Evaluation of the chitosan films of essential oils fromOriganum vulgare L (oregano) and Rosmarinus officinalis L (rosemary)

Artículo original

 

Evaluation of the chitosan films of essential oils from Origanum vulgare L (oregano) and Rosmarinus officinalis L (rosemary)

Evaluación de las películas de quitosano de los aceites esenciales de Origanum vulgare L (orégano) y Rosmarinus officinalis L (romero)

 

Johannes Delgado Ospina1* ORCID: http://orcid.org/0000-0001-8095-4741
Carlos David Grande2 ORCID: http://orcid.org/0000-0002-6243-4571
Leidy Vanesa Monsalve1 ORCID: http://orcid.org/0000-0003-4459-3306
Rigoberto C Advíncula3 ORCID: http://orcid.org/0000-0002-2899-4778
José Herminsul Mina4 ORCID: http://orcid.org/0000-0003-3767-4431
Mayra Eliana Valencia4 ORCID: http://orcid.org/0000-0001-9001-2045
Jingjing Fan5 ORCID: https://orcid.org/0000-0001-9853-9204
Debora Rodrigues5 ORCID: http://orcid.org/0000-0002-3124-1443

1Universidad de San Buenaventura Cali, Colombia.
2Universidad del Atlántico, Colombia.
3Case Western Reserve University, Estados Unidos.
4Universidad del Valle, Colombia.
5University of Houston, Estados Unidos.

 

*Autor para la correspondencia. Correo electrónico: jdelgado1@usbcali.edu.co

 

 


ABSTRACT

Introduction: Recent years have witnessed an increase in the demand for safe materials derived from biodegradable compounds useful to lengthen the shelf life of foods. Chitosan is a biological macromolecule with antimicrobial and antioxidant properties used alone or combined with natural ingredients such as essential oils for its capacity to lengthen the shelf life of foods. Origanum vulgare L. (Lamiaceae) and Rosmarinus officinalis L. (Lamiaceae) are two plants traditionally used in Colombia for medicinal and food purposes due to their bioactive and organoleptic properties.
Objective: Study the mixture of essential oils of O. vulgare and R. officinalis, Colombian varieties with chitosan films, to incorporate the antioxidant and antimicrobial properties of these films and thus prevent the decomposition and deterioration of foods.
Methods: Chitosan films mixed with essential oils were obtained. The essential oils were extracted from leaves of Origanum vulgare L. and Rosmarinus officinalis L. by hydrodistillation and incorporated with the films at concentrations of 0.4 and 1.5 % (v/v). An evaluation was performed of their mechanical properties, total phenolic content (TPC), antioxidant capacity and antibacterial activity against E. coli K-12 MG 1655 and B. subtilis 102.
Results: It was found that incorporation of the essential oils with the chitosan matrix reduces the mechanical properties of the films (tensile strength), increases their opacity (Y) and total phenolic content (TPC), and improves their antioxidant and antibacterial properties.
Conclusions: Results suggest that the presence of rosemary and oregano essential oils may increase the antioxidant and antimicrobial properties of the films, both of which are useful for food preservation. Therefore, they are recommended as a food packaging method. However, further experimentation should consider the forms of interaction between the films and the food item in question to better understand their effect on organoleptic properties.

Key words: antioxidant, antimicrobial, chitosan films, essential oils, Origanum vulgare L., Rosmarinus officinalis L.


