**1. Introduction**

Plastics are synthetic polymers derived from fossil fuels, which because of its production cost and versatility are one of the most popular food packaging materials [1,2]. Despite that, the non-biodegradable nature of these materials has generated serious pollution problems [3]. For these reasons, the interest on developing edible films and coating that can totally or partially replace the use of synthetic polymers derived from petroleum, has grown [4,5]. Edible films can be obtained from proteins, carbohydrates, glycoproteins, lipids or their mixtures [6,7]. One of the most widely used natural biopolymers with a great potential in the production of biodegradable materials is starch [1,8]. Starch is the main energy reserve of higher plants, a natural resource, renewable, abundant and low cost [4]. Starch films offer several advantages for use as food packaging, being colorless, odorless, tasteless, transparent, biodegradable, non-toxic and with low oxygen permeability [9]. On the other hand, compared with synthetic polymers, starch films are highly hydrophilic with poor mechanical properties [3,4]. It has been reported that the different properties of starch-based films vary with the botanical source [10–15]. These variations are mainly due to the amylose content in the native starches (18%–30%) [16]. The food industry is constantly looking for new sources of starches that may offer different or better functional properties [1,17]. Regarding to this, Stevenson et al. [18] evaluated the functional properties of starches isolated from immature apples of six cultivars, their results indicated that the amylose content was 26.0% to 29.3%. In this sense, apple starches may form films with acceptable properties (due to its amylose content) and comparable to those obtained from conventional sources such as corn and wheat. Apple is a climacteric fruit, whose ripening process is regulated by ethylene [19]. Within its growth and development, the fruit is characterized by significant concentrations of starch, which become almost zero at the time of commercial harvest [18,19]. One of the most common practices in apple orchards is the fruit thinning. The objective of this process is to achieve the development of high-quality fruits with regular yields and high blooming return [20,21]. Besides, it is important to mention that in order to obtain a good commercial quality harvest, it is required that only between 5% and 30% of the flowers end up as mature fruits [22]. When fruit thinning in apple orchards is done manually (hand-thinning), it is recommended to do it at 28 DAFB. However, late manual thinning (60 DAFB) is a common practice in the region [20], which may be extended up to 70 DAFB. Although its benefits are well known, this practice is usually delay due to the farmers fear, that the damages by frost in orchard whose fruits has been previously thinned, diminish the harvest yield. Apples removed during the fruit thinning are considered as a waste and in the best case, they are used as animal feed or as compost [21]. An alternative for the use of the fruits eliminated by this practice may be the isolation of their starch for the development of biodegradable films. The packaging plays an important role in the food industry, it must to contain and protect the food against external and internal factors. Biodegradable films can control the mass exchange between the food and the surrounding medium increasing the extension of shelf life and improving the quality of foods [23,24]. There is increasing interest in developing biodegradable packaging that can be used as active packaging [25]. According to these, starch-based films can develop activities as carriers and active substances-releasing agents (antioxidants, antimicrobials, flavorings, among others) in to the food matrix by diffusion process, which gives rise to the active films [26,27]. At present, there has been an increasing interest in incorporating antioxidants in starch-based films, mainly plants extracts, which are often characterized by their content of phenolic compounds [1,10,25,28,29]. Ellagic acid (EA) is a phenolic compound found in a wide variety of vegetables, usually in the form of ellagitannins (its precursors). EA is mainly found in fruits such as pomegranates, raspberries and blueberries [30], it shows antimicrobial and anticancer properties, besides inhibiting the formation and growth of tumors in animals [31]. Definitely, one of its most studied properties is its ability to capture free radicals [31–33], due to its high antioxidant capacity, since it is a stable molecule at high temperatures, which melting point is ~362 ◦C [34]. EA has previously been used to deal with melanoma cells in chitosan based-films, presenting anticancer activity at concentrations as low as 0.1% (*w*/*v*) [30]. It has also been applied on candelilla wax based coatings at a concentration of 0.01% (*w*/*v*) to minimize the changes of color and improve the texture of fresh-cut fruits [35] and to lengthen shelf life in avocados [36]. For all these reasons, it can be mentioned that the EA is a substance with varied biological activity, which has already been added to films for medical purposes; however, currently there are no reports on the incorporation on the properties of film formation in apple starches. The objective of this study

consisted in evaluating the effect of incorporating EA, on the physicochemical, mechanical, thermal and antioxidant properties of apple starch-based films.

#### **2. Materials and Methods**

#### *2.1. Sample and Reagents*

Starch was obtained from Golden Delicious Smothee apples harvested at 60, 70, 80 and 90 days after full bloom (DAFB) during the production cycle of 2013 in the orchard "La Campana" at Ciudad Cuauhtemoc, Chihuahua, Mexico. Starch extraction was performed by wet milling according to the method reported by Tirado-Gallegos et al. [17].

