Aims: To study how the antimicrobial and antioxidant activities of Lippia graveolens essential oils with different composition are affected after the microencapsulation process with β-cyclodextrin (βCD).
Methods and results: Three Mexican oregano essential oils (EOs) with different carvacrol/thymol/p-cymene ratios (38 : 3 : 32, 23 : 2 : 42, 7 : 19 : 35) were used in this study. Microencapsulation was carried out by spray-drying. Antimicrobial activities were measured as MBC (minimal bactericidal concentration) using 0·05%/0·10%/0·20% (w/v) dilutions of EOs against Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538. Antioxidant activities were determined by the 2,2′-diphenyl-1-picrylhydrazil (DPPH) method. EOs showed antimicrobial and antioxidant activity, but microencapsulation preserved the antimicrobial activity in all cases and increased the antioxidant activity from four- to eightfold.
Conclusions: Although the Lippia essential oils were from the same species, their composition affects the biological activities before and after the microencapsulation process, as well as encapsulation efficiency. Our study supports the fact that microencapsulation of EOs in β-cyclodextrin preserves the antimicrobial activity, improves the antioxidant activity and acts as a protection for EOs main compounds.
Significance and Impact of the Study: Microencapsulation affects positively EOs main compounds, improves antioxidant activity and retains antimicrobial activity, enhancing the quality of the oils.
In these days of ‘green consumerism’, people are demanding more organic foods and a reduction of chemical preservatives for food conservation (Leite et al. 2006). A possible alternative to chemical-based preservatives used in the food industry could be essential oils (Burt 2004; Fisher and Phillips 2006).
Microencapsulation in βCD is an alternative process to protect the essential oil from light, air and humidity, because these are interactions that can lead to oxidation or volatilization, reducing the biological activities of EOs. In addition to preventing these undesirable reactions, microencapsulation increases the solubility of the oil in water, prevents the release of the oil at an undesired stage and makes the oil easier to handle (Liolios et al. 2009).
The use of oregano essential oil, from different species and diverse geographical sources, as a natural antimicrobial compound has been widely published (Burt and Reinders 2003; Burt 2004). The antioxidant activity of oregano essential oil has also been studied before (Gortzi et al. 2007; Rocha et al. 2007), although most of the work has been carried out with essential oil from the Origanum genus. There are few studies on Mexican oregano that measure the antimicrobial activity of its essential oil (Obledo et al. 2002). How the particular environmental conditions and phenotypical differences in the Mexican oregano plants affect the oregano essential oil composition has been reported, including a comparison between the composition of these oils and their antimicrobial and antioxidant activities (Paredes et al. 2007). However, how the microencapsulation process affects the biological activities when using β-cyclodextrin (βCD) as wall material has not been studied, nor has the effect of the oil’s composition on the encapsulation efficiency. In the earlier studies, comparisons have normally been made between two or more different essential oils from distinct species.
In this work, three Mexican EOs with different chemical compositions were studied, because of the great heterogeneity in the composition that exists between the plants of this species (Lippia graveolens) and the difficulty in homogenizing the thymol + carvacrol content of the EO as this plant is most of the time harvested in the wild. Also, it is important to know how the microencapsulation in βCD, being a process involving heat, affects the EOs composition and biological activities which are both studied in this work, as well as how the antimicrobial activity against two gram-negative and one gram-positive pathogen food related bacteria (Leite et al. 2006) is influenced by these changes.
Materials and methods
Oregano essential oils
As mentioned earlier, three Mexican oregano essential oils with different carvacrol/thymol/p-cymene ratios were used and they are as follows: EO1 (38 : 3 : 32), EO2 (23 : 2 : 42) and EO3 (7 : 19 : 35). EO1 was obtained from an 8-year-old Lippia plantation cultivated in a greenhouse. EO2 and EO3 were obtained from Lippia plants collected in the wild, but from different regions of Chihuahua, México. All the EOs were subjected to a water removal process in a vigreux distillation apparatus. All EOs were harvested in October 2007.
Spray-drying and encapsulation efficiency
Emulsion of the EOs with βCD (CPIngredientes, Guadalajara, Mexico) was prepared according to Baranauskienéet al. (2006), with the exception of using 14·3% (w/w) of essential oil. The emulsion was spray-dried in a Büchi B-191 Mini Spray-Dryer and fed at room temperature with an inlet air temperature of 105°C and a pump flow of 1·1 ml min−1.
The quantity of oil entrapped within the βCD microcapsules was determined by distilling the microcapsules (oil was previously removed from surface) for 2 h in a Clevenger apparatus (Bertolini et al. 2001), which gave us the encapsulation efficiency, expressed as a percentage of oil entrapped in the microcapsules.
Scanning electron microscopy (SEM)
Morphology of the microencapsulated powders was examined by scanning electron microscopy. The microencapsulated samples were deposited onto specimen stubs. A sputter coater was used to cover the samples with gold. The specimen stubs were placed into the electron microscope JEOL JSM-5400LV, using the following operational conditions: objective aperture 10 μm, accelerating voltage 5 kV–15 kV and 1000× magnification.
