Orally disintegrating films based on gelatin and pregelatinized starch: new carriers of active compounds from acerola

Food Hydrocolloids 101 (2020) 105518

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Orally disintegrating films based on gelatin and pregelatinized starch: new carriers of active compounds from acerola Vitor Augusto dos Santos Garcia a, Josiane Gonçalves Borges a, Denise Osiro b, Fernanda Maria Vanin a, Rosemary Aparecida de Carvalho a, * a b

University of S~ ao Paulo, Faculty of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CEP 13635-900, Pirassununga, SP, Brazil University Center of the Guaxup�e Educational Foundation, Av. Dona Floriana, 463, CEP: 37800-000, Guaxup�e, MG, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Antioxidant capacity Gelatin Starch Malpighia emarginata Natural compounds

This study aimed to produce orally disintegrating films (ODFs) based on pregelatinized starch and gelatin with the incorporation of acerola powder and to evaluate the effect of the macromolecule concentration on the properties of the films. No ODFs had insoluble particles, and microstructure analysis by atomic force microscopy showed that higher starch concentrations led to greater surface heterogeneity and roughness. Starch inclusion in the films enhanced the hydrophilicity of the material (producing lower contact angle values). These factors, along with higher levels of starch, probably contributed to the favorable disintegration time, as observed by the lower values from in vitro and in vivo analyses. The FTIR spectra of the ODFs demonstrate predominant presence of carbohydrates and proteins, showing good interaction between them. In addition, the antioxidant capacity results showed that, after 50 days of storage under drastic conditions (75% relative humidity and 40 � C), the ODFs retained at least 60% of their antioxidant capacity. Therefore, the ODF containing acerola powder rep­ resents a promising system for use in active compound delivery.

1. Introduction

et al., 2017), pregelatinized starch and gelatin had high film-forming capacities and short disintegration times. Gelatin is derived from the hydrolysis of collagen and exhibits swelling, gelation and film-formation ability (Ullah et al., 2019). Starch is one of the most widely used poly­ mers due to its abundance, cost-effectiveness and film-forming ability. Ogunsona, Ojogbo, and Mekonnen (2018) reported that starch exhibits great promise for use in drug delivery systems, mainly due to its degradability. Due to the solubility of starches and gelatins in water, solutions resulting from these mixtures are expected to be homogeneous (Pod­ shivalov, Zakharova, Glazacheva, & Uspenskaya, 2017). However, different interactions may occur depending on the system (protein, polysaccharide, solvent) as reported in different studies in the literature (Grinberg & Tolstoguzov, 1997; Podshivalov et al., 2017; Zhang et al., 2013). In the case of the production of orally disintegrating films, another relevant aspect is the characteristics of the incorporated active ingredients. Garcia et al. (2018) recently produced an ODF based on starch and gelatin, with the incorporation of camu-camu powder. In addition to camu-camu, acerola (Malpighia emarginata) is also considered to be a

Polymer-based orally disintegrating films (ODFs) have aroused great interest as vehicles for active compounds. However, most studies have focused on synthetic compounds instead of natural compounds. The natural compounds most often applied in orally disintegrating films are propolis extracts (Borges & Carvalho, 2015; Juliano, Pala, & Cossu, 2007), extracts isolated from ginseng root (Watanabe et al., 2009), alcoholic extracts of ginger (Daud, Sapkal, & Bonde, 2011; Visser et al., 2017), peanut extracts ((Tedesco et al., 2017)), Lagerstroemia speciosa, Phyllanthus niruri, Cinnamomum burmanii Blume, Zingiber officinale Roscoe and Phaleria macrocarpa (Visser et al., 2017) and camu-camu powder (Garcia et al., 2018). Most extracts from natural sources were incorporated due to their anti-inflammatory and anticarcinogenic potential. Different polymers such as starch (Heinemann, Vanin, Carvalho, Trindade, & F� avaro-Trindade, 2017), chitosan (Li et al., 2017), gelatin (Borges, Silva, Cervi-Bitencourt, Vanin, & Carvalho, 2016) and hydroxypropylmethylcellulose (Prabhudessai, Dandagi, Lakshman, & Gadad, 2017) can be used in ODF formulation. According to (Garciados

* Corresponding author. E-mail address: [email protected] (R.A. Carvalho). https://doi.org/10.1016/j.foodhyd.2019.105518 Received 26 July 2019; Received in revised form 13 November 2019; Accepted 13 November 2019 Available online 14 November 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.

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potential source of vitamin C (Cruz et al., 2019), along with other compounds such as phenolics, flavonoids, anthocyanins and carotenoids (Belwal et al., 2018; Chang, Alasalvar, & Shahidi, 2018). Thus, due to its antioxidant capacity, which is mainly contributed by vitamin C, acerola has several benefits for the human body, such as anti-inflammatory, antioxidant, anti-tumor, antimicrobial, and hepatoprotective proper­ ties. In addition, consumers are increasingly looking for natural prod­ ucts, which drives the development of products that use natural active principles as sources of nutrients and functional compounds. Acerola has interesting attributes for consumption, despite be a resistant plant that grows easily in several regions and under different climatic conditions (Cruz et al., 2019), it is considered highly perishable because it rapidly degrades after harvest (Belwal et al., 2018), which made its consumption in natura low. According to Scarpa et al. (2017), there is a growing need to develop systems to attend the special needs of different types of patients. Patel, Prajapati, & Raval (2010) reported that ODFs primarily assist in the administration of medications to pediatric and geriatric patients or pa­ tients who do not have easy access to water. Considering that studies related to the development of orally dis­ integrating films as carriers of natural active compounds are incipient, the objective of this study was to evaluate the viability of incorporation of acerola powder (source of compounds with antioxidant activity) in pregelatinized starch and gelatin based orally disintegrating films through studies of interactions between film components as well as the evaluation of the global acceptance of this new product.

