ABSTRACT
The combination of essential oils (EOs) is a novel alternative to improve their preservative effects and to reduce their organoleptic impact in food. In this context, this work aims to investigate the antibacterial combined effect of two EOs combinations through the calculation of the fractional inhibitory concentration index. The combinations tested consists of Lavandula dentata/Origanum majorana EOs and Thymus serpyllum/Origanum majorana EOs. Their chemical compositions were identified by CG/MS analyses. The main compounds of O. majorana EO were (-) - terpinene-4-ol and trans-4-thujanol. Those of L. dentata EO were β-pinene and 1,8-cineole, and those of T. serpyllum EO were p-cymene and γ-terpinene. Regarding the outcomes, results highlighted partial synergistic and additive interactions. Two combinations of marjoram and thyme EOs had antibacterial activities against S. aureus. The first one corresponded to the quarter of the minimum inhibitory concentration of marjoram and half that of thyme. The second one was the mix of half and quarter of the minimum inhibitory concentration of respectively marjoram and thyme EOs. This last combination also showed an antibacterial effect against E. coli. The quarter and the half of their minimum inhibitory concentration of marjoram and lavender combination, respectively, gave a partial synergy against both strains. Henceforth, these findings could be largely exploited in food preservation through the use of minimal doses of these plant products without affecting the antibacterial and the organoleptic properties in foods.
Introduction
Nowadays, antimicrobial resistance is a worldwide problem and the World Health Organization encourages best practices to preserve antibiotic resources. Currently, the use of synthetic antimicrobial agents for ensuring food safety has led to the outcry of consumers all over the world to develop alternatives to antimicrobial treatments based on phytochemicals such as essential oils (EOs). These molecules are eco-friendly and have shown a high ability to preserve foods for months and even years[Citation1,Citation2]. EOs, which are mixtures of volatile compounds, exhibit a wide spectrum of antimicrobial properties. Antibacterial[Citation3], antifungal[Citation4], antiviral[Citation5], nematicidal,[Citation6] and antimite[Citation7] activities have been demonstrated. Furthermore, EOs could present antioxidant properties[Citation8] and could be used as flavoring materials[Citation9]. According to all these biological properties, their application for food preservation has been suggested, either incorporated into the foodstuff or in the packaging material[Citation1].
However, recent studies have reported that the application of EOs has to involve the use of high concentrations in the food to achieve the same effects as those demonstrated in vitro[Citation10]. An efficient solution to this drawback is to use the synergic effect of EOs combinations. Indeed, an increase of their antimicrobial outcomes has been demonstrated with different EO combinations[Citation11–Citation14]. These mixtures also help reduce their sensory impact that can affect the organoleptic quality of foods[Citation12].
Several authors have tested combinations between EOs against numerous bacteria and fungi, and they have highlighted synergistic, additive, and in a few cases antagonistic interactions[Citation14–Citation16]. Interactions between components have been observed even in a single EO. This is for example the case for the EO of dill (Anethum graveolens L.), cilantro (leaves of immature Coriandrum sativum L.), or coriander (seeds of C. sativum L.); these EOs were more effective than the addition of their fractions. On the other hand, some combinations of sub-fractions were found more effective than the whole oils[Citation17]. Other studies demonstrated the synergistic effect of EO combinations such as Origanum vulgare and Rosmarinus officinalis[Citation18], Lippia multiflora and Mentha piperita[Citation14], and Cymbopogon citratus and Cymbopogon giganteus[Citation19]. The antimicrobial effect of EOs has been tested in several foods, like ragweeds, eucalyptus, juniper mint, peppermint, nutmeg, black pepper, rosemary, sage, clove, and thyme in cheese[Citation20], rosemary, cinnamon, bay, sage, garlic, oregano, and ginger in meat[Citation21]. Thus carvacrol, one of their well-known compounds, was tested in rice[Citation22].
The present study aims at finding new combinations of EOs with a high potential of antimicrobial activities and suitable for food safety and quality. Origanum majorana L. (sweet marjoram), Lavandula dentata L. (fringed lavender), and Thymus serpyllum L. (wild thyme) are three spontaneous aromatic and medicinal plants belonging to the Lamiaceae family. These species grow in several regions of Morocco where they are used as culinary spices for traditional recipes. Therefore, this study was to investigate the separated and combined antibacterial activities of these EOs, against both Gram, evaluating the chemical composition effect.
Material and methods
Plant material
Aerial parts of three plants, belonging to the Lamiaceae family, were collected from Taounate region (Morocco) in August (Origanum majorana L. and Thymus serpyllum L.) and in September (Lavandula dentata L.). Voucher specimens of each plant were deposited at the herbarium of the National Agency of Medicinal and Aromatic Plants (Morocco).