Volatile compounds of pure EOs and those recovered from the microencapsulated oregano essential oils (MOs) were determined using a Hewlett Packard 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a flame ionization detector, and a HP 6890 Series autosampler. Separation was carried out on a 50 m × 0·20 mm ID × 0·33 μm film thickness capillary column HP-FFAP (Hewlett Packard, Folsom, CA, USA). An initial oven temperature of 60°C was increased to 220°C at a rate of 2·5°C min−1 and held for 10 min; the carrier gas was helium at a flow rate of 1·2 ml min−1. Injection volume was 0·5 ml using a split ratio of 1 : 150. The temperatures of the injector and detector were 250 and 280°C, respectively. Retention times and peak areas were automatically computed by the HPGC Chemstation software (Rev. A.06·03). Identification of volatile compounds was carried out according to Castillo-Herrera et al. (2007). The results were expressed in percentage of the peaks area.
Antimicrobial activities assays
Preparation of bacterial cells.
All micro-organisms were cultured in nutrient broth at 37°C for 24 h and then diluted in Müeller–Hinton broth at a density adjusted to 0·5 McFarland turbidity standard [1–2 × 108 colony forming units (CFU ml−1)].
Determination of MBC.
EOs were previously diluted in ethanol to ensure solubility in broth; however, there was no need for a previous dilution of MOs, as βCD increases solubility of the oil in water. From these samples, aliquots were taken and placed in tubes with fresh Müeller–Hinton broth, taking into account MOs encapsulation efficiency to respect the 1 : 1 ratio of essential oil used, so the final samples concentrations were 0·05, 0·10 and 0·20% (w/v). Mixtures were inoculated with the bacterial cell suspensions and incubated at 37°C for 24 h. Because of the turbidity generated by βCD, 0·1 ml of this broth was poured on Müeller–Hinton agar and left to incubate under the same conditions. Two positive controls, one without samples and one with ethanol were used to ensure the quality of the tests. The antimicrobial testing was conducted in duplicate. MBC is the lowest concentration of antimicrobial agent at which colonies failed to grow after incubation (Sato et al. 2006).
Antioxidant activities assays
A determination of the antiradical activity (ARA) of both pure essential oil and its microcapsules was carried out using the DPPH radical scavenging method, with equal quantities of the EOs and MOs powders (0·5% w/w). Thus, 0·5% of EOs was used and 0·03, 0·05 and 0·06% of MO1, MO2 and MO3, respectively, were used. Samples were diluted in 96% ethanol and then deposited on a microplate in the presence of DPPH. DPPH (5·9 mmol L−1) diluted in 100 ml of 80% methanol was used as a target (Kulisic et al. 2004). Samples were left to incubate for 3 h at 27°C in the dark for further absorbance analysis at 520 nm in a microplate absorbance reader.
Data were statistically analysed with the programs spss (ver. 15·0, 2001) and Statgraphics centurion (vers. XV.II, 2006). One-way analysis of variance was performed, assuming that there were no statistically significative differences as the null hypothesis. Tukey test was applied. The probability level used was P = 0·05.
Spray-drying and encapsulation efficiency
Average encapsulation efficiency showed variations according to the EO encapsulated. MO1, which had the higher thymol+carvacrol content, showed a lower encapsulation efficiency (53·90%) when compared to MO2 (75·85%) and MO3 (81·03%) even though the three EOs were samples of Mexican oregano from the same origin.
Morphology and size of the microencapsulated powders
Microencapsulated particles did not show visible fractures, cracks, or pores. Only wrinkles were present on the larger microcapsules. All the small size particles presented a spherical shape, and the bigger microcapsules showed a diversity of shapes, from being ovoid to spherical with surface dents (Figure 1). The size of the microcapsules containing EOs varied, with 0·71–20 μm for MO1 being the smallest size interval of the three samples. MO3 varied in size from 1·42 to 28·14 μm and MO2 varied from 1·07 to 38 μm (the largest particle size).
Composition of the EOs before and after microencapsulation
Compound p-cymene was reduced in the MOs compared to EOs. On the other hand, carvacrol and thymol content, was higher in MOs than in EOs, which can be explained by losses of more volatile compounds, such as p-cymene The composition of EOs before and after microencapsulation is shown in Table 1.
Table 1. Main compounds (%) of EOs before and after microencapsulation
EOs from microcapsules
Values within rows followed by the same letter do not differ statistically at P = 0·05.
Antimicrobial activities of the EOs and its microcapsules
MBCs (Table 2) for E. coli showed no differences when using pure EOs, but microencapsulation decreased the MBC fourfold for MO1, and twofold for MO2 and MO3. Against Ps. aeruginosa, all EOs, MO2 and MO3 showed the same MBCs; however, MO1 decreased the MBC twofold with respect to EO1. MBCs for Staph. aureus were identical for EO2 and EO3 but better for EO1, and the MBCs were lower by twofold after microencapsulation for MO1 and MO2, while remaining the same for MO3 when compared to EO3.