#### *2.2. Preparation of Coating Solutions and Films*

Coating solutions and films were made with the plate casting method using the methodology proposed by Mali et al. [37]. Different aqueous dispersions were prepared with each of the starches. Starch was mixed with distilled water until reaching a concentration of 4% (*w*/*w*). The dispersion was heated on a heating plate (model 6795-220, CORNING, Monterrey, NL, Mexico) and kept under constant stirring (350 rpm) with overhead stirrer IKA (model RW 20 digital, WERKE, Wilmington, NC, USA), the dispersion was held at 85–90 ◦C for 15 min. Subsequently, the starch dispersion was cooled to 70 ◦C and glycerol (2%, *w*/*w*) as plasticizer was added [38]. The heating was maintained 15 min at this temperature under the same conditions of agitation. The filmogenic solutions with EA had the same starch and glycerol concentration and were prepared under the same conditions. However, glycerol was mixed with EA at three concentrations (0.02%, 0.05% and 0.1%, *w*/*w*) based on total mass of film forming solution. This mixture was kept under constant stirring for 12 h before being added to the starch solution. Once the process is complete, the filmogenic solution (film-forming solution) was cooled to 60 ◦C and emptied immediately in polystyrene Petri dishes (Ø = 15 cm) using 40 g/box. Boxes with the filmogenic solutions were dried under laboratory condition (RH ≈ 45% ± 5% and 25 ± 1 ◦C) until the formed film was peeled off easily from the plate (≈ 72 h) [24,39]. Subsequently, the films were conditioned for 48 h in desiccators containing a saturated salt solution of NaBr (RH = 55% ± 5%). After the conditioning, the properties of the films were characterized. The films obtained in the first stage from starch of apples harvested at 60, 70, 80 and 90 DAFB were abbreviated as: FILM-60, FILM-70, FILM-80 and FILM-90, respectively. The films made in the second stage, were obtained from starch of harvested apples at 70 DAFB. In this case, the film without EA was appointed as FILM-Control, while the films with 0.02%, 0.05% and 0.1% (*w*/*w*) of EA were identified as FILM-EA0.02%, FILM-EA0.05% and FILM-EA0.1%, respectively.

#### *2.3. Films Characterization*

#### 2.3.1. Color

The films tri-stimulus color was evaluated with a Minolta colorimeter CR-300 (Minolta, Osaka, Japan) with a diffuse illumination/0◦ viewing geometry and a pulsed xenon arc lamp (PXA). Readings were recorded in the color space in accordance with the CIELAB (*L*\*, *a*\*, *b*\*) scale. Readings were taken in five random points on the surface of the films, using as background white standard used in the calibration of equipment. Moreover, the whiteness index (*WI*) of the films with EA and its control was measured according to the following equation [40]:

$$WI = 100 - \sqrt{(100 - L\*)^2 + a\*^2 + b\*^2} \tag{1}$$

2.3.2. Optical Properties using Ultraviolet–visible (UV-vis) Spectroscopy

The light barrier properties of the films evaluated as transparency was determined according to the methodology proposed by Luchese et al. [39]. Sample rectangles were cut and placed in the cell of a spectrophotometer (Evolution 300, Thermo Scientific, Waltham, MA, USA) and readings of the absorbance of the cell were made with the film at 600 nm taking the filmless cell as blank. Films transparency (*T*) was calculated according to the following equation:

$$Transpancy\ (T) = \frac{A\_{600}}{\varepsilon} \tag{2}$$

where *A*<sup>600</sup> is the absorbance of the cell with the film at 600 nm and *e* is the film's thickness. According to the above, high absorbance values mean less transparency in the films. In the case of the films with EA, a sweep of the absorbance from 200 to 800 nm was performed. Five replicates were performed.

#### 2.3.3. Scanning Electron Microscopy

The surface morphology of the films was evaluated by scanning electron microscope JEOL (JEE400, Tokyo, Japan). The film sample was adhered to the sample holder and covered with gold to make it conductive. Finally, micrographs were taken at acceleration potential of 5 kV and a current intensity of 2 mA.

#### 2.3.4. Thickness Measurement

The film thickness (*e*) was measured using a digital micrometer Mitutoyo (model Coolant Proof 293-348, Kanagawa, Japan) with an accuracy of 0.001 mm, the thickness was measured at 10 points randomly designated on the films.

#### 2.3.5. Moisture Content

The moisture content was determined using the standard method of the International Association of Official Analytical Chemistry (AOAC) [41]. Samples of 0.5 g was weighed and dried in an oven Shel Lab (model 1370GM-2, Sheblon Manufacturing, Inc., Cornelius, OR, USA) at 105 ◦C to constant weight with a variation of 0.0001 g (≈ 2.5 h). This analysis was carried out five times and the moisture content was expressed as percentage of water in the film according to following equation:

$$Moisture\left(\%\right) = \frac{W\_{\bar{i}} - W\_{f}}{W\_{\bar{i}}} \times 100\tag{3}$$

where, *Wi* is the initial weight of the humid film and *Wf* is the final weight of the dry film.
