Exploiting films based on pectin extracted from yellow mombin (Spondias mombin L.) peel for active food packaging

In this study, the potential of yellow mombin (Spondias mombin L.)–extracted pectin as a film-forming matrix for the elaboration of edible coatings was investigated. The films of chitosan, citric pectin, and carboxymethylcellulose (Sigma/USA) and natural pectin were prepared by casting. The film morphological characterization was performed by scanning electron microscopy (SME), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and atomic force microscopy (AFM). Additionally, total phenolic compounds (TPC), total antioxidant activity (TAC), DPPH scavenging capacity, and antimicrobial activity were assayed for the natural pectin–based films. The results showed that the film based on natural pectin presented a 46% degree of esterification that was higher than commercial (34%), showing its capacity of forming gels. The film formed by chitosan presented a very different behavior for loss of mass during TGA when compared to the other films, occurring a mass loss completely at 647.7 °C in 61 min. The value obtained from the concentration of total phenolics (mg GAE/100 g dried) found in the formulated film based on pectin was 3998.99 ± 42.64 and with solubility in water of almost 100%. Additionally, the scavenging of the DPPH radical with a value of 15.48 ± 1.48 μmol TE/g was found on the film based on natural pectin thus showing antioxidant capacity. Finally, this film showed less significant mechanical properties when compared to other films and a good pronounced antimicrobial effect against the gram-negative bacteria tested. Therefore, it could be potentially used for producing food-active packages.


Introduction
Over the last years, the food industry has turned its attention to the development of biomolecule-based edible films as an option for reducing the use of chemical matrices and enhancing packaging properties [25,64]. Edible films are thin coating layers made from safe-to-eat macromolecules, which can be used as primary packaging alternative in order to increase products' shelf life, control respiration, oxygen, and moisture exchange rates, or perform active functions on the product, by the inclusion of different compounds [29,33,47]. Several biopolymers have been considered for the production of such coating materials. Cellulose (bacterial, micro-, and nano-fibrillated), starch, chitosan, protein isolates, gums, and pectin are among the matrices reported in the literature [65,73].
Pectin is one of the major structural polysaccharides found on plant cell walls, and its versatility as an edible, environmentalfriendly and as a biodegradable film-forming agent has been Highlights • Potential of yellow mombin (Spondias mombin L.) extracted pectin as a film-forming matrix for the elaboration of edible coatings • Film based on natural pectin presented a 46% degree of esterification showing a good capacity of forming gel.
• Film showed mechanical properties and antimicrobial effect against the gram-negative bacteria • Film could be potentially used for producing food-active packages highlighted by recent studies [23,64]. Moreover, the biocompatible structure of pectin-based coatings allows the incorporation of several bioactive molecules, such as flavors and antimicrobial and antioxidant compounds that enhance the matrix functional features, already given by pectin itself [21,52]. Citrus fruit peels and apple pomace are known sources of pectin for food applications [69]. In this context, the use of fruit-processing by-products for the extraction of natural pectin appears as an alternative for dealing with the increasing accumulation of agro-industrial residues, as well as for developing added-value outputs from lowcost feedstock [11,71].
Yellow mombin (Spondias mombin L.), or cajá, as it is known in Brazil, is a tropical-native fruit known by its typical dark-yellow skin and sweet flavor pulp. It is commonly found in Northeast Brazil, where it plays a significant role as an income source for small local producers and as raw material for the juicing industry [20,30]. Recent reports have shown that, beyond economical applications, cajá is bioactive-rich, and its flavonoids, carotenoids, vitamins, triterpenes, and total phenolic content can be associated to several biological applications, such as anti-inflammatory, antimicrobial, hypoglycemic, and antioxidant activities [14,60,62].
Therefore, the aim of this study is to evaluate the potential of cajá-extracted pectin as a film-forming matrix for the elaboration of edible coatings. Thus, the casting technique was used to produce edible films using natural pectin from yellow mombin fruit, synthetic citric pectin, carboxymethylcellulose, and chitosan. To the best of our knowledge, this is the first report on the literature regarding such use of fruit-extracted pectin and this kind of performance comparison. Morphological, bioactive, antimicrobial, and physicochemical characterizations were carried in order to evaluate the behavior of the different matrices.

