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Brazilian Journal of Biology

Print version ISSN 1519-6984On-line version ISSN 1678-4375

Braz. J. Biol. vol.78 no.2 São Carlos May/Aug. 2018  Epub Aug 17, 2017

https://doi.org/10.1590/1519-6984.08716 

Original Article

Microencapsulation of essential thyme oil by spray drying and its antimicrobial evaluation against Vibrio alginolyticus and Vibrio parahaemolyticus

Microencapsulação do óleo essencial de tomilho e avaliação de sua atividade antibacteriana sobre V. alginolyticus e V. parhaemolyticus

O. Tomazelli Júniora 

F. Kuhnb 

P. J. M. Padilhac 

L. R. M. Vicentec 

S. W. Costac 

A. A. Boligond 

J. Scapinellob 

C. N. Nesic 

J. Dal Magrob 

S. Lamo Castellvía  * 

aDepartament d’Enginyeria Química, Universitat Rovira i Virgilli – URV, Av. Països Catalans, 26, 43007, Tarragona, Spain

bPrograma de Pós-graduação em Ciências Ambientais, Universidade Comunitária da Região de Chapecó – UNOCHAPECÓ, Av. Atílio Fontana, 591, CEP 89809-000, Chapecó, SC, Brazil

cCentro de Desenvolvimento da Aquicutura e Pesca – CEDAP, Av. Admar Gonzaga, 1188, Itacorubi, CEP 88010-970, Florianópolis, SC, Brazil

dLaboratório de Fitoquímica, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria – UFSM, Av. Roraima, 1000, Prédio 26, Sala 1115, Camobi, CEP 97105-900, Santa Maria, RS, Brazil


Abstract

The aims of this research were first, to evaluate the antibacterial potential of commercial thyme essential oil against V. alginolyticus and V. parahaemolyticys and second, using the spray drying technique to produce microcapsules. chemical compounds of thyme oil and microcapsules were identified and quantified being thymol the chemical component present at the highest concentration. Oil-in-water (O/W) emulsions were prepared and the microcapsules were obtained with a spray dryer using maltodextrin as wall material (ratio 1:4). Thyme oil and the microcapsules exhibited antimicrobial activity against V. parahaemolyticus and V. alginolyticus. The spray drying process did not affect the antimicrobial activity of thyme essentialoil.

Keywords: essential oil; Thymus vulgaris; Thymus zygis; bacterial control; aquaculture

Resumo

Os objetivos desta pesquisa foram avaliar o potencial antibacteriano do óleoessencial de tomilho sobre V. alginolyticus e V. parahaemolyticys e produzir microcápsulas através do processo de secagem por aspersão (spray dryer). Os compostos químicos do óleo essencial de tomilho e das microcápsulas foram identificados e quantificadaos. Foi preparada uma emulsão de óleo em água (O/A) e em seguida foram produzidas microcápsulas em um spray dryer utilizando-se óleo essencial de tomilho e maltodextrina como material de parede na proporção de 1:4 respectivamente. Entre os vários compostos identificados, o timol apresentou maior concentração. O óleo essencial de tomilho e as microcápsulas exibiram atividade antibacteriana sobre V. parahaemolyticus e V. alginolyticus. O processo de secagem por aspersão não afetou a atividade antibacteriana do óleo essencial de tomilho.

Palavras-chave: óleo essencial; Thymus vulgaris; Thymus zygis; controle bacteriano; aquicultura

1. Introduction

In the Americas are produced about 20% of total world production of shrimp (Lightner, 2011). White shrimp (Litopenaeus vannamei, Boone) represents 95% of total production. The countries leading this production are Ecuador, Brazil, Honduras and Mexico (Lightner, 2011). However diseases of viral and bacterial origin, has caused losses in crops, jobs, income and a decrease in exports over the past two decades.

