In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils

Authors


Dr Sofia Cosentino, University of Cagliari, Department of Experimental Biology, S.S. 554 Km. 4,500, 09042 Monserrato (CA), Italy (e-mail: scosenti@vaxca1.unica.it).

Abstract

Essential oils and their components are becoming increasingly popular as naturally occurring antimicrobial agents. In this work the chemical composition and the antimicrobial properties of Thymus essential oils and of their main components were determined. Three essential oils obtained from different species of Thymus growing wild in Sardinia and a commercial sample of Thymus capitatus oil were analysed. The essential oil components were identified by GC/MS analysis. The antimicrobial activity of the oils and components was determined against a panel of standard reference strains and multiple strains of food-derived spoilage and pathogenic bacteria, using a broth microdilution method. The GC/MS analysis showed that the major constituents of the oils were monoterpene hydrocarbons and phenolic monoterpenes, but the concentration of these compounds varied greatly among the oils examined. The results of the antimicrobial assay showed that essential oils extracted from Sardinian Thymus species have an antimicrobial activity comparable to the one observed in other thyme oils. It seems also confirmed that the antimicrobial properties of thyme essential oils are mainly related to their high phenolic content. Among the single compounds tested carvacrol and thymol turned out to be the most efficient against both reference strains and food-derived bacteria. The results of this study confirmed the possibility of using thyme essential oils or some of their components in food systems to prevent the growth of foodborne bacteria and extend the shelf-life of processed foods.

The antimicrobial properties of essential oils derived from many plants have been empirically recognized for centuries, but scientifically confirmed only recently ( Deans & Ritchie 1987; Janssen et al. 1987 ). Practical uses of these activities have long been suggested in humans and animals, but only in the last years has it been reported that some essential oils are capable of inhibiting foodborne bacteria and extending the shelf-life of processed food ( Conner & Beuchat 1984; Kim et al. 1995 ; Smith-Palmer et al. 1998 ).

Among the aromatic plants belonging to the Lamiaceae family, the genus Thymus is noteworthy for the numerous species and varieties of wild-growing plants. Many of these species are typical of the Mediterranean area. The plants are extensively used, fresh and dried, as a culinary herb. The essential oil is utilized as flavor ingredients in a wide variety of food, beverage and confectionery products, as well as in perfumery for the scenting of soaps and lotions. Because of its antiseptic, antispasmodic and antimicrobial properties it is also used for medicinal purposes ( Van Den Broucke & Lemli 1981; Panizzi et al. 1993 ).

In recent years, several reports have been published concerning the composition and/or the biological properties of Thymus essential oils ( Adzet et al. 1991 ; Stahl Biskup 1991; Biondi et al. 1993 ; Senatore 1996). These studies have emphasized the existence of marked chemical differences among oils extracted from different species or varieties. These variations are likely to influence the antimicrobial activity of the oil and are generally a function of three factors: genetically determined properties, the age of the plant and the environment.

The aims of this work were: (i) to assess the antimicrobial properties of Sardinian Thymus essential oils and their main components, and (ii) to determine the susceptibility of multiple strains of spoilage and/or pathogenic bacteria isolated from food products to these oils.

Materials and methods

Plant material and distillation

The study was carried out on three essential oils samples obtained from different species of Thymus growing wild in Sardinia and on a commercial sample of T. capitatus oil. The Sardinian species analysed were T. capitatus, which is typically present in the Mediterranean basin and Portugal ( Tateo et al. 1996 ) and T. herba-barona, an endemic plant of the mountain areas of Sardinia and Corsica ( Camarda 1978). The specimens of T. herba-barona were collected from two ecologically different stations in Sardinia (see Table 1). The fresh plants were collected at the flowering stage and processed immediately after harvest. A semi-industrial stainless steel distilling apparatus with recirculation of the condensed water (cohobation) was used. The samples were distilled for two hours and the oils obtained were dried with anhydrous sodium sulphate, and stored in full, dark vials at +4 °C.

