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Article

Enhancing the Extraction Process Efficiency of Thyme Essential Oil by Combined Ultrasound and Microwave Techniques

by
Adina I. Gavrila
,
Ciprian G. Chisega-Negrila
,
Laura Maholea
,
Mircea L. Gavrila
,
Oana C. Parvulescu
and
Ioana Popa
*
Faculty of Chemistry Engineering and Biotechnologies, National University of Science and Technology Politehnica Bucharest, 011061 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(9), 2331; https://doi.org/10.3390/agronomy13092331
Submission received: 17 August 2023 / Revised: 4 September 2023 / Accepted: 5 September 2023 / Published: 6 September 2023
(This article belongs to the Special Issue It Runs in the Family: The Importance of the Lamiaceae Family Species)
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Abstract

:
In this paper, the essential oil (EO) extraction from thyme by the consecutive use of ultrasound and microwave treatments is presented. The aim of this study was to apply an ultrasound pre-treatment of thyme leaves to enhance the thymol content and the extraction yield of the EO obtained by microwave-assisted hydro-distillation (MWHD). Compared with conventional hydro-distillation (CHD), the consecutive use of ultrasound pre-treatment and microwave extraction resulted in a 72% lower extraction time. When the ultrasound pre-treatment (using the ultrasonic processor with an amplitude of 70%) was applied, the EO content was 23% higher compared to the extraction without pre-treatment (2.67 ± 0.06 g EO/100 g DM for the MWHD with ultrasound pre-treatment compared with 2.18 ± 0.07 g EO/100 g DM for the MWHD without pre-treatment). The EO samples were analyzed by GC/MS. The results showed that the major component, thymol, varied from 43.54% (by CHD) to 65.94% (by the consecutive use of ultrasound and microwave treatments).

1. Introduction

Thyme (Thymus officinalis L.) is a plant that belongs to the Lamiaceae family and is used for food flavoring and preservation. This plant can be found in Europe, North Africa, and Asia [1]. Thyme is rich in essential oil (EO), which is part of the secondary metabolites of the plants (up to 2.5% EO for the dried herb [2]). It is involved in the defense mechanism of the herb (repelling the phytophagous organisms) and in attracting pollinators. Among the constituents of thyme EO (carvacrol, p-cymene, α-pinene, γ-terpinene, etc.), thymol is the major one [3]. This compound has been shown to have antioxidant and anti-inflammatory [4], antibacterial [5], antimicrobial [6], antifungal [7], antiparasitic [8], immunological [9], and anticancer [10] properties. Thyme also contains polyphenolic compounds (rosmarinic, caffeic, p-hydroxybenzoic, and procatechuic acids) and carotenoids (β-carotene, lutein, and zeaxanthin) [11]. These bioactive compounds enhance the valuable properties of thymol, providing thyme extracts with a beneficial effect on human health. Also, if added to foodstuffs, thyme EO can improve their quality. For example, Medina et al. proved that thyme EO decreased oxidation in supplemented minced beef [3].
The extraction of EOs from plants can be performed by conventional methods, such as cold pressing [12], hydro-distillation, and steam distillation [13]. These methods present several drawbacks, such as long extraction times, high energy and solvent consumption, etc. [14]. Thus, in recent years, in order to overcome these shortcomings, alternative methods have been developed. The new approaches include microwave-assisted hydro-distillation [15], ultrasound [16] and enzymatic [17,18] pre-treatments before the extraction of EOs, supercritical [19] and subcritical [20] fluid extractions, solvent-free extraction [21], and microwave hydro-diffusion and gravity extraction [22,23].
Microwave-assisted hydro-distillation (MWHD) combined with the ultrasound pre-treatment of aromatic herbs to extract EOs was successfully employed [24,25]. This strategy combines the advantages of ultrasound and microwave extraction. The latter can provide volumetric and selective heating. During microwave irradiation, the heterogeneous extraction mixture is heated as a whole volume and the vegetal material can be heated selectively. Although the microwave heating of extraction mixture occurs rapidly, in the overall rate of the process, the mass transfer is limited [26,27]. On the other hand, the cavitation phenomena can promote the breakage of the cellular tissue, which will increase the mass transfer rate [28].
Our work describes the extraction of EO from thyme leaves by the consecutive use of ultrasound and microwave treatments. Thus, the aim of this study was to apply an ultrasound pre-treatment of the extraction mixture in order to increase the thymol content and the extraction yield of the EO obtained using MWHD. The influence of the ultrasound pre-treatment on the extraction process of EO was studied using both an ultrasound bath and an ultrasonic processor. To our knowledge, MWHD combined with ultrasound pre-treatment to extract EO was not used for thyme leaves. There are only a few studies regarding the ultrasound pre-treatment of thyme, and neither of them reported the comparison between different sonication equipment. Kowalski et al. applied an ultrasonic pre-treatment of the thyme leaves using an ultrasound bath, but the EO was further extracted by conventional steam distillation. In this study, a maximum yield of 2.4% (v/w) was achieved, meaning approximatively 2.15 g EO/100 g of dried plant material [29]. Roldan-Gutierrez et al. used an ultrasonic processor for the pre-treatment of thyme leaves, but the extraction of EO was further performed by steam distillation and superheated water methods [30].