Agro-industrial residues
The cajá residue (Spondias mombin L.) was supplied by Sterbom, a fruit-processing company located in Parnamirim (RN/Brazil). The residue was washed five times with water (2 L of water/residue kg) and submitted to mechanical stirring for 10 min in each washing cycle. Then, they were put to dry in a greenhouse with air circulation of 60°C for 48 h. After drying, the cleansed residues were grinded in a grinding mill (Tecnal TE-680 Type Willye) and sieved (20 mesh) being stored in closed vessels at room temperature.

Cajá pectin extraction
The proceedings to extract the pectin were adapted according to the method used by McCready and McComb [48] and by Kratchanova [37]. Approximately 2.0 g of the sample was deposited in an Erlenmeyer flask of 250 mL. After that, a solution of 1:70 (m/v) with distilled water was prepared, and the mixture's pH was adjusted to 2.5 through the addition of 1.0 M citric acid. The acidified mixture was submitted to a 650-rpm stirring under a temperature of 80°C. After 120 min, the extraction was finished, and the extract purification stage was started to obtain the pectin.
The extract, still hot, was centrifuged for 10 min at 11,200g and the volume of the supernatant measured in a test tube. Then, the supernatant was transferred to a 500-mL beaker and submitted to stirring. The ethanol 99% (Nuclear) was added in the ratio of 1:2 v/v (supernatant/ethanol) to the supernatant. The mixture rested for an hour to guarantee the separation of the pectin and the ethanol. The pectin was separated through vacuum filtration, by using a quantitative filter paper, washed twice and dried in a forced air greenhouse heater at 50°C to constant weight. The first cleanse was done with ethanol 70% (Nuclear) and the second with ethanol 95% (Nuclear).

Film preparation
In order to prepare the films, a technique called "casting" was used, in which biopolymers were dissolved in acetic acid and water and mixed in a vessel. The synthetic chitosan (Aldrich/ USA)-based film at 2.0% (m/v) was solubilized in 1.0% acetic acid (v/v), with 0.5% (m/m) of glycerol. On the other hand, the citric pectin-based films (Vetec/Brazil), carboxymethylcellulose (Sigma/USA), and natural pectin-based films at 2.0% (m/v) were solubilized in water, together with 0.5% (m/m) of glycerol. The solutions were submitted to magnetic stirring at 45°C for 2 h and, afterwards, the solution's vacuum filtration took place in order to remove the insoluble material.
The filmogenic solution was submitted to the evaporation of the specific solvent according to the type of the used polymer, wrapped in polypropylene plates, and dried in an air circulation greenhouse at 50°C for 24 h. After the solvent evaporation, a thin layer of each film was produced and stored in a desiccator to a further characterization, as described on the following sections.

Scanning electron microscopy
The film samples were analyzed on the scanning electron microscope (Phillips XL -30ESEM, USA) by using a 15-kV electron beam. The electronic micrographics were taken with magnifications of 150 and 800 times.

X-ray diffraction
The films' crystallinity was evaluated through XRD spectroscopy (XRD-6000, Shimadzu, Japan), Cu-kα radiation, 30.0 kV-tension, and 15 mA-electrical current, with a rate of 2.0 degrees per minute to a 2θ-continuous scan with an interval of 4.0-70.0°.

Analysis of the structural links through Fourier transform infrared spectroscopy
The FTIR analysis was used to detect the presence of the main constituent groups of films. The analysis of the FTIR used spectral range from 400 to 4000 cm −1 , achieved by using the equipment Shimadzu IR Tracer 100 (Perkin Elmer, EUA).
In addition, on the film based on the pectin extracted from cajá peel, the FTIR was used for the determination of the degree of esterification (DE) of the pectin, calculated according to the methodology proposed by Fellah [26], using Eq. (1): In Eq. (1), ECG refers to esterified carboxyl groups and TEC to total of esterified carboxyl groups and carboxylate groups.