filmogenic solution), 40:60 (0.8 g of gelatin þ 1.2 of starch/100 g of filmogenic solution), 20:80 (0.4 of gelatin þ 1.6 g of starch/100 g of filmogenic solution) and 100:0 (2 g of starch/100 g of filmogenic solu­ tion). The gelatin was hydrated (30 min) at room temperature, and the pregelatinized starch was dispersed in distilled water under magnetic stirring (30 min, Big squid, IKA, USA). The polymers were solubilized separately in a thermostatic bath (90 � C for 30 min, MA 127, Marconi, ~o Paulo, Brazil). The gelatin and starch dispersions were mixed and Sa homogenized under magnetic stirring (3 min, Big squid, IKA) and held at ~o Paulo, 90 � C for 10 min in a thermostatic bath (MA 127, Marconi, Sa Brazil). Then, sorbitol was added and homogenized under magnetic stirring (3 min). The solution was cooled to 30 � C (room temperature), and the acerola powder (4 g/100 g of filmogenic solution) was incor­ porated under magnetic stirring (4 min). The solution was poured into acrylic plates (12 � 12 cm) and dried in a circulation oven (30 � C for 24 h, MA 35, Marconi). A digital micrometer (0.001 mm, IP - 65 model, Mitutoyo, Japan) was used to determine the film thickness by taking 10 random mea­ surements of the film surface. Except for the “mucoadhesiveness” and “disintegration time in vitro and in vivo analyses”, three ODF formula­ tions were produced for each condition, and from each of these formu­ lations three samples were taken. Therefore, the total number of replicates for each condition was equal to nine. 2.3. Characterization of ODFs 2.3.1. Visual aspect The visual aspect of the ODFs was evaluated in relation to the filmforming ability, handling and homogeneity according to (Garciados et al., 2017).

2. Materials and methods 2.1. Materials

2.3.2. Scanning electron microscopy (SEM) The surfaces and the internal structures of the films were analyzed using a scanning electron microscope (Model TM300, Tabletop Micro­ scope Hitachi, Japan) at 15 kV. Samples of the ODFs were previously stored in desiccators with silica gel (10 days, 25 � 5 � C). The samples were fixed onto aluminum probes using double-sided carbon tape (Ted Pella) for analyses.

Acerola pulp was obtained from the DeMarchi Company (Campinas, Brazil), and maltodextrin was donated by Corn Products Ingredients Industrials Ltda. (DE 12, Mogi Guaçu, Brazil). Pigskin gelatin Type A ~o Paulo, Brazil), pre­ (Blom 260, Mesh 40, Gelita do Brasil Ltda., Sa ~o Paulo, gelatinized cassava starch (AMIDOMAX 3600, Cargill Ltda., Sa ~o Paulo, Brazil) were used to produce the Brazil) and sorbitol (Vetec, Sa ODFs. The chicken skin used for mucoadhesiveness testing was acquired from a local market in Pirassununga (Brazil). For analysis of the anti­ oxidant activity, DPPH� (2,2-diphenyl-1-picrylhydrazine, Sigma Aldrich) and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox­ ylic acid, Sigma Aldrich) were used. To prepare the phosphate buffered ~o Paulo, Brazil), potassium saline solution, sodium chloride (Synth, Sa chloride (Synth), phosphate monopotassium (Vetec), disodium phos­ phate (Synth) and hydrochloric acid (Synth) were used.

2.3.3. Atomic force microscopy The ODF surfaces were evaluated using an atomic force microscope (NT-MDT, Solver Next, Germany). Film samples (1 cm2) were fixed with double-sided tape and analyzed in semi contact mode using the NSG01 tip with a constant force of 5 N/m, a resonance frequency of 150 kHz and a scanning rate of 0.3 Hz. The images were analyzed using Image Analysis 3.1.0.0 software.

2.2. Methods

2.3.4. X-ray diffraction The crystallinities of the films were evaluated using a MiniFlex 600 diffractometer (Rigaku, Japan) equipped with a Cu-Kα spot point source operating at 40 kV and 15 mA. The scans were performed from 2� to 80� (2θ) at rate of 1� /min and a step of 0.02� . Before the analysis, the ODFs were conditioned in a desiccator containing a saturated NaBr solution (at a relative humidity of ~58%) at ambient temperature for 5 days.

2.2.1. Production of acerola powder The acerola pulp was thawed in a refrigerator (5 � 2 � C) for 24 h. After this period, maltodextrin (5%, carrier agent) was added to the pulp under mechanical stirring (IKA RW 20, USA) at 500 rpm (30 min). For ~o Preto, Brazil) in­ drying, a Spray Dryer MSD 5.0 (Labmaq, Ribeira strument was used, which was equipped with an injector nozzle with a 2.0 mm diameter orifice and operated at a fixed flow rate of 20 mL/min. The drying air inlet temperature was set at 120 � C (Righetto & Netto, 2005), and the outlet air temperature was 80 � 5 � C.

2.3.5. Contact angle An optical tensiometer (Attension Theta Lite, KSV Instrument, USA) at room temperature (25 � 2 � C) was used for contact angle determi­ nation. Prior to analysis (5 days), the ODFs (3 cm � 2 cm) were stored in a desiccator containing saturated NaBr solution. For the analyses, the ODFs were fixed to the base of the apparatus, and a drop of deionized water (5 μL) was deposited using a microsyringe (Hamilton). The con­ tact angle was determined by the software (Attension Theta Lite, Version 4.1.9.8) after 10 s of contact.

2.2.2. Production of ODFs The ODFs were produced according to Garcia et al. (2018) and the concentrations of the macromolecules (2 g of gelatin þ starch/100 g of filmogenic solution) and the plasticizer (0.4 g of sorbitol/100 g of fil­ mogenic solution) were kept constant. The ODFs were produced with the following ratios of gelatin þ starch: 100:0 (2 g of gelatin/100 g of filmogenic solution), 80:20 (1.6 g of gelatin þ 0.4 of starch/100 g of filmogenic solution), 60:40 (1.2 g of gelatin þ 0.8 g of starch/100 g of 2

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2.3.6. Surface pH The surface pH was evaluated according to Prabhu et al. (2011) using phosphate buffered saline solution (pH 6.8). The phosphate buffer was €ger, Kopf, Loretz, Albrecht, and Bernkop-Sch­ prepared according to Fo nürch (2008). Samples of the ODFs (3 cm � 2 cm) were placed in contact with the buffered solution (0.5 mL), and the pH was determined after 1 min using a pH meter electrode (3210, WTW, Germany).

ABTS●þ, and the solution was left in the absence of light for 6 min. The absorbance was determined using a Lambda 35 spectrophotometer (PerkinElmer, Shelton, USA) at 734 nm. Solutions with Trolox concen­ trations between 250 and 2500 μM were used to construct the calibra­ tion curve, and the results were expressed in μM Trolox equivalent/g of film. 2.4.2. FRAP assay Antioxidant activity determination by the ferric reducing/antioxi­ dant power method (FRAP) was performed according to Benzie & Strain (1996). ODFs samples (0.02 g) were dispersed in water (10 mL volu­ metric flask) and held for 10 min in an ultrasonic bath (Ultra Clear, 1400 A, Unique). Aliquots (150 μL) of the solubilized films were added to 2850 μL of the FRAP reagent. The solutions were kept in a thermostatted bath (MA-127, Marconi, Brazil) at 37 � C (30 min). The absorbance was determined at 593 nm using a Lambda 35 spectrophotometer (Perki­ nElmer). The calibration curve was prepared by using Trolox as the standard (99.9–549.4 μM), and the results were expressed in μM Trolox equivalent/g of film.