2. Materials and Methods

2.1. Materials

Fresh thyme (stems and leaves) was purchased from Hofigal (Bucharest, Romania). The fresh leaves and stems were chopped into pieces of 1–2 cm, then dried in an air flow-heating oven (Memmert UNE 500 Universal Oven, Memmert GmbH + Co. KG, Schwabach, Germany) at 60 °C to a constant weight. Part of the dried vegetal material was used as such, and another part was ground using an electric grinder and screened to a particle size under 0.1 cm. The ground thyme was dosed in samples of 100 g (in sealed plastic container) and stored at 4–5 °C until extraction of EO. The water content of the dried thyme leaves was 7.7% (w), being determined with the help of a moisture analyzer (PMB 202 Moisture Analyzer, Adam Equipment Co., Ltd., Bletchley, UK). For the GC-MS quantification of thyme EO, the following standards were used: thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene from Merck (Merck & Co., Inc., Darmstadt, Germany).

2.2. Methods

2.2.1. Essential Oil Extraction Procedure

The extraction of EO from thyme was performed by MWHD using a multimode microwave oven (Plazmatronika, Plazmatronika NT SP Z.O.O., Wroclaw, Poland), which has a frequency of 2450 MHz. This apparatus is described in our previous work, Calinescu et al. [17]. During the experiment, temperature, time, and power were controlled via an operating console. Steam produced in the reactor carrying the thyme EO was directed to a modified Neo Clevenger trap with a 10 mL graduated tube. The extractions were carried out at different ratios of solvent to plant: 8/1, 10/1, and 12/1 (v/w). The particle size of vegetal material was varied, as follows: pieces of 1–2 cm or particles under 0.1 cm. The solvent used was distilled water and the mixture was subjected to extraction until no EO was obtained (80 min). The protocols for the MWHD method are described in our previous work [17]. Comparative extraction of thyme EO by conventional hydro-distillation (CHD) method was also performed. The conventional extraction was carried out for 180 min (after this time no EO was obtained) at a solvent to plant ratio of 12/1 (v/w). The separated EO was kept at 4 °C until GC-MS analysis. The extraction yield was expressed as grams of EO per 100 grams of dry matter (g EO/100 g DM).

2.2.2. Ultrasound Pre-Treatment of Plant Material

Before MWHD, the vegetal material mixed with the solvent (distilled water) was subjected to ultrasound pre-treatment for 30 min. The pre-treatment was performed using either an ultrasound bath (ES375H Bench Top Ultrasonic Tank, Hilsonic Ultrasonic Cleaners, Birkenhead, UK) with a volume of 3 L, a power of 120 W, and a frequency of 40 kHz, either an ultrasonic processor (Vibracell VCX-750, Sonics & Materials, Inc., Newtown, CT, USA) with a power of 750 W, a frequency of 20 kHz, and a titanium horn with a diameter of 13 mm. The sonication of the extraction mixture, using the ultrasonic processor, was applied with a duty cycle of 5 s on and 5 s off for an amplitude of 50% or 70%. The sonication for the ultrasonic bath was applied continuously. Control samples without any pre-treatment were directly subjected to MWHD or CHD.

2.2.3. GC-MS Analysis of the Thyme Essential Oil

The separated EO was analyzed by GC-MS method. The GS-MS apparatus, a Thermo Electron Corporation Focus GC system (Thermo Fisher Scientific S.p.A., Milan, Italy), is described in our previous work Calinescu et al. [17]. The main constituents of thyme EO (thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene) were identified according to retention times of known standards. Identification of the other constituents of thyme EO was performed by comparing the samples’ spectral peaks with spectra from Wiley database. The main constituents of thyme EO were chosen based on preliminary tests that were part of a research project (PN-III-P2-PED-2019-2118, project: “IMUNOSTIM”, no. 381PED/2020). Absolute amounts of the individual components were calculated based on GC peak areas, and were expressed as milligrams per 100 grams of dry matter (mg/100 g DM).

2.2.4. Statistical Analysis

All measurements were performed in triplicate, and the data were expressed with standard deviation (SD) as mean value ± SD for triplicate of samples (n = 3). All the results achieved at different levels of process factors were subjected to univariate one-way ANOVA and multivariate principal component analysis (PCA). Statistical analysis of the data was performed using the multiple comparison Duncan’s post hoc tests in order to determine the significant statistical differences between the averages of the main components of two or more independent groups. To evaluate the strength of linear correlations between dependent variables, the Pearson correlation coefficient (r) was used [31]. The differences were considered statistically significant at p < 0.05. Statistical analysis was conducted using XLSTAT Version 2019.1 (Lumivero, Addinsoft, New York, NY, USA).