Thermogravimetric analysis
The film samples were carried out through thermogravimetric analysis (TGA) and differential thermal analysis (DTA) using an equipment from Shimadzu (Model DTG -60). The samples were placed on an aluminum stand with nitrogen flow of 50 mL/min and heating ratio of 10°C/min until 800°C. The initial temperature of the natural pectin, synthetic pectin, CMC, and chitosan were, respectively, 28.8, 27.9, 29.2and 35.7°C. The masses were of 2.0, 4.0, 3.0, and 2.0 mg, following this order.

Atomic force microscopy
The morphological surface of the films was analyzed by using an atomic force microscope of the Shimadzu brand, Model SPM-9700. The analysis was performed on the dynamic mode (tapping mode) with a scan speed of 1.0 Hz and with the resolutions of X, Y, with 0.2 nm and Z with 0.01 nm with a piezoelectric tube component.

Evaluation of films' tensile strength
The mechanic properties of the films were determined in triplicate through tensile tests by using a dynamometer (Tenso Lab Automatic 3000 Mesdan). The films were cut in strips of 30 mm of width and 60 mm of length, and each extremity was fixed in a clamp appropriated to the test. As an experiment execution parameter, an initial distance between the clamps and a speed of 10 mm/min were adopted. The samples were pulled until a total rupture happened. The tension and deformation on the rupture were determined directly on the tension versus deformation curve.

Humidity and hygroscopicity determination
The humidity determination was carried out according to the NREL -Determination of Total Solids in Biomass protocol [68].
The samples' hygroscopicity was expressed through the water absorbed by the film samples (0.2 g) stored for 7 days in a desiccator with a NaCl solution and relative humidity of 75 In Eq. (2), a is the mass of the empty plate (g), b is the mass of the plate plus the mass of the initial sample (g), and c is the mass of the plate (g) plus the mass of the final sample.

Solubility
The sample solubility was determined according to Hosseini et al. [34]. Samples with 1 × 3 cm 2 dimensions were weighted and diluted in milliliters of distilled water with constant stirring for 6 h. The solutions were, then, filtered, and what remained of the films was dried in a greenhouse at 105°C to constant weight [34]. The solubility in water was determined by using Eq. (3).
In Eq. (3), A 0 is the film initial dried mass (g) and A 1 is the film final mass (g).

Evaluation of the total phenolic compounds
The quantity of total phenolic compounds (TPC) was determined by using the Folin-Ciocalteau method adapted by Fujita et al. [27]. The samples' absorbance was measured at 750 nm and the results expressed in milligrams of gallic acid equivalent per 100 g of dried sample (mg GAE/100 g dried film), according to the calibration curve using a concentration range of gallic acid from 10 to 200 μg/mL.

Total antioxidant activity test
The total antioxidant activity (TAC) was determined by the phosphomolybdenum method with modifications [38]. For this purpose, 100 μL of each sample was added to test tubes containing 100 μL of ammonium molybdate (40 mM), 100 μL of sodium phosphate (280 mM), and 700 μL of distilled water. Then, the tubes were closed and the samples incubated in a greenhouse at 100°C for 90 min, and, after being cooled, the absorbance was recorded by using a spectrophotometer at 695 nm. In order to express the results in milligrams of ascorbic acid equivalent per gram of sample (mg AA/g), a standard curve was used by using ascorbic acid concentrations in a range of 25 to 250 mg/mL.