2.3.7. Mucoadhesiveness The mucoadhesiveness of the ODFs was determined using a TA.XT2 plus texturometer (Stable Microsystems SMD, England) with a 20 kg charge cell. Chicken skin (tissue model) was used to simulate the buccal mucosa (Wong, Yuen, & Peh, 1999). The tissue model was fixed onto the base of the texturometer and the ODF (2.0 cm of diameter) was fixed onto the probe at the top of the texturometer, 5 samples of different formulations were evaluated. The skin was compressed into the ODF at a constant strength (1.0 N) and kept in contact for 10 s. The ODF and tissue were separated at a constant speed (0.5 mm/s) until reach a dis­ tance of 15.0 mm (Peh & Wong, 1999). The mucoadhesiveness was considered to be the strength necessary to separate the ODF from the model tissue.

2.4.3. DPPH� free radical antioxidant capacity The ODFs were stored in desiccators at 75% relative humidity (NaCl) and maintained in a BOD incubator (MA 415, Marconi, Brazil) at 40 � C (Daud, Sapkal, & Bonde, 2011). The sequestering activity was deter­ mined by the free radical sequestration method DPPH� (Brand-Williams, Cuvelier, & Berset, 1995) on days 1, 8, 22 and 50. Previously, the films were solubilized in distilled water, and samples (0.2 g ODFs) were dispersed in water (25 mL volumetric flask) and kept in an ultrasonic bath (Ultra Cleaner, 1400 A, Unique) for 10 min to achieve complete solubilization. Aliquots (2 mL) of the solutions (solubilized films) were added to 2 mL of the DPPH � radical (0.2 mM), termed A1. Solution A2 was prepared with 2 mL of the ODF solution þ2 mL of distilled water, and solution A3 consisted of 2 mL of DPPH� þ 2 mL of ethanol (Li, Miao, Wu, Chen, & Zhang, 2014). The absorbances (A1, A2, and A3) were determined after 3 h of spectrophotometer reaction at 517 nm, and the antioxidant capacity was determined according to Eq. (1). �� � � A1 A2 Antioxidant ​ capacity ð%Þ ¼ 1 100 (1) A3

2.3.8. Disintegration time in vitro and in vivo For the in vitro disintegration time ODFs samples (3 cm � 2 cm) were fixed onto a supporting slide frame (Garsuch & Breitkreutz, 2010) and placed in a Petri dish. A drop of water (200 μL) was deposited onto the ODF surface (Janβen, Schliephacke, Breitenbach, & Breitkreutz, 2013). The time required for disintegration and hole formation in each sample was quantified as the disintegration time. For theses analyses formula­ tions were produced in triplicate, and from each formulation 4 samples were taken, thus, 12 samples for each condition. The in vivo disintegration time was determined according to (Gar­ ciados et al., 2017), and the experiment was conducted in accordance with the ethical principles approved by the Research Ethics Committee ~o Paulo (USP, Brazil), under number CAAE of the University of Sa 16840613.0.0000.5422 (number of the approval report, 359.878). The group of panelists consisted of 2 men and 8 women between 25 and 29 years old, and they were trained to evaluate the disintegration time of the ODF. The group used a nonstructured scale ranging from fast (score 0) to slow (score 9). The samples were served and evaluated according to Garcia et al. (2018).

2.5. Sensory evaluation of ODFs The sensory evaluation of ODFs containing different ratios of gelatin: starch (100:0, 80:20, 60:40, 40:60, 20:80, 0:100) and incorporated with acerola powder was performed by 55 untrained tasters (between 22 and 30 years old). The parameters (comfort, taste, mouth-feel properties and global acceptability) were analyzed according to Garcia et al. (2018) and in accordance with the ethical principles approved by the Research Ethics Committee of the University of S~ ao Paulo (USP, Brazil) under number CAAE 16840613.0.0000.5422 (number of the approval report, 359.878). The ODFs were randomly encoded and delivered to the tasters inside red-lit booths. The tasters were asked to place the ODFs in the oral cavity (between the tongue and palate), and before the analysis, each taster was instructed to moisten his or her mouth with water. The ODFs were evaluated using a 9-point hedonic scale (1 - Extremely dislike, 5 Neither like nor dislike, 9 - Extremely like) for all parameters.

2.3.9. Fourier transform infrared (FTIR) spectroscopy The spectra in the infrared region were evaluated using a spec­ trometer (Spectrum One, PerkinElmer, USA). The ODFs were kept in desiccators containing silica gel (10 days). Then, sixteen scans were performed with a resolution of 2 cm 1 in the spectral range from 650 to 4000 cm 1 (Cilurzo, Cupone, Minghetti, Selmin, & Montanari, 2008). The FTIR absorbance spectra were mathematically treated, and their baselines were corrected, followed by normalization to the carbohydrate peak (1180 e 880 cm 1); then, their second derivatives were calculated, and the Savitzky-Golay (SG) filter was applied when necessary. The Gaussian function was used to fit all the deconvoluted curves using the second derivatives (R2 > 0.98) (Baker et al., 2014). All mathematical procedures were performed in the Origin Pro 9.0 software obtained from OriginLab. 2.4. Antioxidant activity

2.6. Statistical analysis

2.4.1. ABTS assay The ODFs (0.1 g ODFs) were dipped in water (10 mL volumetric flask) and kept in an ultrasonic bath (Ultra Clear, 1400 A, Unique, Indaiatuba, Brazil) for 10 min to determine the ODF solubilization. The antioxidant activity was determined by the free radical method ABTS●þ according to Re, Pellegrini, Pannala, Yang, and Rice-Evans (1999). Al­ iquots (30 μL) of the solubilized films were added to 3 mL of free radical �þ

Statistical analysis of the data was performed using analysis of variance (ANOVA), and in the case of a significant difference, the Duncan test (p < 0.05) was performed using SAS 9.3 software.