Vibrio spp. mainly located in shrimp digestive tract, gills, cuticle and occasionally in its hemolymph are considered opportunistic pathogens, endemically associated with all stages of life (Lightner, 1993, 2011; Morales-Covarrubias, 2008). V. parahaemolyticus, V. alginolyticys, V. harveyi and Photobacterium daunselae are the mainly responsible of high levels of shrimp mortality in farms (Morales-Covarrubias, 2008). V. parahaemolyticus is linked to a recent illness in penaeid shrimp production known either as early mortality syndrome (EMS) or as acute hepatopancreatic necrosis disease (AHPND). V. parahaemolyticus affects post larvae with 20-30 days after stock and often cause 100% of mortality (De Schryver et al., 2014). In the state of Santa Catarina (Brazil) four shrimp farms were monitored and a constant presence of Aeromonas hydrophila, V. algynolyticus, V. parahaemolyticus and Pseudomonas luteola was reported (Padilha et al., 2013).

Controlling bacteria in shrimp, even in conditions of absence of symptoms are essential. They can cause mortality in conditions of stress and weaken the shrimp immune system (Padilha et al., 2013). Therefore, aquaculture industry needs to apply new strategies to control Vibrio spp. infections. An effective alternative to antibiotics and other synthetic compounds are plant compounds such as phenols, polyphenols, alkaloids, quinones, and also components present in essential oils such as terpenoids (Navarrete et al., 2010; Citarasu, 2010). Thyme essential oil has been proved to have antimicrobial, antifungal and antiviral activity (Dorman and Deans, 2000; Burt, 2004; Pina-Vaz et al., 2004; Rota et al., 2008, Nazzaro et al., 2013, Hernández-Hernández et al., 2014). The main problem of the essential oil components is that their biological activity can be lost by volatilization or degradation triggered by high temperatures, oxidation and UV light. Microencapsulation is one of the most efficient methods to protect solids, liquids or even gases against the surrounding environment into microscopic particles protected by a wall material (Soliman et al., 2013).

Spray drying quickly removes water by vaporization from oil-in-water (O/W) emulsions enabling high retention of volatiles (Badee et al., 2012) and protecting the microencapsulated compounds against reaction with the environment (Madene et al., 2006; Gharsallaoui et al., 2007). Microcapsules consist of a core surrounded by a single or a multilayered wall. Microcapsules with essential oil can be added into the shrimp feed as an ingredient or directly onto the pellet. For this purpose, the materials selected to produce the microcapsules need to be food grade and the powder easily adsorbed by the shrimp. Therefore, casein protein was selected as the emulsifying and maltodextrin as the wall material (Ramakrishnan et al., 2013). This mixture has proved to be very effective for the microencapsulation of oil/fats and volatiles (Sheu and Rosenberg, 1998; Ramakrishnan et al., 2013). The objectives of this research were i) to characterize the main components of thyme essential oil ii) to evaluate their antibacterial effect against V. alginolyticus and V. parhaemolyticus. iii) to use spray drying to microcapsule these components and iv) to study their antimicrobial effect against V. alginolyticus and V. parhaemolyticus.

2. Material and Methods

2.1. Identification and quantification of chemical compounds of pure and microencapsulated thyme essential oil by gas chromatography (GC-FID) -mass spectrometry (MS)

Gas chromatography was performed using a 6890N GC-FID system (AGILENT TECHOLOGIES, Santa Clara, California, USA), equipped with DB-5 capillary column (30m × 0.32 mm; 0.50 mm) and connected to a flame ionization detector (FID). GC-MS analyses were performed on an AutoSystem XL GC-MS system (AGILENT TECHNOLOGIES) operating in the EI mode at 70 eV, equipped with a split/splitless injector (220 °C). The relative amounts of individual components were calculated using their GC peak areas (Boligon et al., 2013).