Evaluation of the antimicrobial activity
Strains that are standards of the gram-positive bacteria Staphylococcus aureus ATCC 25923 and of the Pseudomonas aeruginosa ATCC 10145 were tested. The inoculum was prepared by taking three to four colonies of the strain, isolated in Muller-Hinton agar, and diluted in saline solution at 0.85% until reaching turbidity correspondent with the 0.5 tube of the Mac-Farland standard [44].
To the evaluation of the antimicrobial activity, the determination of the minimum inhibitory concentration (MIC) was carried out through the broth microdilution technique [6,32]. The tests were performed in Muller-Hinton broth contained in microtiter 96-well plates. An aliquot of 10 mL of each solution at a concentration of 0.1 mg/mL was added in each well of the plate containing Muller-Hinton broth and microorganisms to a final volume of 200 mL by well. The solution control was carried out on the Muller-Hinton broth of the microorganism suspensions. The plates were covered with a plastic film and incubated at 35°C for 24 h. The reading was performed on an ELISA reader at the wavelength of 650 nm [32]. The smallest solution concentration able to inhibit the microbial growth was considered the MIC.

Results and discussions
3.1 SEM of the synthetic films (citric pectin, CMC, and chitosan) and to the natural film based on pectin The scanning electron microscopy (SEM) analysis was used after the construction of the films as an alternative to correlate the physicochemical properties with the morphologic structure, detecting possible imperfection on the matrix and the presence of pores on the films. The micrographics of the films' surfaces are shown in the Fig. 1.
The analysis related to the tested magnifications (150 and 800 times) to each type of film was possible to observe the existing differences in each film, which showed themselves as homogeneous and smooth, except the film based on natural pectin. Figure 1a and b related to the magnifications of 150 and 800 times to the natural pectin-based film reveal the presence of pores on the matrix. Padua and Wang [56] affirmed that when produced through the casting technique, the films present varied forms, different from the drying process, causing specific morphologic characteristics between the two sides: the superior part that is in contact with the air on the contact surface is homogeneous and bright, with a smoother structure without a defined characteristic; and the inferior part in contact with the polystyrene plate presents an opaque and irregular distribution, with globular deposits present on the structure.
When the drying process takes place, the side that is on the contact surface is more homogeneous and brighter, resulting in a smoother microstructure without a defined characteristic. On the other hand, the side that is in contact with the air is more irregular and opaquer, with a microstructure presenting globular deposits. According to Monterrey-Quintero and Do Amaral Sobral [49], concerning to the nature of these globular deposits, there is a hypothesis that these black points were microbubbles encrusted on the matrix or spaces occupied by the glycerol before the drying stage [49].
The synthetic pectin-based film showed a smooth surface, with less porosity and a well-homogenized material as shown in Fig. 1c and d. The same behavior was observed to the CMC-based film, as seen in Fig. 1e and f. Additionally, the chitosan-based film presented a structure with precipitates, with an opaque and whitish aspect as observed in the Fig. 1g and h. One of the reasons is the solubilization of the chitosan to the formation of the film, which was not so effective.
3.2 XRD of the synthetic films (commercial citric pectin, CMC, and chitosan) and of the natural film based on pectin The analysis of the X-ray dispersion is important to understand the amorphous nature and crystallinity of the studied material. The diffractogram of the film based on the pectin extracted from the cajá peel revealed a peak with an overlapping (Fig. 2). At 15°, it is possible to see the shoulder that receives this nomenclature since it was not quite intense as the peak, and, in 23°, a relatively expressive peak is observed, revealing a structure of a slightly crystalline polymer. Andrade et al. [5] investigated the use of the commercial pectin. When compared to this study, it is possible to notice a difference on the intensity on the peak at 23°that indicates that the film of pectin extracted from the cajá peel has a more crystalline material when compared to the commercial pectin, and this may be justified by the presence of cellulose at the cajá peel and, consequently, in its extract.
In Fig. 2, considering the curve that is the result of the CMC-based film analysis, two peaks of X-ray diffraction can be observed. Both on the CMC and on natural film based in pectin, an extended peak of 16.9°and another one on the 23.6°angle were noted. These peaks of X-ray diffraction are a characteristic of cellulose I [59,66]. These results show that the microcrystalline cellulose did not have its crystalline structure modified; only the cellulose swelling in water have been formed into inter-crystalline, in which the swelling agent enters the amorphous regions of the cellulose with the crystalline regions not being modified [7].
According to the diffractogram of the film based on chitosan, an only broadband found around 15-30°could be observed, with a maximum peak of 2θ = 23°, a typical behavior of semi-crystalline polymers. This result is compatible with the diffractograms obtained by Luo et al. [42]. The presence of a broadband indicates that the chitosan polymeric basis Fig. 2 Diffractograms of the microstructural characterization to the natural film based on pectin, the film based on commercial citrus pectin, the film based on CMC, and the film based on chitosan followed by the N-glycosidics that link the monomer impedes these structures to have a crystalline ordering in long interatomic distances. The chitosan presents a lack of ordering of the N-terminal of its structure contributing to its amorphous character, the hydrogen bridges act as secondary bonds causing changes on the angle of bonding among the chitosan molecule [39].

