Microencapsulation of Grapefruit Oil with Sodium Alginate by Gelation and Ionic Extrusion: Optimization and Modeling of Crosslinking and Study of Controlled Release Kinetics.

Microencapsulación de aceite de pomelo con alginato de sodio por gelificación y extrusión iónica: optimización y modelado de la reticulación y estudio de la cinética de liberación controlada.

Presentación: 16/07/2020

Aprobación: 03/09/2020

Liliana M. Cáceres

Centro de Investigación en Química Orgánica Biológica, Facultad Regional Resistencia (FRRe), Universidad Tecnológica Nacional (UTN) - Instituto de Modelado e Innovación Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Nordeste (UNNE). Resistencia, Chaco – Argentina

lmarielc@yahoo.com.ar

Gustavo A. Velasco

Centro de Investigación en Química Orgánica Biológica, Facultad Regional Resistencia (FRRe), Universidad Tecnológica Nacional (UTN) - Instituto de Modelado e Innovación Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Nordeste (UNNE). Resistencia, Chaco – Argentina

gavelasco@arnet.com.ar

Eliana P. Dagnino

Centro de Investigación en Química Orgánica Biológica, Facultad Regional Resistencia (FRRe), Universidad Tecnológica Nacional (UTN) - Instituto de Modelado e Innovación Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Nordeste (UNNE). Resistencia, Chaco – Argentina

epdagnino@gmail.com

Ester R. Chamorro

Centro de Investigación en Química Orgánica Biológica, Facultad Regional Resistencia (FRRe), Universidad Tecnológica Nacional (UTN) - Instituto de Modelado e Innovación Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Nordeste (UNNE). Resistencia, Chaco – Argentina

mandhy@hotmail.com

Abstract

Essential grapefruit oil with high concentrations of limonene is used in food, cosmetic and pharmaceutical industries due to its antimicrobial properties, fragrance, and flavour. To facilitate its manipulation and protect it from adverse environmental factors, the microencapsulation is used. The objective of this work was to optimize the microencapsulation process of grapefruit essential oil using external ionic gelation coupled to extrusion with sodium alginate and calcium chloride. We achieved the best encapsulation conditions with calcium chloride concentration at 7.4% w/v and a crosslinking time of 58 minutes, obtaining a yield of 62% and an efficiency of 100% with an oil loading capacity of 10% w/w. The chemical adsorption of calcium as well during the crosslinking process was studied, observing a significant fit with the Elovich equation. And an adjustment of the controlled release of the essential oil was obtained to the empirical kinetic model of Korsmeyer and Peppas.

Keywords: sodium alginate, encapsulation, essential oils, ionic gelation, crosslinking.

Resumen

El aceite esencial de pomelo con alto conteniendo de limoneno tiene aplicaciones en la industria alimenticia, cosmética y farmacéutica debido a sus propiedades antimicrobianas, su aroma y sabor. Para facilitar su manipulación y protegerlo de los agentes ambientales externos se lo microencapsula. El objetivo de este trabajo fue optimizar el proceso de microencapsulación del aceite esencial de pomelo utilizando gelificación iónica externa acoplada a extrusión con alginato de sodio y cloruro de calcio. Los mejores resultados de encapsulación se obtuvieron con una concentración de cloruro de calcio de 7,4% p / v y un tiempo de reticulación de 58 minutos, obteniendo un rendimiento del 62% y una eficiencia del 100% con una capacidad de carga de aceite del 10% p / p. Se estudió la adsorción química de calcio en el proceso de reticulación observando un ajuste significativo con la ecuación de Elovich, y se obtuvo el ajuste de la liberación controlada del aceite esencial con el modelo cinético empírico de Korsmeyer y Peppas.

Palabras claves: alginato de sodio, encapsulación, aceite esencial, gelación iónica, reticulación.

Introduction

Grapefruit oil is an essential oil (EO) known as a sub product of the citrus industry, of great interest for its antimicrobial properties. It is a growth inhibitor of Escherichia coli, Staphylococcus aureus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. diacetylactis, Leuconostoc mesenteroides subsp. dextranicum and Lactobacillus plantarum (Vasek, et al., 2015; Ribeiro Santos et al., 2017), making it appropriate for the food and pharmaceutical industry.