RESUMEN

Introducción: En los últimos años se ha incrementado la demanda de materiales seguros derivados de compuestos biodegradables con el fin de prolongar la vida útil de los alimentos. El quitosano es una macromolécula biológica con propiedades antimicrobianas y antioxidantes que se utiliza sola o combinada con ingredientes naturales como los aceites esenciales por su capacidad para prolongar la vida útil de los alimentos. Origanum vulgare L. (Lamiaceae) y Rosmarinus officinalis L. (Lamiaceae) son dos de las plantas tradicionales utilizadas en Colombia con fines medicinales y alimenticios debido a sus propiedades bioactivas y organolépticas.
Objetivo: Investigar la mezcla de los aceites esenciales de O. vulgare y R. officinalis, variedades colombianas con película de quitosano, para introducir las propiedades antioxidantes y antimicrobianas de estas películas con el fin de evitar la descomposición y el deterioro de los alimentos.
Métodos: Se obtuvieron películas de quitosano incorporadas con aceites esenciales. Se extrajeron los aceites esenciales mediante hidrodestilación de las hojas de Origanum vulgare L. y de Rosmarinus officinalis L. y se incorporaron a las películas en concentraciones de 0,4 and 1,5 % (v/v). Se evaluaron sus propiedades mecánicas, el contenido total de fenoles (TPC), la capacidad antioxidante y la capacidad antibacteriana frente a E. coli K-12 MG 1655 and B. subtillis 102.
Resultados: Se encontró que la introducción de los aceites esenciales en la matriz de quitosano disminuye las propiedades mecánicas de las películas (tensile strength), aumenta la opacidad (Y) y el contenido total de fenoles (TPC), mejora las propiedades antioxidantes y antibacterianas de las películas.
Conclusiones: Los resultados sugieren que la presencia de los aceites esenciales de romero y orégano pueden aumentar las propiedades antioxidantes y antimicrobianas de las películas, propiedades útiles para la preservación de alimentos, por lo que se recomiendan como un método para aplicaciones de empaque. Sin embargo, futuros experimentos deben considerar las interacciones de las películas con el alimento para comprender su efecto en las propiedades sensoriales.

Palabras clave: antioxidante; antimicrobiano; películas de quitosano; aceites esenciales; Origanum vulgare L.; Rosmarinus officinalis L.


 

Recibido: 21/05/2017
Aprobado: 05/10/2018

 

 

INTRODUCTION

The food sector is constantly searching for chemical-free preservatives and low-cost effective methods to preserve food due to their advantages such as biodegradability and safety.(1) Different methods have been used for spoilage control due to microbial pathogens and oxidation, including fungicides, bactericides and modified atmospheres.(2) However, synthetic microbicides are associated with the formation of highly toxic chlorinated and sulfur compounds that create a cumulative effect and resistance in the microorganisms.(3) For that reason, alternative methods have been developed to inhibit microbial growth and oxidation, based on natural and safe materials such as chitosan films, including natural ingredients.(4) Chitosan has been described as a promising candidate for protective films in the food industry, because it has antimicrobial properties, is non-toxic and comes from renewable resources.(5)

By the other side, researchers have suggested that essential oils obtained from different aromatic plants have antimicrobial and antioxidant properties, which inhibit the growth of pathogens in food,(6) especially by their phenolic and terpenoid contents.(7)

The use of chitosan-essential oil composites to reinforce antioxidants and antimicrobials properties, has recently been reported.(8) In Colombia, the exploration of many plants for essential oil extraction is a very active area of research that could solve many economic lost from post-harvesting activities. From the traditional plants, Origanum vulgare L. (Lamiaceae) and Rosmarinus officinalis L. (Lamiaceae), are used in medicine and food applications due to their biological and organoleptic properties.

R. officinalis (Rosemary) is used as a poultice to treat eczema or accelerate the healing of wounds, and orally as an antiseptic, anti-inflammatory, and antispasmodic agent. O. vulgare (Oregano) is used orally in the treatment of respiratory diseases (laryngitis, tonsillitis, cough) and digestive condition (bile stimulant, eliminates intestinal gas), and externally it is used as operative and if swollen glands.(9)

Therefore, in the present study, we investigated the blending of essential oils of O. vulgare and R. officinalis Colombian varieties with chitosan films, to introduce antioxidant and antimicrobial properties of chitosan films to prevent spoilage and deterioration of food.

 

 

METHODS

Chitosan characterization

Chitosan of medium molecular weight (190-310 kDa) and 64.17 % of deacetylation was used (Sigma Chemical Co.). Fourier transform Infrared Spectroscopy (FTIR) was used to identify the functional groups of chitosan films. The FTIR analysis was performed in a wave range between 400 and 4 000 cm-1 by means of a Nicolet® 6700 spectrophotometer using the KBr disc.