Films' FTIR analysis
The FTIR specter of the films studied are shown in the Fig. 3. It can be seen the existence of bands of the groups O-H between 3100 and 3500 cm −1 . The second band, centered around 2900 cm −1 , corresponds to the vibrations of the C-H bond. The absorption bands between 1100 and 1200 cm −1 are associated to the C-O-C bond and the C-C bonds [35].
The pectin behavior depends both on the quantity of ionic groups bonded to it and on its distribution throughout the main chain. Thus, it is important to determine the degree of esterification (DE) of the pectin in order to predict its behavior in the solution [43].
The DE determination of the sample of natural and industrial pectin film through the integration of the bands of the esterified (COOR) and free (COO─) carboxylic groups shown on the infrared specter (FTIR) in Fig. 4 is in accordance with Fellah et al. [26]. In the region between 1600 and 1800 cm −1 are the characteristic bands of the pectin, i.e., between 1790 and 1730 cm −1 associated to the esterified carboxylic groups (-COOCH 3 ) and the bands between 1660 and 1590 cm −1 that are associated to the free carboxylic groups (-COOH).
The sample of the film based on natural pectin extracted from the cajá's peel presented a degree of esterification (DE) equal to 46%, while for the commercial citric pectin was 34%; thus, both are classified as low-methoxylation pectin (LM). According to Turquois et al. [72], the degree of methoxylation of 50% is used as a parameter of reference, since pectins are, commercially, classified as high-methoxy (HM) when they contain over 50% of their carboxylic groups esterified and low-methoxy (LM) when values equal or under 50% of these groups are esterified [72]. It should be highlighted that de Souza et al. [70] found, by extracting the pectin with organic acid, a degree of esterification (DE) of 41% that is quite near for the results shown in the present study then indicating a satisfactory DE value. Compared to the industrial pectin, the natural one has a higher degree of esterification; thus, it is capable of forming gel more easily.

Thermogravimetric analysis
The thermoanalytical techniques are based on analysis of a physical property that varies according to time or temperature. Such properties can be mass, enthalpy, etc. Thermogravimetric analysis (TGA) is a technique used to monitor the mass variation of a substance according to its temperature, while DTA corresponds to its first derivative [16].
In the present study, a thermogravimetric analysis of the four films produced using CMC, chitosan, and commercial and natural pectin was performed. Figure 5 shows the mass behavior of each polymer when submitted to a temperature of up to 800°C.
As can be seen in Fig. 5a, the initial mass of the natural pectin is of approximately 2.0 mg, which decreases almost linearly with time and with temperature increase (red curve). At 6.0 min (temperature of 81°C), its mass was practically zero. The behavior of the commercial pectin (Fig. 5b) was very similar to the natural pectin. At a temperature of 94.6°C , the mass decomposes itself almost totally in a time of 7.3 min. According to Fig. 5c, the CMC had lost almost all its mass when a temperature of 94°C was reached, at 7.1 min. On the other hand, the film formed by chitosan (Fig. 5d) On the first stage, there is a mass loss of 8.2% due to water loss. Next, 4.6% of the mass is lost. On the third stage, 39.8 and 36.6% of the mass is decomposed because of the polysaccharide structure break. Kavianinia et al. [36] found results similar to the ones presented on this study concerned to the first stage. The increase of thermo-stability is associated to the compound crystallization, requiring more energy to break it [4].