3. Results

3.1. Microwave-Assisted Hydro-Distillation (MWHD) vs. Conventional Hydro-Distillation (CHD)

The first step to optimize the extraction process of thyme EO consists of kinetics study of the control samples obtained by MWHD and CHD (Figure 1). As shown in Figure 1, the time required to achieve the reflux temperature (the time after the EO starts to be collected into the Clevenger apparatus) for MWHD (10 min) is lower compared with CHD (60 min). This can be explained by the volumetric heating of microwaves compared with the conventional one, which occurs by convection [27]. The MWHD leads to a rapid increase in EO content in the first 40 min (which corresponds to 83% of the total yield), achieving a maximum amount after 70 min (1.73 ± 0.03 g EO/100 g DM). For CHD, the EO content increases slowly, obtaining 1.65 ± 0.06 g EO/100 g DM after 170 min. Further, the experiments were performed by MWHD.
The thyme EO was analyzed and identified by GC-MS. The results, for both methods (MWHD and CHD), are shown in Table 1.
For both methods, approximately 12 compounds were identified. As shown in Table 1, the main constituents of thyme EO are thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene. These compounds constitute between 93% and 97% of the total amount of resulted EO. Considering the EO composition, there is only a slight difference between the two extraction methods. Thymol is extracted more efficiently by MWHD, while γ-terpinene is extracted more efficiently by CHD. This difference can be explained by the bioconversion of γ-terpinene in thymol, which is influenced by different factors, one of them being the extraction method [32]. The same behavior can be noticed for p-cymene, α-terpinene, and β-pinene, which are extracted more efficiently by CHD. Krause et al. proposed a biosynthetic pathway of thymol, starting from the cyclization of geranyl diphosphate forming γ-terpinene. The latter is oxidized by the P450 monooxygenases enzyme to cyclohexadienol intermediates. These products are unstable being dehydrogenated by a dehydrogenase/reductase enzyme to allylic ketone intermediates. Further, through keto-enol tautomerisms, thymol is formed [33].

3.2. Influence of Solvent to Plant Ratio on the Extraction of Thyme Essential Oil

The next step to optimize the EO extraction from thyme was to study the influence of solvent to plant ratio. To establish the solvent volume required to achieve as much EO as possible, different ratios of solvent to plant material were used. Moreover, to avoid degradation of thyme leaves due to direct microwave irradiation, an adequate amount of water is required. As shown in Figure 2a, the amount of EO increases with the increase in the water volume. The most efficient solvent to plant ratio for the extraction of thyme EO by MWHD is 12/1 (v/w).
The resulted EO, for all solvent to plant ratios, was analyzed by GC-MS. The main compounds of thyme EO identified by GC-MS are shown in Figure 2b. It can be noticed that the EO component content is similar for all three ratios and the amount of thymol is directly proportional with the solvent to plant ratio. Thus, a high amount of water is required to entrain the constituents with high boiling points. The ANOVA analysis showed that by increasing the solvent to plant ratio from 8/1 to 10/1, the EO content increases significantly (p < 0.05). By the further increase in the solvent to plant ratio, the EO content increases non-significantly (Figure 2a). Regarding the thymol extraction, the ANOVA analysis showed that by increasing the solvent to plant ratio, the thymol concentration increased significantly (p < 0.05, Figure 2b).
Although the amount of EO is similar for both 10/1 and 12/1 ratios, further experiments were carried out for a 12/1 (v/w) solvent to plant material ratio since the amount of the targeted compound (thymol) is higher and the specific energy (see Table 5) is lower compared with the other ratios used (969 ± 28.90 mg thymol/100 g DM for 12/1 ratio compared to 926 ± 16.93 and 824 ± 20.67 mg thymol/100 g DM for 10/1 and 8/1 ratios, respectively).