3

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A1

B1

C1

D1

A2

B2

C2

D2

A3

B3

C3

D3

A4

B4

C4

D4

A5

B5

C5

D5

A6

B6

C6

D6

Fig. 1. Orally disintegrating films containing acerola powder: (A) ODFs images, (B) micrograph of the surface, (C) micrograph of the internal structure and (D) atomic force microscopy images of the surface in 3 dimensions with different gelatin:starch ratios of (1) 100:0; (2) 80:20; (3) 60:40; (4) 40:60; (5) 20:80 and (6) 0:100.

3. Results and discussion

gelatin:starch ratio (Fig. 1A). These characteristics were related to the ease solubilization of acerola powder in the film-forming solution. In addition, the formulations tested (different gelatin:starch ratios) were shown to have high film-forming abilities, indicating that gelatin and pregelatinized starch, whether isolated or combined, were macromole­ cules that can be used to produce orally disintegrating films. Similar

3.1. Visual assessment The ODFs with the addition of acerola powder presented no insoluble particles and homogeneity after the drying process, independent of the 4

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Table 1 Thickness, roughness, contact angle, surface pH and mucoadhesiveness of orally disintegrating films containing acerola powder with different gelatin:starch ratios. Gelatin: Starch ratio

Thickness (mm)

Roughness

Contact angle (� )

Surface pH

Mucoadhesiveness (N)

100:0

0.069 � 0.005a 0.070 � 0.004a 0.068 � 0.005a 0.069 � 0.005a 0.071 � 0.005a 0.071 � 0.006a

34.4 � 2.6c 37.5 � 3.4c 44.2 � 4.1c 86.3 � 9.8b 112.1 � 8.4a 85.7 � 10.2b

107.7 � 4.6a 104.0 � 5.6ab 102.5 � 2.0ab 101.8 � 2.2ab 89.8 � 9.0c 89.1 � 8.6c

5.8 � 0.4ab 5.6 � 0.1b 5.6 � 0.1b 5.7 � 0.3b 5.9 � 0.2a 6.1 � 0.2a

0.54 � 0.07a

80:20 60:40 40:60 20:80 0:100

0.58 � 0.03a 0.60 � 0.08a 0.63 � 0.12a 0.64 � 0.03a 0.63 � 0.14a

*Different lowercase letters in the same column indicate significant differences (p < 0.05). Fig. 2. X-ray diffraction of orally disintegrating films containing acerola powder with different gelatin:starch ratios: 1 ¼ 100:0; 2 ¼ 80:20; 3 ¼ 60:40; 4 ¼ 40:60; 5 ¼ 20:80 and 6 ¼ 0:100.

results were reported by Garcia et al. (2018) for ODFs based on starch and gelatin with the incorporation of camu-camu powder. A number of studies have reported the production of gelatin-based (Castro et al., 2017; Heinemann et al., 2017) and modified starch-based ODFs (Pimparade et al., 2017). According to the results, these polymers have desirable characteristics for the production of ODFs.

�pez, García, parallel organization of the double helices (Castillo, Lo Barbosa, & Villar, 2019; Flores-Silva et al., 2017; Lourdin et al., 2015). 3.3. X-ray diffraction Regardless of the formulation, no crystallinity peaks were observed in the X-ray diffractograms of the ODFs (Fig. 2). According to the results, the produced ODFs exhibited pseudoamorphous behavior, with peaks close to 20� . Similar results were observed for gelatin and starch films (Aguilar-M� endez, Martin-Martínez, Ortega-Arroyo, & Cruz-Orea, 2010; Wang et al., 2017). Wang et al. (2017) evaluated gelatin and starch films and related the peaks close to 20� as the reconstitution of the three-dimensional collagen structure. The authors attributed this pseudoamorphous behavior to the gelatin-plasticizer interaction and the gelatin-starch interaction.

3.2. Microscopy On surface micrographs (Fig. 1B), ODFs produced with gelatin alone (100:0, gelatin:starch ratio) showed a more homogeneous surface (Fig. 1B1), and the addition of starch (Fig. 1B5) resulted in the formation of a more heterogeneous structure. (Garciados et al., 2017) also reported alteration of the conformation in polymeric ODFs based on gelatin and pregelatinized starch blends. In ODFs with higher gelatin concentra­ tions, more homogeneous structures were probably associated with the maintenance of the α-helix and triple-helix structures, which were characteristic of the collagen, as observed in the spectra of infrared re­ gion (Fig. 4). Garcia et al. (2018) observed similar behavior for gelati­ n/starch films containing camu-camu powder; i.e., the effect was associated with the structural characteristics of the macromolecules used in the production of the ODFs. Raza, Kar, Wani, and Khan (2019) produced ODFs of hydrox­ ypropylmethylcellulose, carboxymethylcellulose, alginate and gelatin with the incorporation of losartan (synthetic compound) and found that the films had smooth surfaces and smooth or transverse striations, indicating a uniform drug distribution. In relation to the internal structure of the ODFs (Fig. 1C), hetero­ geneous structures and the formation of discontinuity zones were observed. Borges et al. (2016) evaluated ODFs composed of gelatin and lecithin with different concentrations of ethanol extract of propolis and observed that the increase in the extract amount resulted in the forma­ tion of a greater number of pores on the surface and internal micrographs. The roughness values of the orally disintegrating films with added acerola powder can be observed in Table 1. The higher the starch con­ centration in ODFs the higher the roughness. Additionally, a significant difference in roughness was observed for the ODFs produced with the isolated polymers, being higher for the starch film. The internal architecture of native starch granules was characterized by “growth rings”, which correspond to concentric semicrystalline shells, separated by amorphous regions. The shells consist of a regular alternation of amorphous and crystalline coverslips with a repeating distance from ~9 to 10 nm. The crystalline lamellae are formed by the

3.4. Thickness, contact angle, surface pH and mucoadhesiveness For all formulations, no significant differences were observed in relation to the thickness (Table 1), indicating that the mass control performed during the production process was efficient. In general, an increase in the starch concentration resulted in a reduction in the con­ tact angle value (Table 1), indicating that higher starch concentration in the ODFs produced more hydrophilic characteristics (gelatin:starch ra­ tios of 20:80 and 0:100). According to Arkles (2006) and Rios, Dodiuk, Kenig, Mccarthy, & Dotan (2007), surfaces with contact angles under 90� show hydrophilic characteristics. The hydrophilicity of the material can control the release and disintegration of the film, and in this way, ODFs with higher hydrophilicity values tend to have lower disintegra­ tion times and therefore produce a faster release of the compound. (Garciados et al., 2017) and Garcia et al. (2018) produced ODFs based on pregelatinized starch and gelatin with and without incorpo­ ration of camu-camu powder and observed a reduction in the contact angle values with the increase in the starch concentration, reporting values < 90� . The values of the surface pH were not influenced by starch concen­ tration and acerola powder incorporation (Table 1), and agree with the range reported in the literature, indicating that the consumption of ODFs does not cause oral mucosa irritation. (Garciados et al., 2017) deter­ mined that ODFs based on pregelatinized starch and gelatin had a sur­ face pH close to 6.8. For ODFs with the addition of acerola powder, the 5

V.A.S. Garcia et al.