Table 1.  Quantitative and qualitative composition (% w/w) of the four Thymus essential oils studied
Components *T. capitatus (commercial)T. capitatusT. herba-baronaaT. herba-baronab
  1. * = Listed in order of elution

  2. a= collected from ‘Limbara’ (northern Sardinia)

  3. b= collected from ‘Gennargentu’ (central Sardinia)

α-thujene1·0
α-pinene25·20·81·91·1
camphene0·20·60·60·9
β-pinene0·4
myrcene0·90·80·7
α-terpinene0·41·31·0
p-cymene4·126·427·65·2
limonene1·41·50·7
1,8-cineole1·21·5
γ-terpinene0·68·46·14·6
linalool10·37·45·83·3
1 terpinen-4-ol0·61·0
α-terpineol2·60·9
borneol1·00·53·8
bornyl acetate1·0
α-thujene1·0
thymol46·129·350·346·9
carvacrol2·810·82·920·6
β-caryophyllene1·73·41·31·1
Total98·095·999·490·2

GC/MS analysis

The essential oils were chromatographed using a Hewlett Packard 5890 series II gas chromatograph (Hewlett Packard, Avondale, PA). It was fitted with a Mass Selective Detector HP 5971 A, an HP 7673 autosampler and a split-splitless injector, and connected to an MS ChemStation HP vs. C.00·07. A DB5MS fused silica column (5% phenyl-methylpolyxiloxane, 30 m × 0·25 mm i.d. film thickness 0·25 μm) (J & W Scientific Fisons, Folsom, CA) was employed. The injector and interface were operated at 200 °C and 280 °C, respectively. The oven temperature was programmed as follows: 60 °C raised to 180 °C (3 °C/min), and held for 15 min. Helium was the carrier gas at 0·9 ml/min; the sample (1 μl) was injected in the split mode (1 : 20). MS conditions were as follows: ionization voltage of 70 eV, scan rate 1·6 scan/sec, mass range 40–500, ion source temperature 180 °C.

The essential oil components were identified by comparing their relative retention times and mass spectra with those of authentic samples (analytical standards from Aldrich, Acros and Fluka; purity ≥97%). Sample solutions were prepared in n-hexane (GC grade, Merck) at 1·0% (w/w).

Reference strains

The reference strains used in all antimicrobial assays were: E. coli ATCC 25922, E. coli O157:H7 ATCC 35150, Ps. aeruginosa ATCC 27853, S. aureus ATCC 25923, S. epidermidis ATCC 12228, E. faecalis ATCC 29212, Yersinia enterocolitica ATCC 9610, Candida albicans ATCC 10231 (Difco laboratories); B. cereus ATCC 11778, Listeria monocytogenes ATCC 7644, Salm. typhimurium ATCC 14028 and Sacc. cerevisiae ATCC 9763 (Oxoid).

Bacterial strains were grown in nutrient broth (NB) and incubated at 37 °C. Yeasts were grown in Sabouraud broth (SB) and incubated at 25 °C.

Food-derived strains

Susceptibility to the oils and components was also determined against the following microorganisms isolated from different food products: E. coli (102 strains), S. aureus (64 strains) and B. cereus (45 strains). These species were chosen because they may be responsible for foodborne diseases and/or for spoilage of contaminated products.

Antimicrobial assay

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the oils and their components were determined using a broth microdilution method. All tests were performed in NB or SB supplemented with tween 80 detergent at a final concentration of 0·5%. Serial doubling dilutions of each oil or component were performed in a 96-well microtitre plate (Nunc, Copenhagen, Denmark) over the range of 3·5–900 μg/ml.

Overnight broth cultures were prepared in NB or SB and adjusted so that the final concentration in each well following inoculation was approximately 5·0 × 105 cfu/ml. The concentration of each inoculum was confirmed using viable counts on Tryptic Soy Agar (TSA) plates for bacteria and Sabouraud Dextrose Agar (SDA) for yeasts.

Positive and negative growth controls were included in every test. A microbial susceptibility control test was performed with gentamicine for bacteria and with ketoconazole for yeasts.

The plates were incubated aerobically at 37 °C for 24 h and the MICs and MBCs determined. Bacterial growth was indicated by the presence of turbidity and a ‘pellet’ on the well bottom. MICs were determined presumptively as the first well, in ascending order, which did not produce a pellet. To confirm MICs and to establish MBCs, 10 μl of broth was removed from each well and inoculated on TSA plates. After aerobic incubation at 37 °C overnight, the number of surviving organisms was determined. The MIC was the lowest concentration which resulted in a significant decrease in inoculum viability (>90%), while the MBC was the concentration were 99·9% or more of the initial inoculum was killed.

Each experiment was repeated at least three times for each oil or compound at each test concentration and the modal MIC and MBC values were selected.