EO extraction
EOs were obtained using the method recommended by the European pharmacopoeia (2014)[Citation23]. Accordingly, the fresh aerial parts (leaves and stems) of each plant were subjected separately to hydrodistillation for 3 h using a Clevenger apparatus. The obtained EOs were stored at 4°C in dark until use.
Gas chromatography/mass spectrometry analysis
The chemical composition of the studied EOs was investigated by GC/MS analysis. The analytical GC/MS system used was an Agilent GC-MSD system (Agilent Technologies 6850/5973) with helium (high purity) as the carrier gas at a constant linear velocity of 36 cm/s. The transfer, source, and quadrupole temperatures were 245°C, 230°C, and 150°C, respectively, operating at 70 eV ionization energy and scanning the m/z range 50–550. The column used was an Agilent DB5 MS capillary column (30.0 m × 0.25 mm × 0.25 µm film thickness) programmed from 60°C to 245°C at 3°C/min. EO samples were diluted with hexane (Sigma Aldrich) (1:3000). The injected volume was 2.0 µL, in splitless mode, and the injector temperature was 250°C. Identification of the individual components was based on comparison with NIST MS Search database 2012 where possible and by Adams terpene library[Citation24].
Antimicrobial bioassays
Agar-disc diffusion method
The antimicrobial effect of EOs was tested against two bacterial strains: Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922, using the disc-diffusion method according to CLSI[Citation25]. Briefly, Mueller Hinton Agar (MHA) plates were inoculated with a bacterial inoculum already adjusted to 0.5 McFarland standard[Citation26]. The discs (filter paper, 6 mm of diameter) impregnated with 10 µL of each EO were placed in the inoculated agar surface. After incubation at 37°C for 18–20 h, diameters of the growth inhibition zones were measured. Each assay was carried out in triplicate.
Determination of minimum inhibitory concentration
The MIC was obtained in 96-well microplates using the broth microdilution assay, as described by Balouiri et al.[Citation27] with slight modifications. Firstly, EO was serially diluted in Mueller Hinton broth (MHB) supplemented with agar 0.15% (w/v)[Citation28] used as an emulsifier, to achieve the final concentrations of 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, and 0.01562% (v/v). The 12th well was considered as a growth control. Then, 50 µL of bacterial inoculum was added to each well at a final concentration of 106 CFU/mL. After incubation at 37°C for 18–20 h, 5 µL of resazurin was added to each well as bacterial growth indicator. After further incubation at 37°C for 2 h, the bacterial growth was revealed by the change of coloration from purple to pink. The MIC value was determined as the lowest concentration that prevented a change in resazurin color. Experiments were carried out in triplicate.
Determination of minimum bactericidal concentration
The MBC was determined by spreading 5 µL from negative wells on Luria Bertani agar plates. The MBC value corresponded to the lowest concentration of the EO yielding negative subculture after incubation at 37°C for 24 h[Citation27].
Determination of fractional inhibitory concentration
The antibacterial effects of two EO combinations (O. majorana and L. dentata) and (O. majorana and T. serpyllum) against bacterial strains were evaluated using the checkerboard method followed by FIC index calculation[Citation14,Citation29]. In this test, dilutions of both EOs were prepared in MHB supplemented with bacteriological agar (0.15% w/v). Along the x-axis across the checkerboard plate, 50 μL of each concentration of the first EO was added into each well, i.e. from the first to the 11th well. As for the y-axis, 50 μL of each concentration of the second EO was added into each well. The well (12th –A) was considered as a growth control.
Bacterial inoculum was then added into all of the wells to achieve a final concentration of 106 CFU/mL. The 96-well plate was then sealed and incubated at 37°C for 18–20 h. After incubation, 10 µL of resazurin was added to each well to assess bacterial growth, and after further incubation at 37°C for 2 h, the FIC index values were calculated using the following formula:
where
and
The ∑ FICI values are interpreted as follows: ≤ 0.5= synergistic; 0.5–0.75 = partial synergy; 0.76–1.0 = additive; >1.0–4.0 = indifferent (non-interactive); > 4.0 = antagonistic[Citation29].
Results
Chemical composition of EOs
Origanum majorana, Thymus serpyllum, and Lavandula dentata EOs were extracted with yields of 1.2%, 0.9%, and 1.1% (v/w), respectively. shows the chemical composition of L. dentata EO. As can be seen, 56 compounds accounting for 99.9% of the total oil were identified. The monoterpenes were the major class, especially the oxygenated monoterpenes. The major compounds of this EO were β-pinene (25.8%), 1,8-cineole (10.8%), fenchone (9.8%), and α-pinene (8.6%).
Table 1. Chemical composition of Lavendula dentata essential oil by GC/MS analysis.