1150 1107 1078 1050 1020 993 962 930

1236 1200

1455 1408 1375 1335 1298

1550

1661 1628

1725

3292

F')

3070 2930 2878

Food Hydrocolloids 101 (2020) 105518

E') D') C') B') A')

F) E) D) C) B) A) Fig. 3. Disintegration time in vivo and in vitro of orally disintegrating films containing acerola powder with different gelatin:starch ratios. Different lowercase letters under the same parameter (in vivo and in vitro values) indicate a significant difference (p < 0.05) between the different formulations of the ODFs.

3600

3200

2800 1800

1600

1400

Wavenumber (cm-1)

1200

1000

(a)

1,0

authors observed a reduction in the surface pH, but nevertheless, the pH remained close to neutral (7.0). Yehia, El-Gazayerly, & Basalious (2009) produced mucoadhesive buccal films and reported that the surface pH was 4.5–6.5. In vivo ex­ periments showed that the films did not cause any irritation in the tasters’ mucosa. Upon comparing the developed formulations, no significant differ­ ences in mucoadhesiveness were observed (Table 1). The amino group contains the –CH2OH and OH groups (Nagar, Chauhan, & Yasir, 2011) and the gelatin CH3, CH2, NH2 and COOH groups (Bialopiotrowicz & � czuk, 2002). In addition, vitamin C contains OH, CO and –O groups Jan (Yoon, 2014). By considering the main functional groups present in the ODFs, it can be suggested that both have the capacity to form hydrogen bonds with the mucosa. According to Andrews, Laverty, and Jones (2009), polymers with functional groups such as COOH (carboxyl), OH (hydroxyl), NH2 (amide) and SO4H (sulfate groups) may be more favorable for inter­ acting with the oral mucosa. Similar results of mucoadhesiveness (Table 1) were reported for ODFs of gelatin (0.5 N) and those with the incorporation of an ethanol extract of propolis (0.8 N) (Borges & Car­ valho, 2015).

A B C D E F

Absorbance

0,8

0,6

0,4

0,2

0,0 3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

(b) Fig. 4. FTIR absorption spectra (A, B, C, D, E and F), second derivative of the FTIR (A 0 , B0 , C 0 , D0 , E ‘and F0 ) spectra (a) and normalized spectra (b) of ODFs containing acerola powder with different gelatin:starch ratios of (A) 100:0; (B) 80:20; (C) 60:40; (D) 40:60; (E) 20:80 and (F) 0:100.

3.5. Disintegration time

extract of propolis and reported disintegration time values lower than 26 s. Pimparade et al. (2017) reported that the disintegration time of modified starch films remained between 6 and 11 s, according to the film thickness (0.06–0.11 mm) and added compounds, such as citric acid. Similar to the results observed in the in vitro tests, the in vivo results (Fig. 3) indicated that the increase in the starch concentration caused a reduction in the disintegration time of the ODFs. Pimparade et al. (2017) reported that the normal flow of saliva in a healthy person was 0.34 mL/min, but it may increase in the presence of stimulating agents; furthermore, the ascorbic acid present in acerola was considered a salivary-stimulating agent (Siddiqui, Garg, & Sharma, 2011). According to Lam, Xu, Worsley, and Wong (2014), the flow rate, composition and pH of saliva can influence the drug absorption into the oral cavity, mainly due to the aqueous environment provided by the greater amount of saliva. This could explain why ODFs with camu-camu powder (Garcia et al., 2018) present fast disintegration times (<3.1), when used a nonstructured scale ranging from fast (score 0) to slow (score 9), than those of ODFs with acerola powder. Camu-camu has a lower pH (as a

The disintegration times (in vitro and in vivo) of the ODFs with the addition of acerola powder can be observed in Fig. 3, lower values were obtained by the formulation produced with only starch (0:100 gelatin: starch ratio). However, regardless of the starch concentration, the disintegration time was <20 s, indicating that all ODFs exhibited rapid disintegration. The reduction in the disintegration time with increasing starch concentration was correlated with the increased hydrophilicity of the matrix, as determined by contact angle values (Table 1). The same effect was observed by (Garciados et al., 2017) and Garcia et al. (2018), in which the increase in the starch concentration resulted in a reduction in the disintegration time of gelatin and starch ODFs with and without addition of camu-camu powder, respectively, with reported values of <30 s. However, it was worth noting that, according to Jyoti, Gurpreet, Seema, & Rana, 2011 and Mahboob, Riaz, Jamshaid, Bashir, and Zul­ fiqar (2016), films with a maximum disintegration time of 60 s were classified as fast. Borges et al. (2016) produced gelatin-based ODFs with ethanol 6

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Food Hydrocolloids 101 (2020) 105518

Table 2 Band assignments for infrared spectra of orally disintegrating films containing acerola powder with different gelatin:starch ratio. Bands (cm 1)

Functional group

3280

Stretching N–H (amide A) Stretching O–H

3070 2930, 2878

C–N stretching vibration) C–H stretching

1730

C¼O stretching

1661

Amide I (predominantly the C– –O stretching vibration of the α-helix) O–H bend, C– –O stretching

1455

Amide I (C– –O stretching vibration of the triple helix) C¼C stretching Amide II (out-of-phase combination of N–H bending and C–N stretching) C¼O stretch and C–C ring stretch C–H bend

1408

COO stretching C¼C aromatic ring stretching C–O–H bend

1628 1550

CH2 bending

1375

1335

symmetric stretching of carboxylate Stretching C–O, C–O–H bend; symmetric in-plane bending of –CH3 deformation N–H C–O–H bend CH2 bending

1298

C–O–H bending

1285

CH2OH (side chain) related mode Amide III (in phase combination of N–H bending and C–N stretching) C–O–H bending C–O–H bending

1236

1200 1150, 1107

1078

C–C(¼O)–O stretch C–O–C stretching C–O–H bending C–O–C stretching C–O–H bending

Table 2 (continued ) Bands (cm 1)