Results

Chemical analysis

Table 1 shows the results of GC-MS analyses. The compounds are listed in order of their elution time on the DB5MS column. A total of 19 components have been identified. Significant quantitative differences between the oils were apparent for seven compounds: the alcohols α-terrpineol and linalool, the two isomeric phenols carvacrol and thymol, their precursor α-terpinene and p-cymene, and α-pinene.

The concentration of these components varied greatly among the oils examined, but particularly that of carvacrol (from 2·8 to 20·6%), p-cymene (from 4·1 to 27·6%) and α-pinene (from 0·8 to 25·2%). In all samples, however, the sum of the two phenols and their precursors constituted the bulk of each essential oil: 53·6%, 74·9%, 86·9% and 77·3% of the total oil, respectively. As for the alcohols, linalool was present in appreciable amounts in all samples (from 3 to 10%) whereas α-terpineol has been identified almost exclusively in the commercial oil sample (2·6%).

Antimicrobial assay

The variability in the concentration of the main components present in the essential oils analysed led us to evaluate the antimicrobial activities of the oils and of the isolated authentic compounds. Table 2 reports the MBC of the oils and components tested against selected reference strains. Although the MIC and MBC results varied between test organisms, in most cases the MIC was equivalent to the MBC, indicating a bactericidal action of the oils.

Table 2.  MBC (μg/ml) of the Thymus essential oils and their main components against the reference strains tested
Gram negativeGram positiveYeasts
Microorganism:
ATCC number
E. coli
25922
E. coli O157:H7
35150
Ps. aeruginosa
27853
S. typhimurium
14028
Y. enterocolitica
9610
S. aureus
25923
S. epidermidis
12228
E. faecalis
29212
L. monocytogenes
7644
B. cereus
11778
C. albicans
10231
S. cerevisiae
9763
  • a

    = collected from ‘Limbara’ (northern Sardinia)

  • b

    = collected from ‘Gennargentu’ (central Sardinia)

Thymus oils
T. capitatus (comm.)900900>900900900900900900900450450450
T. capitatus900900>900450450900900900450900450450
T. herba-baronaa450450>900450900900900900450900450225
T. herba-baronab450450>900450450225450450225225225225
Components
α-pinene>900>900>900>900>900>900>900>900>900>900>900>900
p-cymene>900>900>900>900>900>900>900>900>900>900>900>900
γ-terpinene>900>900>900>900>900>900>900>900>900>900>900>900
linalool>900>900>900>900>900>900>900>900>900>900>900>900
α-terpineol450>900>900225>900900>900900>900900225225
thymol225450>90056·25225225225225450450112·5112·5
carvacrol225225>900225225450450225450900112·5112·5

Generally, the oils exhibited similar levels of antimicrobial activity, but T. herba-barona‘sample b’ appeared to be more efficient. In particular, it showed significant antimicrobial power against S. aureus, L. monocytogenes, B. cereus, C. albicans and Sacc. cerevisiae.

Yeasts were the most sensitive among the microorganisms tested, followed by Gram positives and Gram negatives. S. typhimurium among Gram negatives and L. monocytogenes among Gram positives were the most susceptible strains. Only Ps. aeruginosa was resistant to all oils at the highest concentration tested.

Of the main compound tested, α-pinene, p-cymene, γ-terpinene and linalool did not show any activity against the reference strains at the highest concentration tested (900 μg/ml), whereas α-terpineol appeared to possess some activity against the yeasts and two Gram negative bacteria (E. coli and S. typhimurium). The phenols carvacrol and thymol had the most potent antimicrobial activity against all strains tested, with the exception of Ps. aeruginosa.

The data obtained from the screening against food-derived strains are reported in Table 3. Overall, E. coli isolates were the most susceptible to the oils, followed by B. cereus and S. aureus. The oil of T. herba-barona‘b’ exhibited the strongest antimicrobial activity against all strains tested, as demonstrated by the higher percentage of isolates having a MBC equal or lower than 450 μg/ml.