Analysis of the films by atomic force microscopy
The understanding of the microstructures of a film is necessary in order to understand its rheological properties. In addition, these rheological properties are sensitive to molecular structure variations, which are responsible for the relations' function-structure to the polysaccharide solution systems [75].
The high viscosity of a film may, for example, form a solution not so homogeneous, and if there is the formation of air bubbles, it makes it difficult to remove them [17]. Therefore, the films forming solutions with low viscosity are more attractive [50]. In order to form a layer over a solid surface and the viscosity of the filmogenic solution of the film, the formation of depressions generated by gravity should be avoided, and the uniformity of capillarity on the film should be allowed [58].
A substantial rugosity rise can be observed on the film based on natural pectin (Fig. 6a, b), when compared to the film based on commercial pectin (Fig. 6c, d). One of the justifications can be the degree of esterification: the film based on natural pectin had a DE of 46%, while the film based on synthetic pectin had a DE of 34%. The pectin varies considerably in its capacity of forming gels, the source from which it was extracted, the differences of the polygalacturonic acid chain size, and the degree of esterification of its carboxylic groups. The extraction proceeding, the location of the pectin on the plant tissue, and the presence of 15 neutral sugars are responsible for its final characteristics [10].
The sample of the film based in the CMC (Fig. 6e, f) presented a surface without rugosities, generating a more homogeneous morphology. The sample of the film based on chitosan (Fig. 6g, h), after the overlaying, had a change on its rugosity and on the distribution of the filmogenic solution, being the low solubilization of the chitosan one of the reasons for it, then taking place the formation of gelatinized granules of chitosan.

Determination of the film's tensile strength
The tensile strength and the elongation are mechanical properties presented by a film. The tensile strength is the maximum tension supported by the film until its rupture. The elongation is the measure of the film malleability. It is a characteristic that the film presents by deforming itself before its rupture. Low elongation values implicate in brittle films [45].
The tension properties are responsible for the material resistance when a tension is applied causing deformation by stretching. The characteristic curve of tension versus deformation of flexible films shows that, at the beginning, the material offers tensile strength, provoking stretching. From the right point, the rise of strength reduces to a same deformation rise until the pour point, from which it is possible to stretch the film without a response of the strength rise. With the rising stretching, the material resists until its rupture takes place [46].
As shown in the Fig. 7, the film based on chitosan presented a higher value of tensile strength when compared to the others, with an average of 24.2 N, while the film based on CMC presented an average of 12.8 N. For the natural pectinbased film, the difference was less evident, with an average of 10.1 N. And, lastly, the synthetic pectin-based film presented the lowest strength value, with an average of 5.4 N.
As in the results of the tensile strength test, the chitosanbased film presented a greater elongation in relation to the others, with a value of 56.2%. The commercial pectin film presented a stretching value of 28.6%, with a small difference when compared to the natural pectin-based film that presented a value of 22.3%. The CMC-based film was the one that showed the lowest stretching, 18.6%. The tensile strength, stretching on the rupture, and elasticity module are common indicators used to describe the mechanical properties of the films and are related to their chemical structure and the mechanical strength being these important parameters, since they describe the structural and mechanical performance [25].