Functional group

Assignment

1050

ester bond C–O–C stretching and C–O–H bending

1020

C–O–C bending

993

Stretching C–O

Dry extract (proteina,b) and gelating,h Dry extract (vitamin Cd, carbohydratesa,b,i); starchg,i and sorbitole,f Dry extract (proteina,b, carbohydratesa,b,i), starch7.9 and gelating,h Dry extract (carbohydratesa,b,i); gelating,h and starchg,i Dry extract (carbohydratesa,b,i) and starchg,i

Assignment Dry extract (proteina,b) and gelating,h Dry extract (carbohydratesa,b,i, proteina,b, alcoholsa,b, fatty acidb, vitamin Cd and phenolic compoundsa,b,c), sorbitole,f, gelating,h, Water molecules adsorbedi and starchg,i Dry extract (proteina,b) and gelating,h Dry extract (carbohydratesa,b,i, fatty acidb; proteina,b, alcoholsa,b and phenolic compoundsa,b,c, vitamin Cd), sorbitole,f, gelating,h and starchg,i Dry extract (phenolic compoundsa,b,c, fatty acidb, vitamin Cd) Gelating,h,j and dry extract (proteina,b,j)

962 930

Dry extract (phenolic compoundsa,b,c, vitamin Cd), sorbitole,f, Water molecules adsorbed in the noncrystalline region of starchg,i Gelating,h,j and dry extract (proteina,b,j) Dry extract (phenolic compoundsa,b,c) Gelating,h, j and dry extract (proteina,b, j, phenolic compoundsa,b,c) Dry extract (phenolic compoundsa,b,c, vitamin Cd) Dry extract (carbohydratesa,b,i, fatty acidb; proteina,b, alcoholsa,b and phenolic compoundsa,b,c, vitamin Cd), sorbitole,f and starchg,i Dry extract (proteina,b) and gelating,h Dry extract (phenolic compoundsa,b,c) Dry extract (phenolic compoundsa,b,c, vitamin Cd) Dry extract (fatty acidb; proteina,b), gelating,h Gelating,h

skeletal mode vibrations of R1,4 glycosidic linkage, (C–O–C) skeletal mode vibrations of R1,4 glycosidic linkage, (C–O–C) C–H bend and C–C ring bend skeletal mode vibrations of R1,4 glycosidic linkage, (C–O–C)

Dry extract (carbohydratesa,b,i) and starchg,i Dry extract (vitamin Cd) Dry extract (carbohydratesa,2.9) and starchg,i

a

Türker-Kaya & Huck, 2017. Movasaghi et al., 2007. c Abbas et al., 2017. d Yang & Irudayaraj, 2002. e Pourfarzad et al., 2018. f Murrieta-Martínez et al., 2019. g Zhang et al., 2013. h Riaz et al., 2018. i Kizil et al., 2002. j Bridelli, 2017. b

function of the higher concentration of ascorbic acid) and, consequently, stimulates saliva production and reduces the time required for disintegration. In other studies in the literature, Cilurzo et al. (2008) and Sayed, Ibrahim, Mohamed, & El-Milligi, 2013 produced maltodextrin and hydroxypropylmethylcellulose films, respectively, and reported a good correlation between the results obtained in vitro and in vivo. In the pre­ sent study, in vivo and in vitro results presented a Pearson correlation equal to 0.8.

Dry extract (proteina,2, carbohydrates1,b,i, fatty acidb; phenolic compoundsa,b,c, vitamin Cd) and starchg,i Dry extract (proteina,b) and gelating,h Dry extract (phenolic compoundsa,b,c, carbohydratesa,b,i, proteina,b, vitamin Cd) and starchg,i Dry extract (carbohydratesi) and starchg,i Dry extract (carbohydratesa,b,i) and starchg,i Dry extract (carbohydratesi) and starchg,i Dry extract (proteina,b) and gelating,h

3.6. FTIR spectroscopy Fig. 4 shows the absorption spectra in the Fourier transformed me­ dium infrared region (spectra A, B, C, D, E, F) and the respective second derivatives (spectra A’, B’, C’, D’, E’, F’) of theODFs. The FTIR spectra of the films presented wide bands due to signal overlap, which was mainly attributed to the functional groups of the most abundant macromole­ cules (Baker et al., 2014). Negative peaks of the second derivative of the absorption spectra were used to identify the overlapping signals. The FTIR spectra after baseline correction were normalized to match the maximum broadband intensity between 1180 and 880 cm 1. This band is mainly the result of the stretching vibrations of C–C and C–O, the angular C–O–H of carbohydrates from acerola and starch (Türker-Kaya & Huck, 2017), and the C–OH stretching vibrations of sorbitol (Pour­ farzad, Ahmadian, & Habibi-Najafi, 2018). According to Castro and Cassella (2016), the stretching vibrations of sorbitol were centered C–OH at ~1046 and ~1084 cm 1. The ODFs had a complex chemical composition of ~62% acerola powder, ~6% sorbitol and percentages of starch and gelatin ranging from 0 to ~31%. The acerola powder must be considered a complex sample, since it is rich in carbohydrates, proteins, fatty acids, phenolic compounds and vitamins, mainly vitamin C (Moura et al., 2018; Prakash & Baskaran, 2018). The FTIR spectra of ODFs can be divided into regions of stretching bands (ν) and angular (δ) characteristics. From 3700 to 3000 cm 1 was νO-H and νN-H, and from 3000 to 2500 cm 1 was νC-H. The shoulder at

Dry extract (vitamin Cd) Dry extract (carbohydratesa,b) and starchg,i Dry extract (vitamin Cd), Dry extract (vitamin Cd, carbohydratesa,b,i) and starchg,i Dry extract (vitamin Cd, carbohydratesa,b,i); starchg,i and sorbitole,f Dry extract (vitamin Cd, carbohydratesa,b,i) and starchg,i Dry extract (vitamin Cd, carbohydratesa,b,i); starchg,i and sorbitole,f

7

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Food Hydrocolloids 101 (2020) 105518

b)

a)

1750

1700

1650

1600

Wavenumber (cm-1)

1550

1500

1700

1650

1600

1550

Wavenumber (cm-1)

1500

e)

d)

1750

1750

c)

1700

1650

1600

1550

Wavenumber (cm-1)

1500

1750

1750

1700

1650

1600

1550

1500

1700

1650

1600

1550

1500

Wavenumber (cm-1)

f)

1700

1650

1600

1550

Wavenumber (cm-1)

1500

1750

Wavenumber (cm-1)

Fig. 5. Gaussian deconvolution of the FTIR spectra of ODFs containing acerola powder with different gelatin:starch ratios: a) 100:0; b) 80:20; c) 60:40; d) 40:60; e) 20:80 and f) 0:100 in the region of the bands of amide I and II.