Chemical compositions of O. majorana and T. serpyllum EOs were already shown in our previous work[Citation30]. O. majorana oil revealed 21 compounds accounting for 95.7% of the total oil. The main compounds were (-)-terpinene-4-ol (29.1%), trans-4-thujanol (24.6%), and o-cymene (12.6%), with an important amount of monoterpenes, mainly the alcohol ones[Citation30]. T. serpyllum oil contains 36 compounds accounting for 99.6% of the total. Its major compounds were p-cymene (36.2%), γ-terpinene (18.3%), and thymol (17.3%).
Antimicrobial activity
Single antimicrobial effect
The antimicrobial effects of O. majorana, T. serpyllum, and L. dentata EOs were tested using the disk diffusion method. Afterward, quantitative evaluation by MIC and MBC determinations, shown in and , revealed a strong inhibitory effect of O. majorana (MIC: 0.125% v/v) and T. serpyllum (MIC: 0.0625; 0.125 % v/v) EOs against E. coli and S. aureus. While L. dentata oil inhibition was lower (MIC: 0.5% v/v) against S. aureus, E. coli was more resistant to this oil (MIC: 2% v/v). It can be also noted that T. serpyllum EO exhibited the highest antibacterial effect against E. coli with MIC value four-fold lower compared to L. dentata EO and two-fold lower compared to O. majorana EO. As shown in , T. serpyllum EO exhibited the strongest bactericidal effect against both tested strains. O. majorana EO showed a bacteriostatic effect against S. aureus with a MIC/MBC ratio of 8 and a bactericidal effect against E. coli. In contrast, L. dentata EO was more bactericidal against S. aureus than O. majorana but without effect against E. coli.
Table 2. Minimal inhibitory concentration of O. majorana, L. dentata essential, and T. serpyllum oils against S. aureus and E. coli.
Table 3. Minimum bactericidal concentration values of O. majorana and L. dentata and T. serpyllum essential oil against S. aureus and E. coli.
Combined antibacterial effect
On the one hand, results of the binary combined effect between EOs of O. majorana and L. dentata showed that the combination (1/2 MICmarjoram + 1/8 MIClavender) displayed a partial synergistic effect against S. aureus with a FIC index of 0.625 (). The second combination (1/2 MICmarjoram+1/2 MIClavender) displayed an additive effect with a FIC index of 1. Regarding the combined effect against E. coli, two combinations of 1/2 MICmarjoram + 1/4 MIClavender and 1/4 MICmarjoram + 1/2 MIClavender showed a partial synergistic antibacterial effect with a FIC index of 0.75.
Table 4. Fractional inhibitory concentration (FIC) index and outcome of interaction of O. majorana and L. dentata essential oil combination against S. aureus and E. coli.
Table 5. Fractional inhibitory concentration (FIC) index and outcome of interaction of O. majorana and T. serpyllum essential oil combination against S. aureus and E. coli.
On the other hand, results of the binary combined effect between EOs of O. majorana and T. serpyllum showed that all tested combinations displayed a partial synergistic effect against both studied strains with a FIC index of 0.75 (). Two combinations of, respectively, 1/4 MICmarjoram + 1/2 MICthyme and 1/2 MICmarjoram + 1/4 MICthyme were found antibacterial against S. aureus. Only one combination of 1/2 MIC marjoram + 1/4 MIC thyme showed an antibacterial effect against E. coli.
Discussion
The chemical profiles of O. majorana and T. serpyllum EOs were described in our previous study[Citation30]. Two monoterpenes, β-pinene and 1,8-cineole, were the major compounds of L. dentate EO. This composition is in accordance with previous studies[Citation31]. Furthermore, it has been reported that the chemical composition of L. dentata EO varies depending upon the harvest region[Citation31] and other authors reported linalyl acetate and linalool as the main compounds of the Moroccan L. dentata EO[Citation32]. The variability of 1,8-cineole, fenchol, borneol, and camphor contents has also been described for in vitro cultured L. dentata plantlets[Citation33].
The L. dentata EO exhibited weak antibacterial activity, especially against E. coli. Its main products, 1,8-cineole, α-pinene, and β-pinene, are known for their weak antibacterial activity compared to alcoholic and phenolic monoterpenes[Citation34,Citation35]. Even if previous studies have demonstrated the synergistic outcome of 1,8-cineole in combination with hydrocarbon monoterpenes[Citation36], this synergic effect was not observed here. The antibacterial activity of O. majorana EO could be related to its high content of the monoterpenes alcohol, (-)-terpinene-4-ol, and trans-4-thujanol. These results corroborate with previous studies showing the antimicrobial activity of O. majorana against Gram-negative and Gram-positive bacterial strains[Citation37,Citation38]. Regarding the antibacterial activity of T. serpyllum, it is well-known that thymol and p-cymene exhibit strong antibacterial effects[Citation39], and they could be considered as the most active compounds of this oil. This could explain the huge discrepancy between the antimicrobial effect of EOs of O. majorana, T. serpyllum, and L. dentata.