2.2. Bacterial strains and growth conditions

Vibrio parahaemolyticus (ATCC 17802) and Vibrio alginolyticus (ATCC 17749) were obtained from American Type Culture Collection (ATCC, Rockville, USA). Cultures were transferred to brain heart infusion broth (BHI, Merck, Rio de Janeiro, Brazil) supplemented with 2% NaCl (Dinâmica, Diadema São Paulo, Brazil) at 37 °C for 24h. Then, growing cultures were stroked on BHI agar (BHIA, Merck, Rio de Janeiro, Brazil) supplemented with 2% NaCl (Dinâmica). The plates remained at 37 °C for 24 h. For inoculum preparation, 2-4 bacterial colonies were selected and transferred to a sterile 0.9% saline solution, followed by homogenization for 15 s. The turbidity of the solution was adjusted to reach 0.5 on Mc Farland scale (equivalent concentration of 1,5 × 108 cells mL-1)

2.3. In vitro antibacterial activity evaluation of thyme essential oil and microencapsulated thyme essential oil

Broth dilution method (CLSI, 2003) was selected to determine antibacterial activity of V. parahaemolyticus and V. alginolyticus. Stock solutions of 1 mg mL-1 for thyme essential oil were prepared with 2% dimethyl sulfoxide (DMSO). Moreover, a stock solution of 1 mg mL-1 of microencapsulated thyme essential oil was also prepared dissolving microcapsules in distilled water. To prepare this solution, the degree of oil entrapment in the microcapsules was taken into account. Four dilutions (0.53, 0.26, 0.13 and 0. 66 mg mL-1) for thyme essential oil and microencapsulated thyme essential oil were serially prepared in 1.9 mL of BHI broth (Merck, Rio de Janeiro, Brasil). Each test tube was inoculated with 0.1 mL of V. parahaemolyticus or V. alginolyticus and incubated at 37 °C for 24 h. BHI test tubes with and without 2% DMSO inoculated with V. parahaemolyticus or V. alginolyticus were used as positive control, as negative control were used BHI test tubes with and without 2% DMSO and BHI test tubes with and without 2% DMSO supplemented with 0.5% chloramphenicol. Bacterial growth was identified visually by turbidity of the medium in the tubes, compared with the controls and considered as the lowest concentration capable of inhibiting bacterial growth. Negative growth was confirmed by plating 0.1 mL of positive sample onto BHIA plates and incubated them at 37 °C for 24h. Minimum bactericidal concentration (MBC) was considered on the plate, which showed no bacterial growth. All tests were performed by triplicate

2.4. Preparation and emulsion characterization

O/W emulsions were prepared by mechanical stirring. The emulsifier, 4.17 g of casein sodium salt (CAS: 9005-46-3, SIGMA-ALDRICH, Saint Louis, MO, USA), was dissolved in 100.14 g of water and stirred at 300 rpm for 2h. Then, 20.9 g of thyme essential oil (white, Food Chemicals Codex, CAS: 8007-46-3, SIGMA-ALDRICH) was added to the solution (water + casein) and homogenized at ambient temperature with an Ultra-Turrax® (model T18, IKA, Staufern, Germany) at 15.000 and 20.000 rpm for 15, 30, 45 and 60 min to study the influence of homogenizer speed on the emulsion droplet-size distribution (Figure 1). The droplet-size distributions of the O/W emulsions were obtained by laser diffraction measurements using a Malver Mastersizer 2000 equipped with a Hydro 2000 SM dispersion unit (Malvern Instruments Ltd., Worcestershire, England).

Figure 1 Oil droplet size as d3,2 evolution with the time of speed for the production of thyme essential oil O/W emulsion. 