Plants and essential oils

The plants Origanum vulgare L. and Rosmarinus officinalis L. were collected from crops from the southwest of Colombia (Cali, Valle del Cauca), classified e identified with the numbers PM122 and PM134, and presents in vivo collection of native medicinal, aromatic, and condiment plants at the Universidad de San Buenaventura Cali. The essential oils used were extracted of the leaves by the hydrodestillation process using a Clevenger tramp during 2 hours. The essential oils were dried over anhydrous sodium sulfate (Sigma Aldrich) and kept in sealed glass vials at 2 °C until further analysis.(10)

Gas Chromatography analysis of essential oils

The compositions of essential oils were determined by Gas chromatography-mass spectrometry (GC-MS) analysis in a gas chromatography spectrometer AT 6890 Series plus (Agilent Technologies, Palo Alto, California, USA), with a mass selective detector (Agilent Technologies, MSD 5975, Inert XL) (full scan). The analysis was carried out using DB-5MS fused silica capillary column (60 m x 0.25 mm x 0.25 µM, J&W Scientific Inc., Folsom, CA, USA). Temperature program used was: 10 min at 60 °C, then to 250 °C at 5 °C/min, held for 10 min. Other operating conditions were as follows: carrier gas, helium (99.999 %), with a flow rate of 1.1 mL/min; injection volume of 2:1 and split ratio 1:30. The identification of the main components of the essential oil was carried out using electron ionization (EI, 70eV). The masses were identified using the Adams database (Wiley, 138 and NIST05).

Preparation of chitosan films

Chitosan-based films were prepared according to a modified procedure reported by Ojagh.(11) Briefly, chitosan was dissolved in acid acetic (1 % v/v) until rich a concentration of 2 % (w/v) with constant agitation and 40 °C temperature. The solution was filtered through a Whatman No.3 filter paper to remove any undissolved particles. After filtration, glycerol was added to a concentration of 0.75 mL/g of chitosan and then was mixed for 40 min. Sodium lauryl sulfate (SLS) was added to obtain a concentration of 0.2 % (w/v) respect to the essential oils. The SLS was used as an emulsifier to assist dissolution of the oil in the film forming solutions. After 1h of stirring, the essential oils were added to the chitosan solution to reach a final concentration of 0 %, 0.4 % and 1.5 % (v/v), respectively. The solution was mixed vigorously at 7 000 rpm for 4 min using an IKA T25-Digital Ultra Turrax (Staufen, Germany). The solutions were degassed under vacuum for 5 min. The film forming solutions were cast on the center of petri dishes (15 cm diameter) and then dried for 30 hours at ambient conditions (25 °C). Dried films were peeled and stored in desiccators at 25 °C and 51 % relative humidity until evaluation.

Characterization of chitosan films

Mechanical characterization of chitosan films consisted by measurements of tensile strength and strain at break, according to ASTM D882-12.(12) Thermal properties of the films were determined by thermogravimetric analysis (TGA) on a TGA 2920 apparatus. Samples were heated up to 600 °C at a heating rate of 10 °C/min under a dry nitrogen atmosphere (flow rate of 80 mL/min). The glass transition temperature (Tg) was determined by differential scanning calorimetry (DSC) from the midpoint of the inflection tangent from the second heating at 10°C/min. TGA and DSC data were analyzed using TA Instruments' Universal Analysis software.

Colorimetry and opacity test

The measurements were performed depositing the films on the standard using a spectrophotometer (Konica Minolta CM600d. Osaka, Japan) with four repetitions. CIE Lab scale was used. The total color difference (ΔE) and whiteness (WI) were calculated as follow.

ΔE = ([ΔL]2 + [Δa]2 + [Δb] 2)0.5 (1)

WI = 100 - ([100 - L*]2 + a*2 + b*2) 0.5 (2)

Where ΔL = Lstandard − Lsample, Δa = a standard − asample and Δb = bstandard − b sample. Standard plate (L* = 89.63, a* = −0.16, and b* = 3.20) was used as a standard. Four measurements were taken on each film.

The opacity (Y) of the films was determinate according to ISO 2471,(13) calculated as the relationship among the opacity of each sample on the black standard (Yb) and the opacity of each sample on the white standard (Yw). This calculation was made automatically by the color data software CM-S100w Spectra Magic NX (Professional version 2.1).

Antioxidant activity

Total phenol content (TPC) was determined using the Folin-Ciocalteu method.(14) The antioxidant activity of the films was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method.(15)

Bacterial viability assay

Fluorescence imaging was carried out to determine the number of total and dead cells exposed to chitosan and chitosan with essential oils. For this experiment, circular coverslips with a diameter of 12 mm were cleaned with 70 % ethanol.