Furthermore, this type of oil contains a high percentage of limonene (higher than 70%), which makes it attractive for the cosmetic and cleaning industries, and also applicable as a food additive and industrial solvent as well (Bakkali, et al., 2008). Citrus essential oils represent 0.27% of the total export from Argentina, so it could be considered as a product with great potential for industrial development (Federcitrus, 2018).

Industrial applications of EO are limited by their high degree of volatility, a characteristic that significantly affects the yield of these processes (Soliman et al., 2013). Furthermore, their sensibility to external agents like ultraviolet light, high temperatures, and water, may affect their composition, which facilitates the loss of some specific properties (Lv et al., 2012). As a matter of fact, it is necessary to develop a system that may protect them and facilitate their manipulation (Chen et al., 2017; Kuang et al., 2010).

Microencapsulation is a coating technique which may be used as a system of protection of the agent or coating component, to protect them from external factors. In the case of EO, it also allows small dosages (Benavidez, Cortés, Parada and Franco, 2016; Aghbashlo et al., 2012).

In recent years, there has been a growing interest in microencapsulation of EO with biodegradable materials. Applications were reported in the pharmaceutical industry (Balanc et al., 2016; Goh et al., 2012; Liakos et al., 2014; Liu et al., 2012), in the nutraceutical industry (Mokhtari et al., 2017) and in the food industry (Arriola et al., 2019; Laokuldilok et al., 2016; Lupo et al., 2015) for the immobilization of enzymes or bacteria in bioprocesses. (Ding and Shah, 2007; Shory, 2017; Aguiar et al., 2016).

Several methods of EO microencapsulation have been described for different authors, such as spray drying (Baranauskiene et al., 2006; Aghbasho et al., 2012; Tuyen et al., 2014), coacervation (Dima et al., 2014; Jun-Xia et al., 2011) and ionic gelation (Chan et al., 2000; Liakos et al., 2014; Hosseini et al., 2013).

Ionic gelation by extrusion is an encapsulation technique which presents great adaptability to different types of active principles, whether hydrophobes or hydrophilics, and is a low-cost procedure (Arriola et al., 2019).

Optimization of EO microencapsulation with alginate has been currently studied for its implementation in the food industry considering its antimicrobial properties, smell and taste (Noppakundilograt et al., 2015; Soliman et al., 2013; Goitia Funes and Amurrio Derpic, 2016). Furthermore, sodium alginate is one of the polymeric materials widely used as a matrix to encapsulate active principles due to its biocompatibility, biodegradability and inherent nature (Hosseini et al., 2013; Chuah et al., 2009). It is composed of β-D-mannuronate and α-L-guluronate units linked by 1-4 β-(1–4) guluronic acid units (Liakos et al., 2014; Levic et al., 2016). The alginate has the capacity of producing ionically cross-linked hydrogels, known as the “egg-box model” and generally accepted as the gelation mechanism (Lertsutthiwong et al., 2008). The crosslinking mechanism involves an intermolecular union between divalent cations (for example, calcium) and carboxyl groups in the residues of the guluronic acid of linear polymer chains (Belscak-Cvitanovic et al., 2016). The concentration of calcium chloride solution and the crosslinking reaction have an effect on the efficiency and yield during the encapsulation process. The matrix obtained after crosslinking can lead to a controlled release (Bal et al., 2013) with a release mechanism of the active components sustained in time (Finch, 2005).

In this study, we optimized the microencapsulation process of the grapefruit essential oil, a regional product with a high percentage of limonene, through external ionic gelation with sodium alginate by extrusion. We applied a two-factor experimental design with three levels in order to obtain a high concentration of EO. The concentration of calcium chloride and the crosslinking time were analyzed in terms of yield and efficiency of microencapsulation. We analyzed the controlled release as well, and the characteristics of the microcapsules.

Material and Methods

Material

It was employed a sodium alginate solution (Aldrich with 39% L-guluronic acid and 61% of mannuronic acid, molecular weight of 120,000-190,000 g/mol) and anhydrous calcium chloride (Cicarelli). Grapefruit essential oil was provided by Mager Enterprise (from the province of Corrientes, in the northeast of Argentina).