2.5. Spray drying

O/W emulsion was mixed with maltodextrin (dextrose equivalent 16.5-19.5 CAS number 9050-36-6, SIGMA-ALDRICH, Saint Louis, MO, USA), ratio 1:4 and stirring at 300 rpm for 2h. To obtain the microcapsules, the O/W emulsions were spray-dried using a laboratory scale dryer (Mini Spray Dryer – B290, BÜCHI, Flawil, Switzerland). The emulsion was fed into the spray-dryer at room temperature with a flow rate of 7.5 mL min-1. The inlet and outlet temperatures were maintained at 110 °C and 70 °C, respectively. The dried powder was collected and stored in opaque, airtight container at 4 °C for further analysis.

2.6. Powder particle size and microcapsule surface analysis

The particle size distribution (d3,2) of the spray-dried powders was determined using a Malvern Mastersizer 2000 (Malvern Instruments Ltd., Worcestershire, England) equipped with a Scirocco 2000 sample dispersion unit. Each sample was analyzed in triplicate and the data were reported as averages. The outer morphology of the microcapsules was characterized by electronic microscopy. An environmental scanning electron microscope (ESEM, FEI, Quanta 600) was used to analyze the surface morphology of the dried powders. Digital images were taken at an accelerating voltage of 20kV.

2.7. Total and surface oil determination

Total oil content of the microcapsules was determined by distilling 5 g of powder, dissolved in 150 mL distilled water for 3 h in a Clevenger-type apparatus. Two mL of ethyl ether (VETEC, São Paulo, Brasil) were added to extract the essential oil from the water phase. The solution was slowly brought to a boil and allowed to distill. The resulting oil was collected in a pre-weighed Erlenmeyer and the ethyl ether was allowed to evaporate at room temperatures for 24 h (Fernandes et al., 2014a). The oil encapsulation retention (%) was calculated using Equation 1:

Oil retention (%)= Total oil in the powderInitial oil load (dry basis) (1)

To determine the surface oil a modified method described by Varavinit et al. (2001) was selected. Twenty mL of hexane were added to 2 g of powder and the mixture was stirred at 300 rpm for 10 min. The suspension was filtered through a dried cellulose filter and was washed with 20 mL of hexane three times. The filter was kept in a desiccator under vacuum conditions to vaporize all residual solvent until obtaining constant weight. Finally, the oil encapsulation efficiency (OEE) was calculated using Equation 2 (Ramakrishnan et al., 2013):

OEE = Total oilSurface oilTotal oil × 100 (2)

2.8. Statistical analysis

Data analyses were performed using R (R Core Team, 2014) and the add-on package‘nls’. Three replicate experiments were run, with duplicate analysis in each of them.

3. Results and Discussion

3.1. Identification and quantification of chemical compounds of thyme essential oil by GC-FID

Twenty-one compounds were identified in thyme essential oil representing 99.65% of the total oil composition (Table 1). The main compounds in thyme essential oil were thymol (60.45%), p-cymene (19.58%), γ-terpinene (4.12%) carvacrol (3.08%) and β-caryophyllene (2.4%) (Table 1). Thymol and carvacrol are phenolic monoterpenes with antimicrobial activity and the ability to affect the outer membrane of Gram-negative bacteria such as V. parahaemolyticus and V. alginolyticus strains (Helander et al., 1998; Burt, 2004; Nazzaro et al., 2013).

Table 1 Composition of thyme essential oil and microcapsules obtained by spray drying. 

Compound RIa Thyme essential oil Microcapsules
RIb Oil % RIb Oil %
α-thujene 931 933 1.05 931 0.87
α-pinene 939 938 0.68 937 0.73
α-camphene 953 953 0.93 950 0.81
β-pinene 980 981 0.59 983 0.37
β-myrcene 991 989 1.04 992 1.83
α-terpinene 1019 1019 1.63 1019 0.79
p-cymene 1029 1027 19.58 1026 15.23
1.8 cineole 1033 1042 1.03 1035 1.46
γ-terpinene 1061 1060 4.12 1059 1.73
linalool 1098 1097 1.87 1098 1.95
camphor 1143 1143 0.03 1140 0.01
borneol 1166 1165 0.25 1161 0.47
terpin-4-ol 1177 1179 0.09 1176 0.28
α-terpineol 1189 1189 0.16 1185 0.09
thymol 1290 1293 60.45 1289 59.74
carvacrol 1298 1300 3.08 1298 8.12
thymol acetate 1355 1354 0.11 - -
β-caryophyllene 1418 1419 2.4 1423 1.54
α-humelene 1454 1453 0.31 - -
germacreno D 1480 1481 0.18 1483 0.13
α-cadidene 1538 1538 0.07 - -
Total identified (%) 99.65 96.15