3.3. Influence of Ultrasound Equipment on the Extraction of Thyme Essential Oil

Prior to the extraction of thyme EO by MWHD, the extraction mixture was subjected to ultrasound pre-treatment for 30 min using two types of equipment: an ultrasound bath (ES375H Bench Top Ultrasonic Tank, Hilsonic Ultrasonic Cleaners, Birkenhead, UK) and an ultrasonic processor (Vibracell VCX-750, Sonics & Materials, Inc., Newtown, CT, USA).
It can be noticed (Figure 3a) that the ultrasound pre-treatment of the extraction mixture leads to higher amounts of EO compared with the experiments without pre-treatment, for both the ultrasound bath (approximatively 6% higher—1.83 ± 0.09 g EO/100 g DM) and the horn (approximatively 21% higher—2.10 ± 0.03 g EO/100 g DM). This could be due to the cavitation phenomenon, which can stimulate the disruption of cell walls and increase the mass transfer rate. In the ultrasonic bath, the cavitation occurs uncontrollably. The ultrasound energy is unequally dispersed in the bath and has low intensity. On the other hand, for the horn, the cavitation has a high localized intensity, and implicitly, the sonication process is more efficient [34]. Therefore, the higher amount of EO achieved when the ultrasound pre-treatment is carried out using the Vibracell equipment (an increase of approximately 15%) could be due to these differences between ultrasonic baths and horns. In addition, the ultrasound pre-treatment of the extraction mixture has a beneficial effect on the extraction time, achieving a maximum amount of EO in only 50 min compared with the extraction without pre-treatment when a maximum amount is achieved after 70 min. Further, the experiments were performed using the Vibracell ultrasonic processor to pre-treat the extraction mixture.
The GC-MS analyses of the EO obtained by consecutive use of ultrasound and microwave irradiation are shown in Figure 3b. It can be noticed that the major constituents of the EO are similar for all three methods (the extraction with ultrasound pre-treatment using both ultrasonic horn and bath and the extraction without pre-treatment). As shown in Figure 3b, the thymol is extracted more efficiently when the ultrasound pre-treatment is carried out using the ultrasonic probe (1196 ± 15.35 and 1040 ± 35.62 mg/100 g DM for ultrasonic probe and bath, respectively). Instead, p-cymene is obtained in lower amounts (90 ± 7.28 and 103 ± 6.49 mg/100 g DM for ultrasonic probe and bath, respectively). This could be due to the transformation of some compounds during ultrasound irradiation, such as isomerization, oxidation, and degradation of dimmers and polymers [35]. Also, in water, the cavitation phenomenon can lead to the formation of free radicals (H•, OH•) which can cause the transformation of the EO constituents [36]. The ANOVA analysis showed that pre-treatment with the ultrasonic horn led to a significant increase in the thymol content, while using the ultrasonic bath the content of p-cymene increased significantly (p < 0.05). The opposite behavior of p-cymene when the pre-treatment is performed using the ultrasonic bath could be due to the low intensity of the cavitation in such equipment. Since the ultrasound energy for the horn can be focused on specific sample area, the constituents with lower boiling points can be rapidly released, and implicitly, they can be degraded afterwards.

3.4. Influence of Ultrasound Amplitude on the Extraction of Thyme Essential Oil

The influence of ultrasound amplitude on the extraction process efficiency was also studied. Amplitudes of 50 and 70% were chosen. As shown in Figure 4a, the amount of thyme EO is directly proportional to the ultrasound amplitude. The EO content increases by 6% compared with the pre-treatment performed at an amplitude of 50% (2.10 ± 0.03 and 2.22 ± 0.06 g EO/100 g DM for an amplitude of 50 and 70%, respectively). The extraction mechanism involves the diffusion of the solvent through the cell walls and, implicitly, their rinsing, since the walls are disrupted. These phenomena can be enhanced by ultrasonic cavitation [37]. Thus, an efficient ultrasound pre-treatment of the extraction mixture can increase the EO yield.
The thyme EO was also analyzed and identified by GC-MS. The results for both ultrasound amplitudes are shown in Figure 4b. As in the previous experiments (see Figure 2b and Figure 3b, and Table 1) the main constituents of thyme EO (thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene) remain unchanged. As shown in Figure 4b, the targeted compound, thymol, is extracted more efficiently when an amplitude of 70% is used (1196 ± 15.35 and 1370 ± 58.78 mg/100 g DM for an amplitude of 50 and 70%, respectively). Statistical analysis confirmed that by increasing the ultrasonic amplitude from 50 to 70%, the EO and thymol contents increase significantly (p < 0.05). However, the content of p-cymene is inversely proportional with the ultrasound power, achieving higher amounts for an amplitude of 50%. This can be due to an accentuated transformation of some constituents at high ultrasound powers.