~1725 cm 1 was characteristic of νO-H, and the region between 1700 and 1490 cm 1 was attributed mainly to amide I and II protein bands. The complex region between 1490 and 1170 cm 1 contained many overlapping signals, and the region between 1170 and 880 cm 1 pre­ dominantly the vibrations of carbohydrates (Türker-Kaya & Huck, 2017; Movasaghi, Rehman, & Rehman, 2007). The detailed identification of the bands in the FTIR spectra of ODFs was shown in Table 2, in which the positions of the signals were determined from the second derivative spectra. The FTIR spectra of the films demonstrated the predominant presence of carbohydrates and proteins, because in addition to the starch and gelatin, the acerola extract also presented peptides and high concentrations of sugars. In addition, vitamin C, another component richly present in acerola extract (Moura et al., 2018; Prakash & Baskaran, 2018), showed intense signals at ~1730 cm 1 (νC ¼ O) and ~1660 cm 1 (νC ¼ C) (Yang & Irudayaraj, 2002); therefore, it should contribute significantly to the intensities of these bands in the FTIR spectra of the films. The normalization of the spectra allowed for the observation of the variation of the intensity and the areas of the absorption bands in the FTIR spectra as a function of the amount of starch and gelatin added in the composition of the ODFs. The spectra were considerably influenced by the addition of poly­ mers, since together, they represent approximately 31% of the final composition. Starch is a mixture of amylose and amylopectin, and gelatin is a collagen protein concentrate with varying degrees of hy­ drolysis (Kumar et al., 2019). The normalization of the spectra to equate the intensities of the bands between 1180 and 880 cm 1 (which were

attributed mainly to carbohydrates) allowed clear observation of the variation of protein bands. In the spectra of the films with higher starch contents and, therefore, less gelatin, decreases in the amide I bands – O stretch of λ ¼ 1661 cm 1 and λ ¼ 1628 cm 1) and amide II band (C– (angular NH and stretch CH in ~1550 cm 1) were observed. In the 0% starch spectrum (Fig. 4a), signals were attributed to proteins, such as the bands at ~3292 cm 1 (νN-H at amida A) and ~3070 cm 1 (νC-N), at ~1661 and 1628 cm 1 (νC ¼ O de amida I) and at ~1550 cm 1 (νC-N e δN-H de amida II) (Riaz et al., 2018; Zhang et al., 2013). The increase in the amount of starch also contributed to the signal at ~3292 cm 1 due to the presence of νO-H; however, its presence favors the widening of this band due to a greater formation of hydrogen bonds (Kizil, Irudayaraj, & Seetharaman, 2002), and the presence of gelatin contributed to a finer and more focused band (Riaz et al., 2018; Zhang et al., 2013). As described in Table 2, the complex region between 1490 and 1170 cm 1 concentrates the signals of proteins and the compounds present in the acerola extract, such as vitamin C, fatty acids and phenolic compounds. In the spectra only with starch (0:100 gelatin:starch ratio) , the peaks between 1490 and 1170 cm 1 were more intense and defined probably because the reduction of carbohydrates emphasizes the vibrations attributed to proteins and the active components in acerola powder, such as phenolic compounds and vitamin C. Therefore, FTIR spectral analysis of the ODFs showed good interac­ tion between starch and gelatin macromolecules when both were pre­ sent (amylopectin and amylose of the starch and the gelatin collagen peptides), which should also contribute to the uniformity distribution of the other compounds present (Türker-Kaya & Huck, 2017; Movasaghi 8

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Food Hydrocolloids 101 (2020) 105518

Table 3 Area of the bands (%) resulting from the deconvolution of the amides region I and II of the FTIR spectra of orally disintegrating films containing acerola powder with different gelatin:starch ratio. Assignment (Bridelli, 2017; Riaz et al., 2018)

Gelatin

100

80

60

40

20

Starch

0

20

40

60

80

Band (cm Amide II Amide I

1

)

1550 a 1553 1606 1626–1629 1659–1666 1687–1692

β-sheet Triple helix α-helix β-turn/β-sheet

ABTS●þ

FRAP

100:0

1.11 � 0.10B

108.03 � 5.62A

80:20

1.14 � 0.14C

108.78 � 6.28A

%

Area

%

Area

%

Area

%

Area

%

Area

%

26.94

33.08

23.25

32.64

19.25

30.99

18.26

29.84

9.45

26.16

6.60

27.26

45.45 28.39

6.42 3.51 3.73

26.51 14.50 15.41

33.25 16.55 4.72 81.46

40.28 20.05 5.71

29.56 14.95 3.46 71.22

60:40

1.12 � 0.12C

106.78 � 9.48A

40:60

1.09 � 0.10B

20:80

1.11 � 0.04B

110.18 � 10.85A 108.37 � 9.76A

0:100

1.23 � 0.13A

109.83 � 5.85A

1

8

22

50

95.26 � 2.52Aa 93.74 � 3.05Aa 98.46 � 0.85Aa 94.10 � 2.73Aa 98.26 � 0.26Aa 98.53 � 0.06Aa

82.42 � 0.86ABb

69.81 � 0.77Bc 71.75 � 0.79Bc 71.98 � 2.17Bc 75.50 � 1.39Ac 71.99 � 1.00Bc 77.83 � 1.17Ab

65.31 � 1.71ABd

84.81 � 3.55ABb 88.26 � 2.11Ab 85.35 � 1.43ABb 84.02 � 6.24ABb

0.00 41.51 21.00 4.86

24.43 13.63 4.86 62.18

39.32 21.94 7.83

24.43 13.63 4.87 61.19

39.92 22.28 7.95

16.42 10.25 36.12

20.26

this film, the signals must have mainly resulted from the presence of peptides from the acerola powder. The signal at ~1660 cm 1 was also influenced by adsorbed water (δO-H) (Riaz et al., 2018) and vitamin C (νC ¼ C) (Yang & Irudayaraj, 2002). 3.7. Antioxidant capacity of ODFs

DPPH scavenging activity as function of time (Day)