Relative proportions of the essential oil constituents were expressed as percentages.

aRetention indices from literature (Adams 1995);

bRetention indices experimental (base on homologous series n-alkane C7-C30).

3.2. In vitro antimicrobial assays of thyme essential oil

Thyme essential oil exhibited antimicrobial activity against V. parahaemolyticus and V. alginolyticus. The MBC value for thyme essential oil was 0.125 and 0.25 mg mL-1 for V. parahaemolyticus and V. alginolyticus, respectively. There are few studies evaluating the action of thyme essential oil against Vibrio spp. in aquatic organisms farming. Among them Navarrete et al. (2010) and Heo et al. (2012) showed antibacterial activity of this essential oil against fish pathogenic bacteria such as V. parahaemolyticus.

3.3. Emulsion droplet size

The equation that describes the size of the particles in the emulsion as a function of time and the stirring speed is, y=N*eke*t , where y is the droplet size (d3,2), N is the intersection with the y axis, ke is the rate of decrease of particle size, and t is the stirring time (Figure 1). There was not interposition between the confidence interval for N and ke for both treatments, indicating differences between them (Table 2). The decay rate, ke was higher for treatment T2 with stirring speed of 20,000 rpm. Reducing emulsion size results in encapsulated powders with higher retention of volatiles and lower content of non-encapsulated oil at the surface of the powder particles (Ramakrishnan et al., 2013). According to these results, the speed of 20.000 rpm was selected to produce the smaller droplet emulsion size under the experimental conditions.

Table 2 Estimated N (intersection with the y axis) and ke (rate of decrease of particle size) parameters obtained using the equation y = Ne-ke*T

Rotor stator N ke
15.000 rpm (span 1.39) 4.60 ± 0.11a 0.0056 ±0.0007
20.000 rpm (span 1.29) 3.68 ± 0.10 0.0079 ±0.0008

aStandard Deviation (95%).

3.4. Powder particle size

The mean particle size of the dried power produced was 10.37 ± 0.0184 μ, and the spread of particles, calculated as span, was 1.679 showing homogeneity between the particles. Diameter of the microcapsules produced depended on the material properties, the concentration, the viscosity of the encapsulating material, and the operating conditions of the spray dryer (Jafari et al., 2008; Fernandes et al., 2014b). Their value range from 10 to 50 μm (Gharsallaoui et al., 2007). The particles exhibited spherical shape (Figure 2), which is typical of materials produced by spray drying (Fernandes et al., 2014b).

Figure 2 Environmental scanning electron microscope micrograh of thyme oil microcapsules produced with wall material maltodextrin:thyme essential oil load rate of 4:1 at magnification of 245 ×. 

3.5. Total oil, surface oil and oil encapsulation efficiency (OEE)

Contents of surface oil and total oil in the powders enabled determining the degree of oil entrapment in the capsules. Powders obtained in the study were characterized by microencapsulation efficiency at a level from 87.16% (Table 3).

Table 3 Encapsulation performance of thyme essential oil. 

Oil content in the emulsion
(g oil 100 g-1 emulsion)
Total oil
(g oil 100 g-1 powder)
Surface oil
(g oil 100 g-1 powder)
OEEa
(%)
Oil retention
(%)
19.25 11.05 ± 3.44b 1.42 ± 0.93b 87.16 ± 0.67b 57.37 ± 1.20b

aOil encapsulation efficiency;

bStandard Deviation (95%).