3.5. Influence of Thyme Leaf Size on the EO Extraction

In addition to external glands, which can be easily destroyed by sonication, the EO can be found in the internal secretory structures of the vegetal materials. This can lead to a mass transfer resistance. A strategy to overcome this drawback is to mill the herb in order to increase the surface area. Thus, more cells will be directly exposed to the cavitation phenomenon, thus enhancing the mass transfer of the targeted compounds from vegetal matrix to the solvent [37]. As shown in Figure 5a, the particle sizes of the thyme leaves influence to a large extent the EO content. For a particle size under 0.1 cm, higher amounts of EO are achieved for both ultrasound pre-treatment (for both amplitudes applied, i.e., 50 and 70%) and extraction without pre-treatment. Using smaller particles, the EO content is 23% higher when an ultrasound pre-treatment (with an amplitude of 70%—2.67 ± 0.06 g EO/100 g DM) of the extraction mixture is applied, compared with the extraction without pre-treatment (2.18 ± 0.07 g EO/100 g DM). The ANOVA analysis showed that EO content increased significantly (p < 0.05) by decreasing the plant material particles size.
These strategies (the ultrasound pre-treatment using an ultrasonic probe and milling the plant material to a particle size under 0.1 cm) lead to a higher extraction yield compared with other studies. For example, Kowalski et al., [29] after applying ultrasonic pre-treatment using an ultrasound bath, followed by conventional steam distillation method, achieved an extraction yield of 2.15 g EO/100 g DM.
The major component content of thyme EO for a granulation of the leaves under 0.1 cm is similar with the one for a particle size of 1–2 cm. However, the amount of the targeted compound (thymol) is higher for the herb milled to a granulation under 0.1 cm (see Figure 3b, Figure 4b and Figure 5b). This behavior can be noticed for all three methods: the extraction with ultrasound pre-treatment applying both 50 and 70% amplitudes and the extraction without pre-treatment (1542 ± 42.54 and 1764 ± 52.68 mg/100 g DM for an amplitude of 50 and 70%, respectively, and 1396 ± 62.7 mg/100 g DM for the extraction without pre-treatment). As shown in Figure 5b, the thymol content is higher when an ultrasound pre-treatment with an amplitude of 70% is applied, while the α-terpinene content is lower (54 ± 4.91 and 33 ± 5.27 mg/100 g DM for an amplitude of 50 and 70%, respectively). As shown in Figure 4b, this behavior of α-terpinene can be also observed for the extraction with thyme leaves of a 1–2 cm particle size (51 ± 4.5 and 46 ± 4.02 mg/100 g DM for an amplitude of 50 and 70%, respectively). Indeed, according to ANOVA analysis, the decrease at an amplitude of 70% is insignificant (see Figure 4b), but the higher decrease when smaller particles are used can be explained by a more considerable exposure of ultrasound energy. Moreover, the p-cymene content is higher for an amplitude of 70% compared with 50% when the plant is milled (Figure 5b—100 ± 9.9 and 170 ± 14.06 mg/100 g DM for an amplitude of 50 and 70%, respectively), while for the extraction performed with leaves of 1–2 cm particle size, an opposite behavior is observed (Figure 4b—90 ± 7.28 and 63 ± 5.99 mg/100 g DM for an amplitude of 50 and 70%, respectively). This could be due to a possible oxidation of some components (such as α-terpinene and γ-terpinene) yielding p-cymene [38]. However, further research is required for possible oxidation under combined ultrasound and microwave treatments.

3.6. Principal Component Analysis

To evaluate the relation between the extraction method and the composition of the EO, principal component analysis (PCA) was performed. For the multivariate analysis, the chemical compositions of the thyme EO obtained by different extraction methods were determined. The PCA results show two eigenvalues higher than 1, i.e., those corresponding to PC1 (3.55) and PC2 (1.04). The main components of thyme EO in the plane formed by these first two PCs explain 90.68% of the variability, including 70.21% on the first axis and 20.47% on the second axis. The coordinates of variables (factor loadings) on the factor-plane PC1−PC2 are shown in Table 2, with the significant levels marked in bold. The projections of cases (factor scores) on the factor-plane PC1−PC2 are summarized in Table 3.
The PCA bi-plot and the correlation matrix are shown in Figure 6 and Table 4, respectively. The significant values of correlation coefficients (r) are highlighted in bold at a significance level α = 0.05 (two-tailed test).
The PCA bi-plot presented in Figure 6 and correlation matrix shown in Table 4 indicate the subsequent aspects:
  • p-Cymene is weakly correlated with thymol, β-pinene, α-terpinene, and γ-terpinene (−0.034 ≤ r ≤ 0.179);
  • Thymol is highly inversely correlated with β-pinene, α-terpinene, and γ-terpinene (−0.890 ≤ r ≤ −0.815);
  • β-Pinene is highly directly correlated with α-terpinene, and γ-terpinene (0.797 ≤ r ≤ 0.908);
  • The EO obtained by methods 6–9 (highlighted using blue and green circles) had a higher content of thymol and lower contents of β-pinene, α-terpinene, and γ-terpinene compared with the EO obtained by methods 1–5 (highlighted using red circle—discrimination on PC1);
  • The EO obtained by method 9 (highlighted using blue circle) had a higher content of p-cymene compared with the EO obtained by methods 6–8 (highlighted using green circle—discrimination on PC2).
  • Moreover, the highest amount of thymol was obtained by method 9.