Determination of the physicochemical and bioactive analysis of the film based on the cajá peel pectin
Nowadays, the packages used to carry food are not passive components anymore. They are considered a component that has an active role, interacting with the external environment and with the food inside, through the liberation of active molecules that extends food shelf life, protecting against microbial deterioration and oxidation. The active package is an innovative technology that allows the product and its environment to interact in order to extend the product shelf life and/or to guarantee microbial safety, keeping the quality of the packaged food [3].
Thus, considering the quantity and the low cost of the pectin produced as a sub-product of agriculture and food industry and with film-formation properties confirmed with the results obtained through the analysis already presented and by the degree of esterification (DE) that was equal to 46%, the natural pectin tested becomes a candidate to the formulation of films as a matrix by using biopolymers. Table 1 shows the characterization performed of the natural pectin-based film and the importance of the obtained values related to the humidity level, hygroscopicity, solubility, and natural antioxidants, especially phenolic compounds present in vegetal extracts.
The humidity level of the film is related to the formulation constituents and the processing stages. The humidity level (%) found for the film was 16.75 ± 0.12, a value close to the grape peel-based film of 20.5 ± 1.0 [22]. Samples with a high humidity level are more susceptible to the development of microorganisms, while the films with a low humidity level, due to their hygroscopicity, may absorb water in a faster pace. Such facts interfere on the structure of the formed film, as well as on the mechanical properties. Films with low humidity tend to become brittle and improper to application, not acting as a barrier anymore. The presence of humidity in hydrophilic films causes change on permeability of gases and vapors. The humidity rise increases vapor pressure, due to water absorption by the polymer acting, then, as a plasticizer decreasing the energy of activation to the diffusion and penetration of water molecules and increasing the pressure of water vapor [67].
The composition of the characterized film formed by pectin presented solubility in water of almost 100%. Solubility is directly linked to the film structural components, and, consequently, films based on proteins or carbohydrates concentrate are hydrophilic and, normally, hygroscopic and get disintegrated when in contact with water [41]. In some applications, this high solubility may be desired. In semi-ready products destined to be prepared when cooked, it is important that its disintegration takes place in a fast pace as soon as it gets in contact with water [13]. On the other hand, the hygroscopicity value found was 30.67 ± 0.01, very high, confirming that its degradation in water is very easy. Normally, the polysaccharides are highly hygroscopic, causing the disintegration in water [63]. According to Ahn et al. [2], pectin is a hygroscopic polymer soluble in water that is used in food and pharmaceutical industries as a thickener and a coating, and works as the basis for the process of encapsulation due to its gelation, stabilization, and thickening properties.
The antioxidant compounds are substances that when present in small concentrations in relation to the oxidizable substrate may delay or even inhibit substantially the oxidation of a substrate, which means the inhibition and/or decrease of the free radicals effects [51]. These compounds delay or inhibit the formation of reactive species responsible for the chemical oxidization of other molecules. The formation of these reactive species happens in three stages: initiation, propagation, and termination [28]. Thus, the antioxidant properties of the film produced were analyzed through three methods, evaluating the initialization block (total antioxidant capacity), propagation (iron chelation), or termination (scavenging of the DPPH radical).
The phenolic compounds present therapeutic properties and antimicrobial effects, causing structural or functional damages to the bacterial cellular membrane [61]. In the present study, the value obtained to the concentration of total phenolics (mg GAE/100 g dried) found in the formulated film based on pectin was 3998.99 ± 42.64 that was higher than açaí (Euterpe oleracea) (1808.00 ± 28.00 mg EAG/100 g) but lower than acerola (Malpighia emarginata D.C.) pulps (12,466.00 ± 1256.00 mg EAG/100 g) as reported by Paz et al. [57]. The filmogenic solution is totally derived from the natural pectin extracted from the cajá. Thus, there was no insertion of additives to modify the quantities of total phenolic compounds, which means that the value found comes from the fruit.
The value found of the antioxidant capacity of the film may be connected to the processing reactions, one of them being the Maillard Reaction and among its degradation products [74].
Studies proved that the pectin potential activity could be improved through the insertion of natural additives derived from the pomegranate juice added to the pectin matrix [55]. One of the advantage shown by the present study is that there was no need to increment another type of natural additive to the film, since the value of the total antioxidant capacity (314.34 ± 9.40 mg AA/g) was very significant.
The DPPH consists of a nitrogen free radical and proton free radical cleanser. The hydrogen antioxidant donation may eliminate the DPPH and generate its non-radical form, DPPH-H. Through DPPH disappearance proportional to the antioxidant effect, it is possible to determine the antioxidant capacity present on the extracts. The scavenging of the DPPH radical with a value of 15.48 ± 1.48 μmol TE/g found on the film based on natural pectin presents phenolic compounds that are important antioxidant agents since their redox potentials allow them to act as reducing agents, hydrogen donors, and quenchers of singlet oxygen [19]. A study with the addition of green tea (GTE), extract of grape seed (OPC), grape seed polyphenols (GSP), ginger extract (GE), and gingko leaf extract (GBE) improved the DPPH capacity [40]. With antioxidant properties, films that present antioxidants on their formulations may bring benefits, since they are able to delay or inhibit oxidation [24].