The clean coverslips were coated with the thin film by double-sided tapes. Each coated coverslip was deposited in a 12-well flat-bottom microtiter plate (Falon). Aliquots of 2 mL of the bacterial culture ( Escherichia coli K-12 MG 1655 and Bacillus subtillis 102) at 0.5 OD 600 nm in PBS were added into each well and then incubated for 1h (without shaking) at room temperature. After the incubation period, an aliquot of 10 mL solution was placed on a glass slide, stained, and imaged under a fluorescence microscope.(16)

For each well, three replicates were made, and six images were taken per each replicate to yield a total of 18 images per solution. To determine the total amount of live and dead cells in the bacteria-sample suspension, the solution was stained with a live/dead backlight bacterial viability kit (Invitrogen). After staining, the surface was placed on a microscope slide, covered with a coverslip (25 × 25 mm) and imaged using a BX 51 Olympus Fluorescence Microscope (Leeds Instrument Inc.) equipped with a DP72 digital camera under a 40x objective and a Fluorescein isothiocyanate (FITC) filter.

All images were acquired and analyzed using Cell Sens Dimension digital imaging software (Olympus). The percentage of inactive cells was expressed as the percentage ratio of the total number of inactive (red) cells to the total number of bacteria (green) attached. Average and standard deviation values were calculated on the percentages based on that cell count.

 

 

RESULTS

Characterization of chitosan and essentials oils

The functional groups of chitosan films used were identified. There is a band at 1660 cm-1 corresponding to the C=O stretching vibration of amides; a strong band rises at 3434 cm-1 for stretching of OH bonds overlapped with the bands of -NH2 groups. Stretching bands are present between 2926 cm-1 and 2883 cm-1 corresponding to -CH aliphatic. Other groups such as pyranose and C-O-C were found at 1067 cm-1 and 1021 cm -1 respectively, confirming the presence of the chitosan functional groups.

The analysis of the raw materials was complemented by the chemical composition analysis of essential oils. O. vulgare essential oil presented trans-sabinene hydrate (21.1 %), γ-terpinene (18.5 %), thymol (15.9 %), terpinen-4-ol (12.1 %) and sabinene (3.2 %) as the most predominant compounds. The R. officinalis essential oil analyzed, was mainly composed of β-myrcene (27.8 %), camphor (23.9 %), 1,8-cineole (16.2 %) and camphene (5.5 %) as the main compounds (Table 1).

Characterization of chitosan essential oil films

The films enriched with essential oil were thicker than the control films. Therefore, as the concentration of essential oils increases, the thicknesses also increased significantly (p< 0.05). The volatile content did not vary with the increasing of the concentration of essential oil, only films with R. officinalis at 0.4 % concentration was significantly lower (Table 2).

By the other side, tensile strength and strain at break are parameters representing the mechanical properties of the films, which depend on their microstructural characteristics which is affected by their chemical compositions. The addition of essential oils decreased significantly ( p< 0.05) the tensile strength of the chitosan films. Only chitosan-oregano essential oil films at 0.4 % (v/v) presented good tensile strength (8.53 ± 3.70 MPa). On the other hand, strain at break presented similar values between chitosan-oregano essential oil films and the control but was higher in chitosan-rosemary essential oil films (Table 2).

Thermal analysis

Thermal studies can improve the understanding of the polymer structure on the molecular level and all the interactions present.(5) Thermogravimetric analysis (TGA) of the chitosan-essential oil films showed the first thermal event beginning at 48.6 °C, attributed to the loss of moisture and more volatile components of the essential oils.

The treatments did not show significant changes compared to the control until the temperature of 152.48 °C, which is indicative that the addition of essential oil does not affect thermal properties, indicating that chitosan-essential oil films can be extended to other uses such as packaging.

The second important thermal event begins at 152.48 °C corresponding to the degradation of chitosan through the C-O bands(17) and the third important degradation occurs at 248.39 °C attributed to chitosan depolymerization.(18) After the second thermal event, no significant variations between treatments are appreciated, which is due to the formation of covalent bonds of some components of essential oils with chitosan, which modifies the degradation temperature of the films (Fig.).

Color properties

The addition of the essential oils increased significantly opacity parameter of the films. Similar results were reported previously.(8,11) An increase in film opacity, as a consequence of the addition of antioxidant, has also been reported in fish-gelatin films containing borage extract.(19)

Chitosan-rosemary essential oil films did not differ significantly from control in most of the parameters, only there was a significantly increase in the opacity (Y). Films with O. vulgare showed greater changes (a slight yellowish color than the control). Finally, an increase in the oil concentration decreased luminosity (L) and whiteness (WI), while was increased b*, ΔE and, Y (Table 3).