78.63 � 2.51Bb

100

Area

Table 4 Antioxidant capacity of orally disintegrating films containing acerola powder with different gelatin:starch ratio by the free radical sequestration method ABTS●þ, iron reducing power (FRAP) and DPPH radical as a function of storage time. Gelatin: Starch ratio

0

In general, as examined by both the ABTS●þ method and the FRAP method, no significant variations were observed in the antioxidant ca­ pacity of the films with the increase in the starch concentration (Table 4). It was expected that different concentrations of macromole­ cules could confer different protections to the active components, depending on the intrinsic characteristics of each molecule (considering that the concentration of acerola powder added to the films was the same, at 4 g/100 g filmogenic solution). Thus, in the studied range, it could be concluded that the protection of the active principle was in­ dependent of the gelatin:starch ratio. Therefore, the reported antioxi­ dant capacity can be attributed to compounds present in the acerola powder, such as vitamin C ((Garciados et al., 2017)). According to Cerqueira, Medeiros, and Augusto (2007) the antioxi­ dant capacity determined by the FRAP method can be attributed to the presence of vitamin C in the extracts incorporated into the ODFs because, during the reduction of the hydrogen peroxide by Feþ, the hydroxyl radical was favored in the presence of vitamin C. Rambabu, Bharath, Banat, Show, and Cocoletzi (2019) reported that the antioxidant capacity determined by the ABTS●þ, FRAP and DPPH� methods was based on the amount of radicals (specific to each method) reduced by the various antioxidant components of the extract. Thus, the decreased stability, as observed by DPPH� free radical scavenging as a function of time (Table 4), may be related to the degradation of vitamin C, as well as other naturally occurring compounds, under the storage conditions of the films.

65.27 � 0.46ABd 63.37 � 3.22Bd 63.50 � 1.81Bd 68.52 � 0.97Ad 67.55 � 1.11ABc

Being: ABTS●þ, FRAP: μMol de Trolox equivalent/g film and DPPH: DPPH radical scavenging (%). Different capital letters in the same column indicate significant differences (p < 0.05) and in DPPH scavenging activity lower case letters in the same line indicate significant difference (p < 0.05) for the different days of storage of the same ODFs.

et al., 2007). Fig. 5 shows the deconvolution of the amide I and II bands of the FTIR spectra of ODFs containing different concentrations of starch and gelatin. Table 2 shows the area values resulting from the deconvolution of the amide I and II bands. As expected, increases in the intensities of the amide I and II bands were observed for films with higher gelatin contents. However, the increase in the intensities of the amide I and II bands were not proportional to the increase of gelatin concentration, possibly due to the presence of adsorbed water, which has also a signal at ~1660 cm 1 (OH bend) (Riaz et al., 2018). The increase in the amount of starch in the films favored the adsorption of water molecules, which can be proven by the amplification of the O–H band (~3300 cm 1) in films with greater starch contents. The analysis of the amide I band in the FTIR spectra allowed the identification behavior of the secondary structure of the proteins (Bridelli, 2017). The results presented in Table 3 show that the gelatin collagen in the films retained a good portion of its predominantly α-helix structure, which presents an amide I band between 1659 and 1666 cm 1, and a supramolecular triple helix, with a signal between 1626 and 1629 cm 1. The gelatin-free film showed signals of amide I at 1606 cm 1, which were assigned to β sheets (26.51%), at 1626.39 cm 1 of the triple helix (14.50%) and at 1666.45 cm 1 of the α-helix (15.41%). In

3.8. Sensory evaluation The results of the sensorial evaluation of the ODFs with the addition of acerola powder (Fig. 6) presented values between 4 and 5, for all evaluated attributes (comfort, taste, mouth-feel properties and global acceptability). These results indicate that the tasters disliked slightly and neither liked nor disliked the orally disintegrating films with the addi­ tion of acerola powder. It was not possible to determine the best formulation from the results of the sensory analysis, possibly because of the concentration of the acerola powder (4 g/100 g of filmogenic solu­ tion), which was the same for all formulations, and because of films thickness, which also not presented significant difference between the different formulations. According to Scarpa et al. (2018), the perceived stickiness, disinte­ gration time and thickness in the mouth were considered the key attri­ butes for determining the acceptability of ODFs.

9

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Comfort Taste Mouth-feel properties Global acceptability

9 8 7 6 5 4 3 2 1 0

E

C

B

F

A

D Fig. 6. Sensory attributes of the ODFs containing acerola powder with different gelatin:starch ratios: A ¼ 100:0; B ¼ 80:20; C ¼ 60:40; D ¼ 40:60; E ¼ 20:80 and F ¼ 0:100.

4. Conclusion

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The incorporation of acerola (powder) in all formulations studied was feasible. The characteristics of the polymers used affect the prop­ erties of the films. The higher the starch concentration the faster the disintegration time of the films (in vitro and in vivo analyses), which could be related to the more hydrophilic characteristics of starch. All ODFs showed antioxidant capacity after 50 days of storage under drastic conditions. Global analysis acceptance of ODFs indicates that tasters “disliked slightly” and “neither liked nor disliked”, indicating its po­ tential use as carriers of natural active compounds. Acknowledgments ~o Paulo Research Foundation The authors would like to thank the Sa (FAPESP, 2013/03143–7) for the scholarship V.A.S.G. and R. A. Car­ valho would like to thank the National Council for Scientific, Techno­ logical Development (CNPq) for the productivity grant (307143/ ~o de Aper­ 2013–9). This study was financed in part by the Coordenaça feiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. References Abbas, O., Comp�ere, G., Larondelle, Y., Pompeu, D., Rogez, H., & Baeten, V. (2017). Phenolic compound explorer: A mid-infrared spectroscopy database. Vibrational Spectroscopy, 92, 111–118. https://doi.org/10.1016/j.vibspec.2017.05.008. Aguilar-M� endez, M. A., Martin-Martínez, E. S., Ortega-Arroyo, L., & Cruz-Orea, A. (2010). Application of differential scanning calorimetry to evaluate thermal properties and study of microstructure of biodegradable films. International Journal of Thermophysics, 31(3), 595–600. https://doi.org/10.1007/s10765-010-0735-7. Andrews, G. P., Laverty, T. P., & Jones, D. S. (2009). Mucoadhesive polymeric platforms for controlled drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 71(3), 505–518. https://doi.org/10.1016/j.ejpb.2008.09.028. Arkles, B. (2006). Hydrophobicity, hydrophilicity and silanes: Water, water everywhere is the refrain from the rhyme of the ancient mariner and a concern of every modern coatings technologist. Paint & Coatings Industry, 1. October.

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