Table 3.  MBC (μg/ml) of the Thymus essential oils and their main components against spoilage and pathogenic bacteria isolated from food products. The values are expressed as % of susceptible strains
E. coli (102 strains)S. aureus (64 strains)B. cereus (45 strains)
MBC

>900900450225112·5>900900450225112·5>900900450225112·5
  • a

    = collected from ‘Limbara’ (northern Sardinia)

  • b

    = collected from ‘Gennargentu’ (central Sardinia)

Thymus oils
T. capitatus (comm)7015123204415165223814224
T. capitatus62296381191714672
T. herba-baronaa12701266628627224011
T. herba-baronab37612925163491611732446
Components
α-pinene100100100
p-cymene100100100
γ-terpinene100100100
linalool742687138020
α-terpineol956266394645532
thymol7420613443498992
carvacrol294229332847194186711

Among the seven compounds tested, carvacrol and thymol were the most efficient, as expected, followed by α-terpineol and linalool. The hydrocarbons α-pinene, p-cymene and γ-terpinene showed a MBC higher than 900 μg/ml against all strains.

Discussion

The GC-MS analysis showed that the major constituents of the oils examined in our study were monoterpene hydrocarbons and phenolic monoterpenes. These results are in partial agreement with those reported by other authors for Thymus species growing in Italy. In samples of T. herba-barona collected from six ecologically different stations, the phenols thymol and carvacrol were largely prevalent ( Falchi 1967). Recently, Biondi et al. (1993) reported for T. capitatus a high content (86·33%) of carvacrol.

Thyme essential oils have been reported to be highly antimicrobial in numerous investigations ( Akgul & Kivanc 1988; Tantaoui-Elaraki et al. 1992 ; Panizzi et al. 1993 ; Nelson 1997) and have also been shown to be inhibitory to selected food-spoilage organisms ( Conner & Beuchat 1984; Ouattara et al. 1997 ; Smith-Palmer et al. 1998 ).

The results of our study showed that the antimicrobial activity of essential oils extracted from sardinian Thymus species is comparable to the one observed in other thyme oils.

It seems also confirmed that the antimicrobial properties of thyme essential oils are mainly related to their high phenolic content. In fact, T. herba-barona‘sample b’, which was characterized by a total content of phenols higher than the other oils analysed (67·5%), also showed the strongest antimicrobial activity against both reference strains and food-derived bacteria.

Among the seven compounds tested, carvacrol and thymol turned out to be the most efficient, followed by α-terpineol. The outstanding position of carvacrol and thymol as antimicrobial agents has already been pointed out ( Gergis et al. 1990 ; Lattaoui & Tantaoui-Elaraki 1994). α-terpineol has previously been reported as having antibacterial activity ( Carson & Riley 1995), whereas the very low antimicrobial response obtained for linalool is in contrast with other reports ( Ross et al. 1980 ; Onawunmi et al. 1984 ). The lack of antimicrobial activity demonstrated by α-pinene, p-cymene and γ-terpinene is in agreement with previous studies showing that hydrocarbons are the least efficient of essential oil components ( Lattaoui & Tantaoui-Elaraki 1994; Carson & Riley 1995).

Overall, the oils and single compounds were more active vs. yeasts and Gram positive bacteria than Gram negatives, as reported in similar studies ( Farag et al. 1989 ; Lemos et al. 1990 ; Smith-Palmer et al. 1998 ). Among the reference strains tested in our investigation, Ps. aeruginosa was the least sensitive, both to growth inhibition and lethal effect. This bacterium has proved to be resistant to many antimicrobial agents, including thymol and carvacrol ( Didry et al. 1993 ; Sivropoulou et al. 1996 ), but was found to be sensitive to pulegone, isopulegone and piperitone, as well as to oils rich in these compounds ( Sivropoulou et al. 1995 ).

Although this study has mainly identified two components, carvacrol and thymol, which appear to contribute significantly to the antimicrobial activity of the Sardinian thyme oils examined, the possibility that other minor components possess some antimicrobial power still remains. In fact, it has been demonstrated in T. zygis oil that the antimicrobial activity of a mixture containing the same proportion of the six main components was lower than the one exerted by the whole essence, thus indicating the significant role played by the minor components ( Lattaoui & Tantaoui-Elaraki 1994).

In addition, synergistic or antagonistic effects between some components may also affect the observed antimicrobial activity of the oils. Synergistic activity of carvacrol and thymol against some bacteria has been reported ( Didry et al. 1993 ). In our study, the high p-cymene content observed in Sardinian T. capitatus and T. herba-barona‘sample a’ may have antagonized the antimicrobial action of phenols, resulting in a weaker activity of these oils, as compared to T. herba-barona‘sample b’.

The results of this study confirmed the possibility of using thyme essential oils or some of their components in food preservation. Since these natural products and compounds are of GRAS status, the safety concern of using them to prevent the growth of foodborne pathogens or spoilage organisms is minimal.

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