Analysis of the antimicrobial activity to the film based on the peel pectin of cajá
Antimicrobial protection is essential to the coatings to effectively eliminate harmful microorganisms that may be in contact with the film and prevent infectious contaminations that may considerably cause damage to the material to be covered with this film. In the present study, the antimicrobial activity of the natural pectin film against the prominent pathogenics that cause infections was evaluated. Thus, the antimicrobial activity assay result is shown in the Fig. 8.
As can be seen in this figure, among the two investigated pathogens, the most pronounced antimicrobial effects obtained were for the gram-negative bacteria tested. According to Nori et al. [54], the gram-negative bacteria have a cellular membrane chemically complex, and one of its constituents, the lipopolysaccharide, determines the antigenicity, toxicity, and pathogenicity of these microorganisms. Agourram et al. [1] detected the antimicrobial effect on the Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Yersinia enterocolitica by using extracts of pomegranate and apple seeds. Carvalho and Orlanda [15] proved the antimicrobial effect of the phenolic extracts of the buriti fruit and observed the antimicrobial effect on the Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. Extracts from vegetal sources may be used as natural antimicrobial additives to food conservation [8]. Thus, they may be incorporated and/or used for the confection of films that work as coating.
The antimicrobial activity of the phenolic compounds is associated to several mechanisms, like the destabilization of the cellular membrane and the increase of its permeability, that may cause a blockage of the substrates to the microbial growing due to metal chelation and causing the enzymatic inhibition due to the presence of the phenolic hydroxyl grouping bonds with active sites of microbial enzymes [18].
The antimicrobial activity against both types of bacteria may indicate the presence of bioactive compounds with antimicrobial properties in a wide range on the composition of fruit pulps [31]. Most of the studies on antimicrobial and antioxidant activities attributed the antimicrobial activity to the phenolics extract and shows a direct relationship between the two activities [9,12]. Therefore, this is in agreement to the result obtained in the present study for the total phenolic compounds (mg GAE/100 g dried) that was of 3998.88 ± 42.64 (Table 1), thus confirming the antimicrobial action. For this reason, this film may be potentially used to inhibit the adherence of bacteria to the surface.

Conclusion
The bioactive edible films based on the pectin derived from extracts of an agro-industrial residue containing the natural antioxidants were obtained. It is interesting to mention that the film based on natural pectin presented a degree of esterification equals to 46% and the commercial citric pectin 34%, showing its capacity of forming gels. In addition, it was possible to observe a substantial increase of the rugosity of the film based on natural pectin when compared to the commercial pectin, and one of the justifications for it is the degree of esterification. The film presented a good tensile strength and stretching. The film formed by pectin presented itself as almost 100% soluble in water. The ease of being soluble is important to the application of the film in relation to the destined process. The use of extracts of vegetal sources may be used as natural antimicrobial additives and incorporated and/ or used for the confection of films with the function of coating. The phenolic compounds derived from cajá present therapeutic properties and antimicrobial effects causing structural or functional damages to the bacterial cellular membrane tested.
Funding The authors received financial support from CAPES and the Brazilian National Council for Research (Grant 305251/2017-1).

Declarations
Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest
The authors declare that they have no conflict of interest. a b Fig. 8 Antimicrobial effect of the Pseudomonas aeruginosa (a) and Staphylococcus aureus (b) tested on the film based on natural pectin