Oregano (Origanum vulgare leaf Oil) and sage (Salvia officinalis vulgare leaf Oil) essential oils (were supplied by Esencias Lozano, SA, Spain), originating from Europe, were used as the core material. 2,4-toluene diisocyanate (TDI, was supplied by Alfa Aesar) and 1,6-hexamethylenediamine (HDMI, was supplied by Panreac) were used as monomers for polyurea wall-forming materials. Tween 80 (was supplied by Panreac) and Poly (vinyl alcohol) (PVA, Mw, 1500) (was supplied by Sigma Aldrich) as the stabilizing agent.

W/O (water/oil) emulsion was formed between organic solution with TDI oregano/sage oil and aqueous solution of 1% PVA tween 80 as a stabilizer. Table 1 shows the experimental parameters.

The emulsification was carried out at 6000 rpm for 30 min with homogenizer (Benchtop, Pro. Scientific Inc., Oxford). Subsequently HDMI was added, the stirring speed was decreased to 600 rpm. After reaching 30˚C and maintaining it (Fisher Scientific, Spain), and left in reaction for 120 min more, were formed the polyurea microcapsules. 150 mL of distilled water was added into the solution to disperse them. The obtained microcapsules were filtered, washed with 10% ethanol and dried in an oven at 40˚C for 24 hours.

For surface observation, a PHENON scanning electron microscope (FEI Company, United States) was used. Each sample was fixed on a standard sample holder and sputter coated with gold. Samples were then examined with suitable acceleration voltage and magnification.

The particle size distribution of the microcapsules was measured by a Mastersize E-3000 (Malvern, UK). The particle size was expressed as the equivalent volume diameter and three replicates were performed for each batch of microcapsules, to reduce error, an average curve was calculated and analyzed. Particles were dispersed in ethanol at a ratio of 1:40 w/v.

Differential scanning calorimetry (Mettler-Toledo 821, Mettler-Toledo Inc., Schwerzenbach, Switzerland) was used to determinate the glass transition temperatures (Tg) of the dried microcapsules. Approximately 10 mg of sample was prepared in aluminium pans in ambient atmosphere. The curves were obtained according to the following heating program: 0˚C and 220˚C at 5˚C/min.

Thermogravimetric (TGA/SDTA 851, Mettler-Toledo Inc., Schwerzenbach, Switzerland) was used to determinate the stability of the microcapsules in high temperatures. Approximately 70 μl of samples were prepared in aluminium pans. The thermal program was located between 0˚C and 220˚C at 10˚C/min under nitrogen gas with a flow rate of 60 mL∙min⁻¹.

Samples were analyzed in an Infrared spectra VARIAN FT-IR 670 with a 0.10 cm⁻¹ resolution for each infrared spectrum. Spectra were collected in ATR mode.

A trace 1300 GC gas chromatograph equipped with a programmed split/splitless injector, an AI 1310 automatic sampler, and coupled to a single quadrupole mass detector (ISQ LT) was used to perform the GC-MS analysis (all from Thermo Scientific, San José, CA, USA). A Zebron ZB-5MS (30 m × 0.25 mm i.d., 0.25 μm thickness) column was used (Phenomenex Torrance, CA, USA). Helium was used as carrier gas, at a flow rate of 1 mL/min.

The injector temperature was 250˚C. Samples were injected in the split mode 1:30 (w/v). The injection volume was 1 μl. The oven temperature was programmed as follows: 40˚C for 2 min, then 310˚C at 7˚C/min, maintained for 5 min.

Samples were washed with ethanol because the polyurea is insoluble in ethanol; the objective was to remove the superficial oil [22] .

Approximately 1 g of sample was dispersed in 30 ml of ethanol. Washing was performed in three consecutive steps of 10 mL collected extracts separately. The amount of essential oil surface was calculated through a formula used for successive extractions.

For the quantification of the oil it is necessary a calibration curve with patterns. For this, the necessary weights are taken (1 mg dissolved in 1 mL of Ethanol) to make stock solutions of 1000 mg/mL in each of the essential oils.

From these solutions the calibration solutions (n = 4) are performed for each of the compounds (sage and oregano). The concentration range is 0.001 - 1 g/g of essential oil.

The measurements were made with a 7890 A gas chromatograph coupled to mass spectrometer (MS) XL 5975C inert. Agilent Technologies (CA, USA).

For this analysis was necessary to break the microcapsules and allow the release of the oil. 0.1 mg of washed microcapsules were taken and were dissolved in 10 mL sulfuric acid (pH = 0.8).

The monitoring of the release of the essential oil concentration of various compounds present in the oils was followed by analyzing samples collected during 120 hours. The measurements were made with a 7890 A gas chromatograph coupled to mass spectrometer (MS) XL 5975C inert. Agilent Technologies (CA, USA).

The release kinetics for each microcapsule is adjusted to order 1, and from the equation constants it has been found liberation and life time media release.

Oregano and sage essential oils and the microcapsules were tested to determinate antifungal/antibacterial activity, based on Standard ASTM E 2149-13. The contact time of the material with fungi was 24 h and the culture medium used was Sabouraud Dextrosa Agar (Scharlab, Spain). The contact time of the material with bacteria was 24 h and the Culture medium used was plate Count Agar (Scharlab, Spain). The Incubation temperature was 35˚C ± 2˚C.

The morphology of the microcapsules was studied by scanning electron microscopy (SEM). Figure 1 shows the micrograph of oregano microcapsules and Figure 2 shows the micrograph of sage microcapsules obtained by

different conditions.

