with a major content of AES 0.16 ± 0.01 mgAA/mgfilm was obtained. Films formulated from 20% EEDF, showed an

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AA content close to films with AES (0.14 ± 0.01 mgAA/mgfilm). Ascorbic acid is an essential antioxidant in the human diet and is widely used for supplementation [28]. It is known the

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biological activity of vitamin C in humans, and like most nutrients it must be preserved during operations and processes involved in different conservation methods. This micronutrient is

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very sensitive to various forms of degradation such as with temperature, salt and sugar concentration, pH, oxygen, enzymes presence, metal catalysts and its initial concentration. All

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of these factors are related to process techniques and composition of the product being

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processed. In some cases drying and different forms of dehydration cases, nutrient degradation rate is associated with water removal rate, and this, in turn, depends on the

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product processing temperature [46]. For healthy men and women, and in order to promote additional health benefits, a daily intake of vitamin C greater than 100 mg is recommended [47]. Consuming five servings of fruits and vegetables daily would bring about 200 mg. Some multivitamin supplements provide about 60 mg of vitamin C. The results show that 1 gram of film with 20% AES would contain 160 mg AA and 1 gram of film with EEFD would contain 140 mg AA.

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Conclusions

Active and functional ingredients were obtained from green apple skin with antioxidant properties and as a source of polyphenols to be added in hydrocolloid matrices and used in possible food applications. Methylcellulose films with addition of green apple skin extracts turned out to be transparent systems, and their properties depended on the type and the extract

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content. Aqueous apple skin extract (AES) did not have a high content of polyphenols, compared with ethanolic extract obtained by freeze-drying. Nonetheless, it was a simple extraction method and low cost. AES incorporation in films had a predominantly plasticizing effect, observed in the properties associated with water barrier properties, thermal and

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mechanical properties. This is due to sugars presence, increasing the free volume of the polymer network and therefore its flexibility. Edible films formulation with ethanolic extracts

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of freeze-dried skin is an effective alternative to obtain an active material with good barrier and mechanical properties, without providing additional plasticizers to the matrix generating

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shifts of the material glass transition temperature and deteriorating mechanical properties. Edible films formulation with active ingredients becomes a strategy to use agro-industrial

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byproducts providing nutrients and allowing the development of new coating types for use in

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functional foods.

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Acknowledgments

Authors acknowledge Marina Urriza, Claudio Reyes and Javier Lecot for technical assistance. This work was supported by the Argentinean Agency for the Scientific and Technological Promotion (ANPCyT) (Projects PICT/2011/0226 440 and PICT/2012/0415) and the Argentinean National Research Council (CONICET) (PIP 2012-2014114-201101-00024).

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ACCEPTED MANUSCRIPT [27] G. Giomaro et al., “Polyphenols profile and antioxidant activity of skin and pulp of a rare apple from Marche region (Italy),” Chem. Cent. J., vol. 8, no. 1, pp. 1–10, 2014. [28] K. Pallauf et al., “Vitamin C and lifespan in model organisms,” Food Chem. Toxicol., vol. 58, pp. 255–263, 2013. [29] Y. Lu and L. Yeap Foo, “Antioxidant and radical scavenging activities of polyphenols

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ACCEPTED MANUSCRIPT vol. 3, pp. 1–15, 2004. [46] M. R. Ochoa, A. G. Kesseler, B. N. Pirone, C. A. Márquez, and A. De Michelis, “Shrinkage during convective drying of whole rose hip (Rosa Rubiginosa L.) fruits,” LWT - Food Sci. Technol., vol. 35, no. 5, pp. 400–406, 2002. [47] Quest, A.F.G. & Leyton, L. (2012). Vitamin C. Information of Micronutrients Center. Pauling

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. Tan δ curves for MC films without and with AES after drying process. Figure 2. FTIR spectra of the MC films without and with AES and EEFD. ) MC; (

) MC-10% AEP; (

(

) MC-20% EEDF; (

) MC-20% AEP; (

) MC-10% EEDF;

) MC-25% EEFD

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(

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ACCEPTED MANUSCRIPT Table 1. Characterization of obtained extracts

Extract

Polyphenols (mg chlorogenic acid/100g ds)

DPPH

Ascorbic acid (mgAA/mlextract)

Sugar (mg ss/ml extract)

AES

131,3a

78.0a

30.3b

101.6b

EEFD

850,0b

83.0b

0.34a

44.2a

(I, %)

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Different letters indicate significant differences between samples (P < 0.05).

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Table 2. Moisture and solubility values of films Sample

Extract

Moisture

Solubility

concentration

(%)

(%)

0

10.3 (0.5) a

93.8 (3.7) a

10

23.9 (0.4) b

97.3 (0.3) a

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26.4 (0.8) c

25

27.7 (0.9) c

10

24.0 (0.1) b

20

30.8 (0.6) d

25

33.9 (2.2) e

EEDP

AES

96.6 (0.4) a 97.0 (0.6) a 90.1 (9,9) a 100 (0.0) a

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control

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(%)

96.7 (4.4) a

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Different letters indicate significant differences between samples (P < 0.05).

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Table 3. Mechanical and thermal properties and water vapor permeability (WVP) values of films

AES

Elongation

Glass transition

WVP x1011

concentration

modulus

(%)

temperature

(g Pa-1m-1s-1)

(%)

(Ec, MPa)

0

3751.1(36.1)d

9.3(0.5)b

10

1787.5(124)c

2.9(0.3)a

171.5 (0.4)d

6.9 (0.3)c

20

1928.2(153)c

2.4(0.4)a

172.0 (0.1)d

6.3 (0.1)b

25

1867.2(71)c

2.2(0.1)a

165.0 (2.1)c

6.5 (0.2)b, c

10

338.0(18.1)b

21.9(0.8)c

160.5(0.3)b

8.7 (0.3)d

20

161.6(18.9)a,b

23.1(0.4)d

155.1(3.5)b

10.1 (1.2)e

25

95.2(7.3)a

41.6(0.3)e

150.4(0.4)a

--

(Tg, ºC) 190.1 (0.1)e

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EEFD

Elastic

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control

Extract

5.6 (0.5)a

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Sample

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Different letters indicate significant differences between samples (P < 0.05).

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Table 4. Inhibition percentage (%) and polyphenols content (mg

chlorogenic acid/g film)

in

films with AES

Sample

Extract

DPPH

Polyphenols content

concentration

(%I)

(mg chlorogenic acid/ g film)

10

14.3 (0.8)a

3.7 (0.3)a

20

13.8 (1.22)a

3.5(0.4)a

10

18.0 (1.5)a

8.6 (0.5) a

20

19.8 (2.0)a

25

21.0 (1.9)a

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EEDF

8.7 (0.4) a

9.9 (0.7) b

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AES

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(%)

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Different letters indicate significant differences between samples (P < 0.05).

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ACCEPTED MANUSCRIPT Highlights Active edible films with green apple bioactives were developed Extracts obtained from green apple peel contains high amount of polyphenols and antioxidants Edible films with ethanolic extracts of freeze-dried peel was developed like a good active

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material with good barrier and mechanical properties

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Figure 1

Figure 2