3.7. Energy Considerations

During thyme leaf pre-treatment and extraction of EO, the microwave and ultrasound powers were recorded for all experiments. The power input for the electrical heater was measured at the heater power supply using a Wattmeter. Using the above-mentioned recorded values, the total energy introduced into the system was determined. The specific energy was also calculated using the following equation:
ES = Etotal/mEO [kJ/g of EO],
where Es is the specific energy [kJ/g of EO], Etotal is the total energy introduced into the system [kJ], and mEO is the amount of EO obtained by each extraction method [g].
The total energy introduced into the system and the specific energy for each method are shown in Table 5. The energy consumption for MWHD is 1104 kJ. The ultrasound pre-treatment step increases this quantity of energy concordantly with Table 5. For example, the highest increase in energy is given by the ultrasonic bath, the overall process energy increasing from 1104 kJ (for the extraction without pre-treatment) to 1896 kJ (for the extraction with pre-treatment using the ultrasound bath). Regarding the pre-treatment performed using the ultrasonic horn, the energy increase is much lower, between 47 and 82 kJ, as shown in Table 5.
For scaling up the microwave and ultrasound technologies, an important parameter to evaluate is the specific energy. The specific energy for MWHD (when thyme leaves with a particle size of 1–2 cm are used) decreases with the increase in the solvent to plant material ratio (see Table 5). Also, it can be noticed that the pre-treatment performed with the ultrasonic bath is not energetically efficient. In this case, the specific energy is 62% higher as compared with the extraction without pre-treatment. However, as compared with MWHD, using the ultrasonic probe to pre-treat the thyme leaves leads to a specific energy that is 14 and 16% lower for an amplitude of 50 and 70%, respectively. This difference is lower for thyme leaves with a particle size under 0.1 cm (a specific energy that is 6 and 13% lower for an amplitude of 50 and 70%, respectively, as compared with the extraction without pre-treatment).
The plant material grinding step implies energy consumption. In order to achieve a particle size under 0.1 cm (starting from 1–2 cm size) 20 s of milling were required. Thus, the grinding step will add 3 kJ to the energy of the overall process of EO extraction. However, this quantity of energy is insignificant, with the specific energy for the experiments performed with a thyme leaf particle size under 0.1 cm increasing by only 0.25%.
The aim of the current study was to develop a procedure appropriate to a small scale with scale-up possibilities. The reduced cost of combined ultrasound and microwave extraction is evidently beneficial for the proposed methods related to energy and time. The specific energy necessary to perform these extraction methods is 638.2 kJ/g of EO for MWHD and 534.5 kJ/g of EO for MWHD + ultrasonic horn pre-treatment. The extraction time is reduced compared with the conventional extraction. To scale-up these methods, Sairem has already commercialized different industrial microwave-assisted equipment [39] and also the ultrasound pre-treatment can be applied at industrial scale. Relating to the environmental effect, the level of the CO2 fingerprint was calculated based on the supposition that 800 g CO2 will be released for each 1 kWh obtained by combustion of fossil fuel [40]. It was observed that for the conventional extraction, the CO2 emission (553 g CO2/g of EO) is higher compared with MWHD (142 g CO2/g of EO) and MWHD + ultrasonic horn pre-treatment at an amplitude of 70% (119 g CO2/g of EO). According to these calculations, the combined ultrasound pre-treatment with MWHD is an environmentally friendly method.
Table 5. Energy consumption during thyme leaf pre-treatment and EO extraction.
Table 5. Energy consumption during thyme leaf pre-treatment and EO extraction.
Extraction MethodsTotal Energy (kJ)Specific Energy
(kJ/g of EO)
Leaf particle
size of 1–2 cm		CHD41042487.3
MWHD without ultrasound pre-treatment (solvent to plant material ratio of 8/1)1104716.9
MWHD without ultrasound pre-treatment (solvent to plant material ratio of 10/1)1104657.1
MWHD without ultrasound pre-treatment (solvent to plant material ratio of 12/1)1104638.2
MWHD + ultrasonic bath pre-treatment18961036.1
MWHD + ultrasonic horn pre-treatment (50% amplitude)1151.1548.2
MWHD + ultrasonic horn pre-treatment (70% amplitude)1186.7534.5
Leaf particle
size < 0.1 cm		MWHD without ultrasound pre-treatment (solvent to plant material ratio of 12/1)1104508.8
MWHD + ultrasonic horn pre-treatment (50% amplitude)1151.2479.6
MWHD + ultrasonic horn pre-treatment (70% amplitude)1186.7442.8