Antioxidant capacity

Chitosan-essential oil films had higher phenol content as compared to the control film. Furthermore, the phenol content increased with the addition of essential oils in the films, which had been reported by other authors.(20) For instance, at 1.5 % films with O. vulgare essential oil had higher total phenol content than the films with R. officinalis essential oil (0.1098 ± 0.04 mg GAE/g film and 0.0523 ± 0.01 mg GAE/g film, respectively). Although the TPC content is lower than results reported in the literature, the general trend remains.(20)

The method of DPPH radical has been widely used to test the ability of compounds as free radical scavengers or hydrogen donors, for the evaluation of the antioxidant activity.(21) The reduction percentage (radical scavenging) of films increased significantly (p< 0.05) with the addition of essential oils as compared to the control film, however, higher oil concentrations do not provide better results (Table 4).

Antimicrobial activity

The chitosan-essential oil films presented higher microbial inactivation as compared to control films against Gram-negative (E. coli) and Gram-positive (B. subtillis) bacteria (Table 4). Regarding the behavior of both chitosan-essential oil films in the inactivation of E. coli and B. subtillis, chitosan-rosemary essential oil film presented a better microbial inactivation than chitosan-oregano essential oil films for both bacteria, which is important for packaging applications as a barrier for microbial spoilage. From the results, it is apparent that the relationship between the concentration of essential oils in the films and the percentage of inhibition is directly proportional for both essential oils, enhancing the antimicrobial properties. In general, the responses of all the films to bacteria inactivation were higher against the Gram-positive (B. subtillis) than with the Gram-negative (E. coli).

 

 

DISCUSION

Characterization of chitosan essential oil films

Film thickness is an important factor in food packing applications, since affects water barrier and mechanical properties of the films.(22) Films thickness is mainly influenced by the solid content of the film-forming solution, provided by essential oils and chitosan.(21) The incorporation of the oils also improves barrier properties against water, due to the hydrophobic nature of monoterpenes and sesquiterpenes in the oil.

The main function of food packing, is to avoid moisture migration between the food and the surrounding atmosphere,(23) for instance, moisture content affects food packaging processing due to the sensitivity of the films to the water.(24)

Chitosan essential oil films presented higher volatile content than control films, mainly affected by essential oils and water. Some of the chemical compounds of the EO are in free form as observed in the first thermal event occurred at 48.6 °C (Fig.) and others linked in the matrix through stronger bonds that break at a higher temperature (Fig.). The moisture content decreased with the increasing concentration of essential oils. According with Ojagh(11) and Park(22) the decreasing can be attributed to a compactness of film network, because essential oils can cause the formation of covalent bonds between the functional groups of chitosan chains, leading to a decreasing in the availability of hydroxyl and amino groups and limiting polysaccharide-water interactions by hydrogen bonding and resulting in a decrease of moisture content of films.(11)

Mechanical properties

It was observed that the tensile strength and strain at break of the chitosan-oregano essential oil films decreased with the increasing of essential oils concentration. This behavior is similar to that reported by Peng(25) where chitosan films were enriched with lemon essential oil. In contrast, chitosan-rosemary essential oil films showed that the tensile strength decreased and the strain at break increased with the oil concentration increasing. Similar results were reported by Abdollahi.(20) It is well known that with the decreasing crystallinity of the structure, the mechanical properties also decreased by the addition of the essential oils. When the essential oil is incorporated into the polymer, the present oil components may create discontinuities and alterations in the polymer matrix and the polymer chain, leading to a weak mechanical response.(26) Otherwise, it has been found that essential oils added into chitosan films decreased tensile strength but did not affect the elongation percentage which can be attributed to the chitosan type and its interactions with the essential oil components.(27)

The physical properties of chitosan films, such as moisture and thickness were affected by the presence of the oils. In general, the R. officinalis essential oil led to thicker and higher moisture content films.

Color properties

The color of the film is a determining factor for the customer due to its general appearance and consumer acceptance.(20,28) Generally, films were transparent and differences in color could be ascribed to the natural yellow of essential oils.(8) These results suggested that films are adequate in terms of color for food packaging applications.

Thickness, moisture content, and colorimetric analysis are consistent with a good quality film for food packaging applications.