Chemical constitution of grapefruit essential oil

Identification of grapefruit EO components were conducted by gas chromatography-mass spectrometry (GC/MS). A two-capillary column QP 5050 Shimadzu equipment was used: one SE 52 (Mega, Legnano, Italia) chemically bonded (25 m x 0.25 mm internal diameter; 0.25 µm thick stationary phase) column, covered by 5% phenil-polydimethylsiloxane (0.25 µm thick stationary phase) at a column temperature of 60 ºC (8 min), increasing up to 180 ºC at a 3 ºC/min speed, then up to 230 ºC at a 20 ºC/min speed. 250 ºC injector temperature, split injection mode; 1:40 split ratio; 0.2 µl oil injection volume. Mobile phase: Helium, 122.2k Pa (51.6 cm/sec), 250 ºC interface temperature and 40-400 m/z mass range acquisition. Another BP-20 (SGE, Australia) 25 m x 0.25 mm internal diameter fused silica capillary column covered by polyethyleneglycol 20.000 Da (0.25 µm thick stationary phase). Column temperature at 40 ºC (8 min), increasing up to 180 ºC (3 ºC/min), and up to 230 ºC (20 ºC/min). 250 ºC, injector temperature, split injection mode; 1:40 split ratio; 0.2 µl oil injection volume. Mobile phase: Helium, 92.6 kPa (55.9 cm/s), 250 ºC interface temperature and mass range acquisition: 40-400 m/z. Fragmentation patterns in each component were compared to those stored in the software library spectra (Mc Lafferty and Stauffer, 1991; Adams, 2001).

Preparation of solutions and alginate-oil emulsion

A calcium alginate solution was dissolved in deionized water (0.2 µS conductivity, 40 ºC) at a concentration of 1% w/v by soft mechanical stirring. Then, the resulting solution was left standing for an hour in order to de-gas it. After that, the viscosity of the alginate solution was sized with a Ni-Run NDJ-8S digital viscometer using a spindle N° 0. The crosslinking solutions were then prepared with anhydrous calcium chloride in deionized water to 2, 5 and 10% w/v. Separately, 30 g of alginate solution was mixed with the specific amount of grapefruit oil necessary to obtain a 10% w/v load. The DLAB D500 homogenizer used was at 2000 rpm for three minutes to obtain a homogeneous distribution of the emulsion. To assure its stability, the solution was then left standing for an hour, observing if the separation of phases took place (Chan, 2011; Levic et al., 2015).

Microscopic observation

The degree of dispersion and homogeneity of the emulsion was determined by measuring the droplet size and calculating the polydispersibility (Mabille et al., 2000). These statements were applied following the Benavides et al. methodology (2016) with slight modifications. Degasification was then performed by putting the emulsion into an ultrasonic bath with a frequency of 40 khz (Digital Utrasonic Leaner, ARCANO) with water at room temperature (25 °C) for three minutes.

After that, the emulsion was observed with a trinocular microscope Lancet SME F6E equipped with a camera (Touptek UCMOS 01300 KPA, TP601300A model, origin China) and data acquisition system (Toup View: Touptek Photonics of Toupteck Corporation) to size the diameter of the droplets. The polydispersibility of the emulsion was calculated with the following equation (1) (Camino, Sánchez, Patino and Pilosof, 2012):

(1)

Where di is the droplet diameter and is the average droplet diameter of the emulsion.

Statistical analysis: Optimization of the encapsulation process

Experiments were conducted following a 3k central composite factorial design, with k=2, and analyzed according to the response surface methodology to estimate the polynomial quadratic equations of optimization (Kuehl, 2000). Calcium chloride (2, 5 and 10% w/v) and time (30, 45 and 60 minutes) were the factors selected, expecting that both were linked to the yield and efficiency in the obtention of microcapsules. All experiments were performed with a 10% w/w load of essential grapefruit oil and with a sodium alginate polymeric solution to 1% w/v. Two replicates of the design were carried out with nine runs each, fulfilling a total of 18 runs. The statistical analysis was performed with Minitab Statistical Software 18.1 (2017), trial. For this specific design, it was employed the following quadratic model (2):

(2)

Where y is the factor, β0 is the constant, βi are the mean linear coefficients, βii are the mean square coefficients, βij are the mean effect of interactions, x1 and x2 are the independent variables, and ε is the random error (Montgomery, 2003). Analysis of Variance (ANOVA) was then performed with a level of statistical significance of 95% (p<0.05). In addition, the regression coefficient (R2), the p-value, the F-value (Fisher Test) and the lack of adjustment were evaluated in order to determine the adjustment of the model. The two responses (yield and efficiency) were maximized. After that, the model was validated with five runs at the optimal point.