The microencapsulation essential oil efficiency (OEE) based on the values obtained for the total surface oil is one of the quality parameters used to determine the amount of oil successfully encapsulated by spray drying (Ramakrishnan et al., 2013).

Oil retention is defined as the ratio of total oil in the final powder to that of the initial oil in the emulsion on dry basis, and it was 57.37%. A similar result was found by Fernandes et al. (2014a) in rosemary essential oil microencapsulation by spray drying that evaluated the effects of gum arabic, modified starch and inulin in various properties of the powder.

3.6. Identification and quantification of chemical compounds of thyme oil microcapsules by Gas chromatography – mass spectrometry (GC-FID)

Eighteen compounds were detected in thyme oil microcapsules representing 96.15% of the total oil composition. The main compounds in thyme oil microcapsules were thymol (59.74%), p-cymene (15.23%), carvacrol (8.12%), linalool (1.95%) and β-myrcene (1.83%) (Table 1). The total oil content in thyme oil microcapsules after applying the spray drying process was 11.04% (Table 3). Thymol was the major constituent in thyme essential oil and in the microcapsules. The thymol concentrations in thyme essential oil and in the microcapsules were 60.45 and 59.74% respectively (Table 1). The microencapsulation by spray drying had a positive effect on the concentration of carvacrol, another phenolic monoterpene with significant antimicrobial activity, (Dorman and Deans, 2000; Rosato et al., 2007) increasing its concentration in 2.63 times approximately (Table 1). Compounds from the alcohols group, 1,8 cineole, linalool, borneol and terpin-4-ol, increased their relative concentrations after microencapsulation. The greatest increases were to terpin-4-ol and borneol with 3.11 and 1.88 times respectively. According to Baranauskiené et al. (2006) phenolic compounds and monoterpenes alcohols contain a hydroxyl group, which can form hydrogen bonds with relevant sites in the protein molecules, enhancing the retention of volatiles after spray drying. In addition, volatiles compounds without hydroxyl group reduced their relative concentration. This phenomenon may be explained by the relatively high temperatures used during the spray drying process. P-cymene and α-terpinene showed the highest reduction observed was to with 1.28 and 2.0 times respectively.

3.7. In vitro antimicrobial assays of thyme essential oil microencapsulated

Thyme essential oil microcapsules exhibited antimicrobial activity against V. parahaemolyticus and V. alginolyticus. The MBC value was 0.25 and 0.25 mg mL-1 for V. parahaemolyticus and V. alginolyticus, respectively. Therefore, the spray drying process did not have any negative effect on the antimicrobial activity of thyme essential oil and could be used to deliver this compound into shrimp feed without using emulsifiers than can be toxic. In fact, it has been reported that shrimps challenged with V. vulnificus, V. parahaemolyticus and V. cholerae, and subsequently fed directly with oregano essential oil had significantly reduced the numbers of these bacteria in their tissues (Gracia-Valenzuela et al., 2014).

4. Conclusions

The emulsion produced, the wall material used and the spray drying conditions were effective in keeping the major chemical compounds in the thyme essential oil microcapsulated and kept the antimicrobial action against V. parahaemolyticus and V. alginolyticys. Therefore, the production of thyme oil microcapsules by spray drying may be a promising technique to keep the chemical stability and antimicrobial activity of this essential oil and may be used in aquaculture to fight against the disease associated to V. parahaemolyticus and V. alginolyticys.

Acknowledgements

The authors would like to acknowledge FINEP - Recarcina, Ministerio Economía y Competitividad” project CTQ2014-54520-P and Departament d’Enginyeria Química from Universitat Rovira i Virgili from their financial support. In memoriam of Margareth Linde Athayde.

(With 2 figures)

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Received: June 20, 2016; Accepted: October 25, 2016

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