Antioxidant capacity

The antioxidant capacity is very important for food preservation and is generally assigned to natural phenolic compounds.(29) Then, the antioxidant activity is explained by polyphenols acting as antioxidants by donation of a hydrogen atom by interrupting hydroperoxide conversion into radicals or by chelating redox-active metal ions.(30,31) In the case of O. vulgare essential oil, phenols were found in high concentrations such as thymol, carvacrol, carvacrol methyl ether and thymol methyl ether, while in R. officinalis essential oil phenols were found in lower amounts (< 0.1 %). Similar results were reported by incorporating essential oils of Zatari multiflora and Thymus moroderi, attributing this behavior to the presence of large amounts of phenols in the essential oils, which benefit the free radical scavenging, preventing the films oxidation.(32,33)

It should be noted that the small positive value founded for the chitosan film control could be attributed to the reaction of the Folin-Ciocalteu reagent with non-phenolic reducing compounds (sometimes nitrogen and oxygen-based compounds).(32,33) The scavenging mechanism of chitosan is described as a form of stable macromolecule radicals and ammonium (NH4+) groups due to the reaction of free radicals with residual free amino (-NH2) groups.(34) They are also responsible for the percentage inhibition of DPPH found in the films without the addition of essential oil.

The increase in the antioxidant capacity is due to the phenolic and non-phenolic compounds present in the essential oils, for example, R. officinalis essential oil is rich in myrcene (27.8 %), which had greater antioxidant activity, supported on a high free radical scavenging capacity.

The chitosan-oregano essential oil films presented a higher antioxidant capacity as compared to chitosan-rosemary essential oil films but both antioxidant capacities were relatively low.

Antimicrobial activity

Packaging plays a prominent role in food conservation, in the case of fresh or processed food, the microbial contamination occurs mainly in the surface, therefore inhibition of bacteria growth is a concern for maintaining food quality.(23) It has been discussed that the main reason of antibacterial activities of essential oils can be related to the hydrophobicity of essential oils due to the hydrophobic phenolic constituents(35) and also it can be attributed to components such as camphor, carvacrol and some others,(36,37,38) however, it is also widely accepted that a synergistic effect between various components, regardless of their dominance could be the reason for the antibacterial properties.(39) Nevertheless, it is important to highlight that essential oils have different antimicrobial mechanisms that affect microbial cells such as changes in the genetic material of bacteria, attack of phospholipid bilayer of the cell membrane, disrupt of enzyme systems and cytoplasm coagulation by damage in lipids and proteins.(6)

In the case of the antioxidant and antimicrobial properties, the presence of essential oils enhanced both properties. The chitosan-rosemary essential oil films presented higher antimicrobial properties against E. coli K12 MG 1655 and B. subtillis 102. The antimicrobial activity against B. subtillis was in all cases higher than the activity against the growth of E. coli. These results suggest that the presence of Colombian R. officinalis and O. vulgare essential oils can enhance antioxidants and antimicrobial properties of chitosan films, which are very useful to preserve foods. However, further experiments considering the interactions of the films with food are necessary to understand the effect of the sensorial properties of food.

 

Acknowledgements

The authors acknowledge Instituto Colombiano de Investigaciones Científicas (COLCIENCIAS), for economic support to travel to the CWRU University for the realization of this project.

 

 

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Author contributions

Johannes Delgado Ospina, Leidy Vanesa Monsalve and Carlos David Grande: Conceptualization and essential oil characterization and antioxidant activity.

Leidy Vanesa Monsalve, José Herminsul, Mayra Eliana Valencia Mina and Rigoberto C. Advíncula: Chitosan films characterization.

Jingjing Fan and Debora Rodrigues: Bacterial viability assay.

Johannes Delgado Ospina, Leidy Vanesa Monsalve and Carlos David Grande: Writing original draft preparation.

Johannes Delgado Ospina, Debora Rodrigues and Carlos David Grande: Writing review and editing.

Rigoberto C. Advíncula: Funding acquisition.

 

Conflicto de intereses

Los autores expresan que tienen conflicto de intereses.





Copyright (c) 2019 Johannes Delgado Ospina, Carlos David Grande, Leidy Vanesa Monsalve, Rigoberto C Advíncula, José Herminsul Mina, Mayra Eliana Valencia, Jingjing Fan, Debora Rodrigues

Licencia de Creative Commons
Este obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial 4.0 Internacional.