Obtention of microcapsules

Microcapsules were obtained following the methodology described by other authors with slight modifications (Chan, 2011; Benavidez et al., 2016). A syringe pump Apema PC11UBT was used to perform extrusion with a needle of internal diameter of 0.21 mm. For this part of the experiment, 10 ml of the emulsion previously prepared were taken with a syringe that was connected in the pump programmed at a dripping speed of 90 mL/h and fixed 5 cm above the surface of the solution. Afterwards, the drops fell into a calcium chloride solution which was prepared according to the design. The gelling solution was gently stirred with a magnetic stirrer over 30, 45 and 60 minutes. The obtained microcapsules were separated from crosslinking with a steel filter net and rinsed into tert-butyl alcohol in order to eliminate oil residues from the surface. Then, the microcapsules were weighed in a balance analyzer and stored in a caramel recipient at 4 °C.

Yield of the encapsulation process

The yield is the rate between the number of microcapsules obtained and the amount of the emulsion used during the encapsulation process. The percentage yield was calculated with the following equation (3) (González et al., 2005):

(3)

Where wm is the weight of the filtered microcapsules, wp is the weight of the polymer solution (sodium alginate) and wa is the amount of the active components added in order to prepare the emulsion.

Efficiency of the microencapsulation process

The efficiency of the microencapsulation process measures the amount of active components that were encapsulated into the microcapsule.

The calculation was based on the determination by gas chromatography of the microencapsulated limonene mass (Solomon et al., 2012) using the method of internal standards. In order to measure with a chromatography, the microcapsules were filtered with a steel sieve and rinsed with a considerable known amount of tert-butyl alcohol to remove the residual oil from the surface of the microcapsule. A sample of 30 µL of this solution was taken and then 10 µL of methyl isobutyl ketone were added as internal standard. This mixture was analyzed with a Shimadzu GC–14B gas chromatography, Megabore DB-WAX P/N 125-7032 polar column of 30 m long x 0.53 mm of internal diameter x 1 µ thickness, connected to a Flame Ionization Detector (FID). The FID temperature was of 220 ºC and for the injector temperature was of 180 ºC. The temperature of the column remained isothermal in 40 ºC for over two minutes. Then, it increased to 5 °C/min until reaching 180 ºC. Lastly, the temperature remained isothermal in 180 °C for over two minutes. The related areas of the chromatogram in a standard curve were compared in order to determine the limonene mass present on the surface (ws). Afterwards, the weight of the encapsulated limonene was calculated in a specific mass of microcapsules (w1) stating a difference between the weight of the initial (w2) and the superficial (ws) limonene. For this study, the efficiency value was determined as follows (4) (Maji, Baruah, Dube and Hussain, 2007):

(4)

Loss of water during the yield process

The moisture of the microcapsules was determined to evaluate the amount of loss of water during the crosslinking process. Almost 0.8 g of filtered microcapsules were weighed and then placed onto an aluminum tray. In order to determine their moisture, it was employed a Radwag PMR 50 moisture balance analyzer and the measurements were assayed in quintuplicate. The microcapsules used were prepared without oil, with 1% w/v alginate crosslinking in calcium chloride solution to 2, 5 and 10% w/v during the crosslinking time of 30, 45 and 60 minutes.

Morphology of the alginate microcapsules

Microcapsules were observed through an optical microscope equipped with a digital camera in order to measure the sphere diameter. The morphology of the microcapsules was evaluated with a Scanning Electron Microscope (SEM) Jeol 5800 LV. Microcapsules were then placed over the stage and sputtered with a thin coat of gold. Observations were made with different increments at room temperature.