Table 2. Minimum bactericidal concentrations of oregano essential oils and its microcapsules (% w/v)
Antioxidant activities of the OEOs and its microcapsules
Before encapsulation, EOs showed an antiradical activity above 80%. After the encapsulation process, the ARA percentage varied from 38·10 to 45·81% (Table 3). EO1 presented the higher ARA before the encapsulation process and MO1 kept the higher ARA percentage when compared to the other MOs, maintaining 52% of the EO1 antiradical activity. MO2 and MO3 maintained 46%/49% of the EO2/EO3 antiradical activity.
Table 3. Antiradical activities percentage of pure EOs and its microcapsules
Values within the columns followed by the same letter do not differ statistically at P = 0·05.
MO2 and MO3 showed an encapsulation efficiency similar to those achieved with oregano flavour from Origanum vulgare L. (27 : 7 : 30) encapsulated in skimmed milk protein (80·2%) and whey protein concentrate (71·8%) as reported by Baranauskienéet al. (2006). All microcapsules had the same solid/oil proportion and were homogenized and spray-dried within the same parameters. The difference in the encapsulation efficiency is assumed to be because of the EOs physicochemical properties, which are determined by its composition and how the size of the essential oil molecules fit into the matrix.
The particle size of the microencapsulated powder may be as well a significant factor in encapsulation efficiency, as MO1 had the lowest content of oil entrapped within the matrix and the smallest particle size. This could mean that the surface area increased and more EO1 could have adhered to its exterior causing an efficiency reduction.
Changes in the composition of EOs occurred after microencapsulation. In this case, the increase of carvacrol and thymol could be because of losses of the more volatile compounds (data not shown), mainly p-cymene. Normally, these three components comprise the majority in an oregano essential oil and the biological activities studied in this work have been related mainly to thymol and carvacrol.
In most cases, the MOs were more effective for inhibiting bacterial growth than EOs. This improvement could be because of an increment of the antimicrobial compounds given by a decrease of the non antimicrobial compounds, which may have a low volatile point. Also, the antibacterial activity improvement may be related to the increased solubility of the oils in water, improving stabilization and bioavailability of the guest molecule (Polyakov et al. 2004) in broth mixture, drastically modifying the physical, chemical and biological properties of the encapsulated molecules (Mourtzinos et al. 2008). Gortzi et al. (2007) observed an increased inhibition of micro-organism’s growth after microencapsulating essential oil of the Origanum genus in another matrix using a different microbiological technique. A relationship between the thymol+carvacrol composition in the MOs and the MBCs was observed for all the micro-organisms used, as reported by Burt (2004) and Baranauskienéet al. (2006). When the thymol+carvacrol composition is higher, inhibition of the micro-organism’s growth is better. For pure EOs, only Staph. aureus presented this thymol+carvacrol inhibition behaviour using EO1 as the inhibiting agent. Other studies have shown that Ps. aeruginosa is more resistant to essential oils containing thymol and carvacrol (Origanum vulgare) than either of the other two micro-organisms used (Hammer et al. 1999). This study confirmed that observation.
The percentage of oil retained in the microencapsulated powder (v/w) was 7·70% in MO1, 10·84% in MO2 and 11·58% in MO3 when compared to the 14·3% initially added to the emulsion. An ARA remainder of 46, 49 and 52% in the MO2, MO3 and MO1, respectively, with respect to the EOs means that microencapsulation increased the ARA fourfold when using MO2 and MO3 as both maintained almost half of the ARA with 8–10 times less oil. With respect to MO1, the increase in the ARA was above eightfold, as the sample containing these microcapsules had 16 times less oil and maintained half of the EO1 antiradical activity. The increase in the antioxidant activities could be because of the changes in the composition of pure oils because of the spray drying process as well as improved bioavailability and a lower degradation rate of the oils. This behaviour has been observed in different encapsulated essential oils as Gortzi et al. (2007) previously reported.
In conclusion, the results obtained in this study showed that the thymol+carvacrol content affects encapsulation efficiency, because of the physicochemical properties that its composition confers to each EO, and therefore every EO should be considered as a different oil, unless there are similarities in thymol+carvacrol content. Also, microencapsulation increased the antioxidant activity in all cases, meaning that less oil can be used to obtain the same antiradical activity, which improves the quality of the EOs. The antimicrobial activity showed an absence of loss in all cases, depending on the sample and the micro-organism against which it was used. This makes microencapsulated EOs potential agents to inhibit dangerous pathogens as well as water soluble disinfectants for ‘organic’ foodstuffs. This occurs without loss of antimicrobial activity and with an improved antioxidant activity. Further analysis should be carried out to study how the microencapsulated oregano essential oil inhibits micro-organism growth in situ and directly on foodstuffs.
We thank Dr. Israel Ceja Andrade from the CUCEI (Universidad de Guadalajara) for his help in the SEM tests and Winston Smith for his assistance in the language revision of this work. We thank SAGARPA for the partial financial support towards this project (SAGARPA: Clave 821, 2002) and finally, thanks to CPIngredientes for providing the βCD used in this work.