As regards combined antibacterial effects, it was observed that the MIC value of each EO used alone was decreased in their combined applications. In fact, a considerable reduction of the lavender MIC value to its one-eighth and to its quarter against S. aureus, and a reduction of the marjoram and thyme MIC values to their half or to their quarter against both studied strains were reached.
In sum, both antibacterial combined effects highlighted by FIC index calculation against S. aureus and E. coli showed partial synergistic or additive antibacterial outcomes, which could improve the effect displayed by each EO alone. This could be mainly due to the interactions between both major and/or minor components of the combined EOs. In the literature, an increase in the antimicrobial activity of combined EOs has been explained by the combination of major compounds like eugenol and thymol for the mixture of Ocimum basilicum and Lavandula multiflora EOs and with linalool mixed with menthol for the combination of L. multiflora and Mentha piperita EOs[Citation14]. Moreover, it was also suggested that the antibacterial activity of the main components may be modulated by the minor ones, and consequently contribute to the final interactive effect of the combination[Citation17]. It has also been previously reported that the combination of 1,8-cineole with terpene hydrocarbons, such as limonene[Citation40] and aromadendrene,[Citation41] produces synergistic and additive antibacterial effects, respectively. Even if many researchers have focused on the antimicrobial effect of EOs, to date their mechanism of action has not been fully understood although EOs appear to be able to disrupt cell wall and cytoplasmic membranes of fungi and bacteria[Citation42–Citation45]. This could be related, in addition to their complex composition, to the fact that their components can act at many particular cell sites[Citation46]. Ultee et al. have demonstrated a synergistic effect between carvacrol and p-cymene, and they suggested that this effect occurred when p-cymene enabled carvacrol to be more easily transported into the cell[Citation22]. By comparison, it was supposed for the mixture of O. majorana and L. dentata EOs that β-pinene (monoterpene hydrocarbon) leads to an increase in the uptake of (-)-terpinene-4-ol and trans-4-thujanol (monoterpenes alcohol). Concerning the combination of O. majorana and T. serpyllum EOs, a similar observation was made with the mixture of p-cymene and γ-terpinene (monoterpenes hydrocarbon) and (-)-terpinene-4-ol, trans-4-thujanol (monoterpene alcohols).
It is noteworthy that this combination did not show any difference in the inhibitory effect against L. monocytogenes compared to each oil treatment[Citation47]. In the present study, the synergetic partial effect could be explained by the interactions between the four major compounds, (-)-terpinene-4-ol, trans-4-thujanol, p-cymene, and γ-terpinene, following the mechanism of monoterpene hydrocarbons/alcohols cited above.
The combination of γ-terpinene and p-cymene to (-)-terpinene-4-ol had led to a significant antagonism effect against E. coli[Citation48]. The authors suggested that this effect could be due to a solubility decrease of the terpinene-4-ol in the aqueous medium once combined with γ-terpinene and p-cymene[Citation48]. Furthermore, in our previous study of the ternary antibacterial effect between O. majorana, T. serpyllum, and O. compactum EOs, a synergistic magnitude was revealed by the coefficient of the binary interaction between O. majorana and T. serpyllum against S. aureus, while an antagonistic effect against E. coli was observed[Citation30]. This divergence could be related to the used methods, since there are no studies reporting a relationship between the FIC index calculation and the mixture design approach.
In the background, it must be remembered that the preservative effect of EOs against bacteria has been already demonstrated and food preservatives containing EOs have been commercialized since 1990s[Citation49]. Basically, to replace the harmful synthetic preservatives[Citation21], several EOs have been tested in foods[Citation20–Citation22] and they have been used in food packaging and edible packaging films to prevent from food spoilage pathogens[Citation50]. However, several studies have outlined the need of huge concentrations of such plant products to achieve the same effects as those found in vitro[Citation20,Citation47]. Consequently, the investigation of the interactive effects between different classes of phytochemical compounds, especially EOs and their compounds, could be a safe tool to potentiate their effects and thus to overcome the impediment of their applications in food, cosmetic, and pharmaceutical industries[Citation22,Citation51,Citation52] . In fact, mixtures of bactericidal EO compounds, such as cinnamaldehyde, diacetyl, and acetic acids, with other fungicides, such as allyl isothiocyanate, hexanal, thymol, and 2-nonane, were patented as preservative agricultural commodities[Citation53]. In this context, both combinations highlighted in the present study showed partial synergistic antibacterial effect. Both could be used for preserving different kind of foods, especially because lavender, thyme, and marjoram have been already used as flavoring materials.
Acknowledgment
The authors would like to thank Sylvie Baudino for his assistance in the final editing of the English manuscript.
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