Crosslinking calcium adsorption

After the optimization of the process of obtaining microcapsules, the kinetic yield with the optimized values of calcium chloride concentration and the crosslinking time were studied. Then, 1% w/v of alginate solution in deionized water was placed into the syringe pump, without essential oil, and it dripped on the crosslinking solution of calcium chloride 5% w/v. They were cross-linked during 1, 2, 3, 5, 15 and 30 minutes. Microcapsules were then filtered and rinsed in deionized water. Approximately 30 g of microcapsules were weighed and placed into a crystallizer to get dry. After that, they were placed in an oven at 100 °C for over 12 hours. The dried particles were then transferred to a clean and dried crucible and introduced in a muffle furnace at 750 °C for over eight hours or until they turned to ashes. After that process, dried calcium ions were valued by emission spectrophotometry using Jenway PFP07 flame photometer (Industrial Version). The data obtained was precisely revised in order to discover kinetic adsorption. To obtain this, we employed the Elovich model about the application of general processes of chemisorption, using the following equation (5) (Pinzón-Bedoya and Vera Villamizar, 2009):

(5)

Where, qt is the adsorbed amount in time t (mmol/g), a is the initial speed of adsorption (mol/gmin) and b is related to the covered surface and the energy of activation by chemisorption (mmol/g).

Study of the controlled release

The controlled release was performed by placing 8 g of microcapsules obtained during the optimized process in a 50 mL Erlenmeyer with 20 g of tert-butyl alcohol at room temperature with continuous stirring on an orbital shaker. Aliquots of 30 µL were extracted from the solution at regular intervals of 30 minutes until eight hours of release. Then, a diary sample was taken for two days, and after that, a weekly sample was taken until two weeks were completed. The samples were analysed in a gas chromatograph following the exact conditions described above. The experimental data were presented to make the mathematical model after that.

Kinetics analysis of drug release

The mechanism of essential oil release was studied from the matrix to the exterior fitting the data obtained to the semi-empirical model presented as follows (6) (Korsmeyer and Peppas, 1981):

(6)

Where Mt ⁄ M∞ is the fractional solute release, t is the release time, k is a constant characteristic of the macromolecular network system and the substance encapsulated, and n is the diffusional exponent characteristic of the release mechanism.

Results and discussion

Chemical constitution of essential oil

21 components were identified by GC/MS analysis: 9 terpenes, 3 sesquiterpenes, 2 aldehydes, 7 alcohols, and 1 ester (representing 99.10% of the components). The compound present in the highest proportion was limonene (92.6%) near the upper limit of the reported range (76.00-96.00%) for citrus essential oils (Kirbaslar, et al., 2006; Espina, et al., 2011). The complete characterization of the grapefruit essential oil used in this work was carried out in previous studies and published by the authors (Vasek, et al., 2015).

Preparation of solutions and alginate-oil emulsion

The viscosity of the alginate solution at 1% used in the emulsion at 20 °C with a rotor speed of 0.6 rpm was 0.35 kg/ms. This concentration of alginate was established in preliminary studies being ideal for extrusion due to the size of the needle selected. The emulsion prepared with the alginate solution and 10% w/w of grapefruit oil showed stability after an hour of standing, with no phase separation. Levic et al. (2015) reached similar results with grapefruit oil concentrations of 5 and 10% w/v. Chan (2011) demonstrated stability during the same time with oil concentrations from 5 to 40% v/v. Oil drops in the emulsion, observed in the microscope, have an average diameter of 0.0018 mm (Figure 1a), slightly smaller than the diameters obtained by Noppakundilograt et al. (2015) with similar concentrations of alginate emulsion and eucalyptus oil. The polydispersibility calculated with the equation (1) was 0.8, and this value (> 0.5) indicated that it was an emulsion with a broad distribution of diameters. Benavidez et al. (2016) obtained similar polydispersibility values but with lower homogenization speed and less oil concentration, and with a larger average diameter. Additionally, the asymmetry of the sample was calculated and showed a value of 0.36 indicating a positive asymmetry. Thus, it cannot be statistically represented by the sample mean. The mode of the distribution was then calculated, which showed a value of 0.0008 mm. This not-gaussian distribution responded to other distribution models (Sis et al., 2005) and it was a feature of the emulsions prepared at high speeds and with small droplet sizes.

Obtaining of microcapsules

It was possible to obtain spherical microcapsules with a uniform size (Figure 1b) using a dropping distance of 0.05 m and a dropping speed of 90 mL/h (established in preliminary studies).