Figure 1 and Figure 2 evidences that at lower concentrations of polymer (a) and (b) are formed microcapsules but are not entirely closed and their morphology is very irregular, however, at higher concentrations of polymer (c) the obtained microcapsules are spherical, in this case the concentration of oil used also influences the morphology, a higher concentration of microcapsuled oil shows the best morphology (d).

The SEM images analyzed previously demonstrated the spherical shape, and although the size was determined as variable and it could have been estimated, the particle size test offers objective and more precise measurements. Figure 3 represents the size distribution for microcapsules studied in this paper.

Depends on the oil the size distribution changes, oregano microcapsules are bigger than sage microcapsules. Its sizes reach 100 µm while sage size microcapsules only reach 25 µm.

Thermal analysis has been studied in order to determine the microcapsules thermal characteristics and predict their thermal behaviour.

Figure 4 and Figure 5 show the DSC thermograms of the oregano/sage oil, the polymer from the shell and the microcapsules containing both oils and the shell respectively. In Figure 4, it can be observed an endothermic transition at 190˚C, this temperature is attributed to the decomposition temperature of oregano oil. Microcapsules have and endothermic transition at 190˚C and polyurea at higher temperatures than 220˚C. These temperatures demonstrate that drying temperature doesn’t affect the microcapsules.

In Figure 5, it can be observed an endothermic transition at 150˚C; this temperature is attributed to the decomposition temperature of sage oil. Microcapsules have and endothermic transition at 130˚C and polyurea at higher temperatures than 220˚C. These temperatures demonstrate that drying temperature doesn’t affect the microcapsules.

Figure 6 and Figure 7 show the TGA thermograms of the oregano and sage oil, the polymer (shell) and microcapsules.

Oregano oil shows the decomposition temperature at 190˚C, and at 160˚C starts to decompose. This results matches with DSC analysis. In Figure 6, it can be observed three curves corresponding to the oregano oil, polyurea as polymer used as wall material and oregano microcapsules obtained by interfacial polymerization with polyurea shell.

The weight loss starts at 140˚C in microcapsules due to the indoor humidity evaporation, because the outdoor humidity is removed in the drying process to turn them into powder form. In polyurea shell, the weight loss starts at 200˚C, due to the outdoor humidity [23] .

Sage oil shows that it is thermally stable up to 70˚C, and the decomposition temperature is at 95˚C. The weight loss starts at 110˚C in microcapsules due to the indoor humidity evaporation, because the outdoor humidity is removed in the drying process to turn them into powder form.

FTIR test was conducted in order to characterize the microcapsules. Figure 8 shows the spectra for three compounds, oregano oil, the polyurea and the microcapsules (oregano PYA).

When the study is centred in the region between 3800 - 2000 cm⁻¹, two important zones can be observed. The first one is comprised between 3800 - 3100 cm⁻¹ and is attributed to O-H bonding, the second one is located around 3100 - 2600 cm⁻¹ and is assigned to C-H stretching. All the samples show picks at both regions. Thus, it can be attributed to the oregano oil, which is composed in majority of carvacrol and timol [24] -[26] .

When the oil has been encapsulated, the picks moved towards a different wavenumber. The polyurea shell shows a movement of the O-H stretching pick towards a lower wavenumber due to the presence of N-H in the polymer which is located around 3200 - 3300 cm⁻¹. The pick attributed to polyurea oil is more sharpened than the rest of the picks.

On the other hand, Figure 9 shows the spectra for three compounds, sage oil, the polyurea and the microcapsules (sage PYA).

In the sage spectrum it can be observed different picks due to main compounds, at 2950 cm⁻¹ which is attributed to CH₃ stretching, at 1750 cm⁻¹ which is assigned to C=O stretching, in the region between 1390 - 1375 cm⁻¹ it finds C-(CH₃)₂ group and at 750 cm⁻¹ is attributed an aromatic group flexion, all this is characteristic to the camphor compound [24] [26] .

Also appear in the spectrum bands characteristic to the compound 1,8-Cineol at: 1374 cm⁻¹ due CH₃ (CO), 1214 cm⁻¹ produced by stretching of the COC group and between 984 - 843 cm⁻¹ due to stretching of the CH groups [24] .

Table 2 and Table 3 show the main components that were identified in the samples of oregano and sage essential oil by cromatography.

The determination of oils on microcapsules surfaces was performed starting from a calibration curve with different concentrations of oil in ethanol. The compounds selected in the chromatography study were used.

Table 4 shows the results of oil on surface microcapsules.

In the study of oil release was taken into account the percentage of theoretical encapsulated essential oil (w/w) and the oil calculated on the microcapsule surface.

Theoretical encapsulated essential oil is 4 ppm (if all the oil had been encapsulated)

0.1 mg of encapsulated (0.40% w/w) in 10 mL of the solution (pH = 0.8) = 4.0 μg∙mL⁻¹.

Monitoring of the release of microcapsules was studied analyzing the concentration of various compounds distributed along the chromatogram. Polyurea membrane was deteriorated very quickly, therefore the oil release is produced in the first period of time. Figure 10 and Figure 11 showed the controled release of oregano and sage microcapsules respectively.

Given release kinetic equations can be attached to different kinetic orders (order 0, order 1, order 2). The best regression was obtained for one order kinetic release (1)

where:

C = concentration;

T = time.

The constants found for each of the oils in each of the cases were as follows:

From constants has been possible to determinate the half-life time (T_1/2) release (in hours) using an aggressive solution to break the shell (2).

According to the results, it is possible to conclude that the essential oils (oregano and sage) are encapsulated and that the oils are released completely when the membrane is broken, yielding different half-lives of retention of oils in the solutions before complete volatilization.

Oregano and sage essential oil were tested as an antimicrobial and antifungal. They presented an excellent reduction at the bacteria and fungi test. Results are showed in Table 5.