4. Conclusions

The purpose of this study was to investigate the influence of the ultrasound pre-treatment of thyme leaves, before MWHD, on the thymol content and on the EO extraction yield. Comparative extractions, without pre-treatment, by both MWHD and CHD were also performed. The influence of several parameters (solvent to plant ratio, particle size of the vegetal material, ultrasound equipment to pre-treat the extraction mixture, and amplitude of the ultrasonic processor) on the extraction of thyme EO by MWHD was investigated. The composition of the EO resulted from all methods was analyzed by GS-MS, the major constituents being thymol, γ-terpinene, p-cymene, α-terpinene, and β-pinene. The results showed that the ultrasound pre-treatment of the extraction mixture enhances the EO content, which can be extracted from thyme leaves. Moreover, by combining the strategies of ultrasound pre-treatment using an ultrasonic probe and milling the plant material to a particle size under 0.1 cm, a maximum yield of 2.67 ± 0.06 g EO/100 g DM was achieved. These strategies were also favorable for the targeted constituent, thymol, leading to a maximum content of 1764 ± 52.68 mg/100 g DM. However, the ultrasound pre-treatment requires additional energy to the MWHD. The energy consideration study showed that the ultrasound pre-treatment using an ultrasonic probe leads to a lower specific energy (442.8 kJ/g of EO) compared with the extraction without pre-treatment (508.8 kJ/g of EO). On the contrary, the energy consumption of the ultrasonic bath was high, leading to a higher specific energy compared with the extraction without pre-treatment, although the EO content was higher. This means that the use of an ultrasonic probe is a better and greener choice to pre-treat vegetal materials.

Author Contributions

Conceptualization, A.I.G. and I.P.; methodology, A.I.G. and I.P.; software, I.P. and O.C.P.; validation, A.I.G. and I.P.; statistical analysis, O.C.P.; formal analysis, C.G.C.-N., L.M. and M.L.G.; investigation, C.G.C.-N., L.M. and M.L.G.; data curation, A.I.G., O.C.P. and I.P.; writing—original draft preparation, C.G.C.-N., L.M. and M.L.G.; writing—review and editing, I.P.; supervision, A.I.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 13 02331 g001
Figure 1. Kinetics study of microwave-assisted hydro-distillation (MWHD) vs. conventional hydro-distillation (CHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm.
Figure 1. Kinetics study of microwave-assisted hydro-distillation (MWHD) vs. conventional hydro-distillation (CHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm.
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Figure 2. Influence of solvent to plant ratio on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a particle size of 1–2 cm: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a–c) within graph show the significant difference between groups analyzed by ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
Figure 2. Influence of solvent to plant ratio on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a particle size of 1–2 cm: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a–c) within graph show the significant difference between groups analyzed by ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
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Agronomy 13 02331 g003
Figure 3. Influence of ultrasound pre-treatment on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w), a particle size of 1–2 cm, and an amplitude of 50%: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a–c) within graph show the significant difference between groups resulted from ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
Figure 3. Influence of ultrasound pre-treatment on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w), a particle size of 1–2 cm, and an amplitude of 50%: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a–c) within graph show the significant difference between groups resulted from ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
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Figure 4. Influence of ultrasound amplitude on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm using the Vibracell ultrasonic processor: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a,b) within graph show the significant difference between groups from one-way ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
Figure 4. Influence of ultrasound amplitude on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm using the Vibracell ultrasonic processor: (a) essential oil (EO) content; (b) major compounds identified by GC-MS. Different letters (a,b) within graph show the significant difference between groups from one-way ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
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Figure 5. Influence of leaf particle size on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w): (a) essential oil (EO) content; (b) major compounds identified by GC-MS for a particle size under 0.1 cm. Letters (a–c) within graph show the significant difference between groups’ ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
Figure 5. Influence of leaf particle size on the extraction of thyme EO by microwave-assisted hydro-distillation (MWHD) for a solvent to plant ratio of 12/1 (v/w): (a) essential oil (EO) content; (b) major compounds identified by GC-MS for a particle size under 0.1 cm. Letters (a–c) within graph show the significant difference between groups’ ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
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Figure 6. Projections of variables (β-Pinene, α-Terpinene, p-Cymene, γ-Terpinene, Thymol) and methods (1–9) on the factor-plane PC1–PC2.
Figure 6. Projections of variables (β-Pinene, α-Terpinene, p-Cymene, γ-Terpinene, Thymol) and methods (1–9) on the factor-plane PC1–PC2.
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Table 1. Chemical composition of thyme essential oil (EO) for microwave-assisted hydro-distillation (MWHD) and conventional hydro-distillation (CHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm. The letters a and b within table show the significant difference between groups analyzed by ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
Table 1. Chemical composition of thyme essential oil (EO) for microwave-assisted hydro-distillation (MWHD) and conventional hydro-distillation (CHD) for a solvent to plant ratio of 12/1 (v/w) and a particle size of 1–2 cm. The letters a and b within table show the significant difference between groups analyzed by ANOVA (p < 0.05) and Duncan’s post hoc t-tests.
RT
(min)		CompoundCASBP (°C)Component Content (mg/100 g DM)
MWHDCHD
9.043α-Pinene80-56-815513 ± 0.2 b15 ± 0.7 a
9.205Camphene79-92-515916 ± 0.2 b26 ± 1.2 a
9.941Sabinene3387-41-51637 ± 0.1 b11 ± 0.5 a
10.039β-Pinene127-91-316533 ± 0.3 b41 ± 1.8 a
10.568α-Terpinene99-86-517357 ± 1.1 b68 ± 2.9 a
10.613p-Cymene99-87-617788 ± 3.0 b110 ± 4.8 a
10.783Eucalyptol470-82-61766 ± 0.1 b11 ± 0.5 a
11.245γ-Terpinene99-85-4183523 ± 37.4 a573 ± 24.9 a
13.071Terpinen-4-ol562-74-3219-14 ± 0.6 a
14.657Thymol89-83-8232969 ± 28.9 a689 ± 29.9 b
16.724β-(E)-Caryophyllene87-44-52544 ± 0.1 b11 ± 0.5 a
17.616γ-Cadinene39029-41-92713 ± 0.1 b4 ± 0.2 a
RT—retention time; CAS—CAS registry number; BP—boiling point.
Table 2. Factor loadings.
Table 2. Factor loadings.
VariablesPC1PC2
β-Pinene−0.9660.045
α-Terpinene−0.888−0.201
p-Cymene−0.1500.985
γ-Terpinene−0.930−0.079
Thymol0.950−0.063
Table 3. Factor scores.
Table 3. Factor scores.
MethodMethod DescriptionPC1PC2
1MWHD without ultrasound pre-treatment,
solvent to plant ratio of 8/1,
leaf particle size of 1–2 cm		−2.3820.174
−3.0410.252
−1.7230.095
2MWHD without ultrasound pre-treatment,
solvent to plant ratio of 10/1,
leaf particle size of 1–2 cm		−1.5060.710
−2.0390.830
−1.0750.821
3MWHD without ultrasound pre-treatment,
solvent to plant ratio of 12/1,
leaf particle size of 1–2 cm		−1.4770.196
−1.9410.433
−0.653−0.170
4MWHD + ultrasonic bath pre-treatment,
solvent to plant ratio of 12/1,
leaf particle size of 1–2 cm		−1.3880.539
−1.6630.672
−1.1130.406
5MWHD + ultrasonic horn pre-treatment (50% amplitude), solvent to plant ratio 12/1,
leaf particle size 1–2 cm		−1.267−0.454
−1.339−0.264
−0.271−0.698
6MWHD + ultrasonic horn pre-treatment (70% amplitude), solvent to plant ratio of 12/1,
leaf particle size of 1–2 cm		0.601−1.865
0.472−1.727
0.895−2.018
7MWHD without ultrasound pre-treatment,
solvent to plant ratio of 12/1,
leaf particle size < 0.1 cm		1.846−0.559
1.737−0.486
2.101−0.754
8MWHD + ultrasonic horn pre-treatment (50% amplitude), solvent to plant ratio of 12/1,
leaf particle size < 0.1 cm		1.798−0.479
1.699−0.384
2.169−0.915
9MWHD + ultrasonic horn pre-treatment (70% amplitude, solvent to plant ratio of 12/1,
leaf particle size < 0.1 cm		3.1201.964
3.1112.181
3.3281.502
Table 4. Correlation matrix.
Table 4. Correlation matrix.
Variablesβ-Pineneα-Terpinenep-Cymeneγ-TerpineneThymol
β-Pinene10.7970.1790.908−0.890
α-Terpinene0.7971−0.0340.742−0.815
p-Cymene0.179−0.03410.055−0.194
γ-Terpinene0.9080.7420.0551−0.832
Thymol−0.890−0.815−0.194−0.8321
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Gavrila, A.I.; Chisega-Negrila, C.G.; Maholea, L.; Gavrila, M.L.; Parvulescu, O.C.; Popa, I. Enhancing the Extraction Process Efficiency of Thyme Essential Oil by Combined Ultrasound and Microwave Techniques. Agronomy 2023, 13, 2331. https://doi.org/10.3390/agronomy13092331

AMA Style

Gavrila AI, Chisega-Negrila CG, Maholea L, Gavrila ML, Parvulescu OC, Popa I. Enhancing the Extraction Process Efficiency of Thyme Essential Oil by Combined Ultrasound and Microwave Techniques. Agronomy. 2023; 13(9):2331. https://doi.org/10.3390/agronomy13092331

Chicago/Turabian Style

Gavrila, Adina I., Ciprian G. Chisega-Negrila, Laura Maholea, Mircea L. Gavrila, Oana C. Parvulescu, and Ioana Popa. 2023. "Enhancing the Extraction Process Efficiency of Thyme Essential Oil by Combined Ultrasound and Microwave Techniques" Agronomy 13, no. 9: 2331. https://doi.org/10.3390/agronomy13092331

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