In this study four strains of foodborne bacteria Escherichia coli, Salmonella enteritidis, Bacillus subtilis and Staphylococcus aureus were examined for sensitivity to essential oils of eight different chemotypes of basil - Ocimum basilicum L. (T₁ -T₈).

Different concentrations of these essential oils were prepared by dissolving the oils with ethanol (1:1 and 2:1) and antimicrobial effects of oils on bacteria were examined by using disk method.

All tested oils displayed some antimicrobial activities except essential oils from T₃ and T₆ chemotypes. However, the efficiency differed and depended both on the chemotype of basil and concentration of the oils, as well as the test microbial strains. Some essential oils at lower concentrations showed biostatic, whereas highest doses proved biocide effect. The most sensitive bacteria was E. coli, the most resistant bacteria were S. aureus and S. enteritidis. The highest activity was demonstrated by essential oil of T₇ chemotype of basil.

Key words: essential oils, Ocimum basilicum, E. coli, S. enteritidis, B. subtilis, S. aureus.

The history of using and researching medicinal herbs and essential oils is very long. Through humankind history some herbs were used because of their medical properties, some because of their magic and talisman powers and others as antimicrobial substances in food, drug and cosmetic products. Essential oils are volatile substances with intensive aromatic odor. In the middle of this century essential oils attach little importance because of expansion of synthetic products (1). However, the interest to essential oils is risen again today because of harmful effects of some synthetic pharmaceutical products.

Sweet basil (Ocimum basilica) is known as an aromatic medicinal herb having curative value (2). Its essential oils are widely used in flavourings including those for confectionery, baked goods, herb liqueurs meats and sausages, as well as in oral care products and certain perfumes. It has been reported that volatile substances from this specie showed antifungal properties against Aspergillus orysae, Alternaria tenuis, Microsporum gypseum, Penicillium chrysogenum and Penicillium notatum (3,4). Some essential oils at lower concentration are fungistatic, whereas higher doses prove to be fungicidal. Reuveni et al., (1984) studded the fungistatic activity of essential oils from Ocimum basilicum chemotypes against two fungi belonging to different taxonomic classes: Fusarium oxysporum f. sp. vasinfectum and Rhizopus nigricans. They concluded that the different antifungal activity of basil oils is determined mainly by the percentage of the main components, cineol, linalool, methylchavicol and eugenol. In addition, Dube et al., (1988) also investigated the antifungal activity of essential oils of basil, and found out that it is evidently a potent mycotoxic agent endowed with properties of killing aflatoxin-producing strains of fungi.

There are also a small number of studies on the microbistatic and microbicidal effects of essential oils on yeasts found in foods. Kivanc and Akgul, (1987) examined the inhibitory effects of twelve essential oils of Turkish spices on six strains of yeasts - C. tropicalis, H. anomala, K. apiculata, P. membranaefaciens, R. glutinis, S. cerevisiae. By agar diffusion method, oregano, savory and thyme oils inhibited completely the growth of all yeasts. The oil of basil was also fairly active since it was not affected only one or two yeasts.

Many laboratory results indicated that thousands of examined essential oils have stronger or slightly effect on bacteria. It was observed that the chemical compositions of essential oils are changeable depending on the plant species and climatic conditions, and therefore their antimicrobial effects may vary enormously (8,9). It has already been reported (10) that a number of O. basilicum varieties differ only in the chemical composition of their oils. Hasegawa et al. (1987) studded characteristic components in basil oils. They pointed out that variation in chemical composition of basil oils is thought to be due to polymorphism in Ocimum basilicum L., which is caused by interspecific hybridisation.

The aim of our research was to examine the antibacterial effects of essential oils of eight different chemotypes of basil on foodborne pathogens: Escherichia coli, Salmonella enteritidis, Bacillus subtilis, Staphylococcus aureus.

For experiments eight different chemotypes of basil were used. The plant materials were provided from the collection of Institute of Medicinal herbs, Belgrade. These chemotypes were distinguished by their qualitative and quantitative properties. They differ in color of leaves, flowers and stalks, in size and shape of leaves and in the same manner in content and composition of their essential oils. Chemotypes of basil T₁, T₂, T₅ and T₈ are colored types - with pink and red pigmentation. Chemotypes T₃, T₄, T₆ and T₇ were types with green leaves and stalks. The dominant components of essential oil of pink types were eugenol, methyl cinnamate and camphor. On the other hand the main components of essential oils of green types were methyl chavicol and linalool.

Essential oils were obtained from the leaves and flowers of Ocimum basilicum by hidrodistillation through Clevenger's apparatus, Ph.Jug.IV (12). The yields of the oils were recorded and the percentages of essential oils were calculated.

For the purpose of establishing the influence of tested essential oils on bacteria indicators of food contamination the disc plate method was used. For disc method essential oils were prepared by dissolving the oils with 96% ethanol in two concentrations (1:1 and 2:1). The 10 ml of prepared essential oils were used for each disc surface.

The test organisms, bacteria from ATCC collection of Institute for immunology and virology "Torlak"-Belgrade were: Escherichia coli ATCC (25922), Salmonella enteritidis ATCC (13076), Bacillus subtilis ATCC (6633), Staphylococcus aureus ATCC (25923). Inoculum was obtained from an actively growing culture with bacterial biomass of a 10⁷ cell·ml^-1.

The Muller-Hinton agar was used with 0.2 ml inoculum per 20 ml of the medium. Tree discs of filter paper (Schleicher & Schuell) with 5 mm in diameter were aseptically transfer onto the agar surface of each plate and incubated at 37°C during 24 h. Relative effectiveness of each of antibacterial substances against the test organisms were accomplished by measuring the diameter of the inhibition zone. As control the same amount of ethanol were used for each disc in place of the essential oils.

For obtaining the microbial or microbiostatic effects of essential oils on tested bacteria pieces of agar from inhibition zone were aseptically transfer into 5 ml sterile nutrient broth. The results were recorded by measuring turbidity after incubation at 37°C.

Identification of components was carried out on GC-MS. Gas chromatograph Hewlett Packard HP 6890 on DB-5 column. The content of components was determined by gas chromatography on FID, on the column of the same polarity.

The percentages of tested essential oils were varying in concentration from 0.5 to 1%, depending on chemotype of basil. Essential oils derived from eight different chemotypes of basil had different antibacterial effects (Tab. 1).

By using disc method we found out that essential oils caused the different inhibition zones on tested bacteria. However, it was established that essential oils of different chemotypes of basil T₁, T₂, T₄, T₅, T₇, and T₈ with concentration of 1:1 had antibacterial effects on following bacteria: E. coli, S. enteritidis, B. subtilis and S. aureus. Essential oil of chemotype T₅ was shown biocide effect on E. coli, and S. enteritidis, T₇ on S. enteritidis and T₈ on B. subtilis. Essential oils of others chemotypes with this concentration showed biostatic effects. Higher concentration (2:1) of essential oils of chemotypes T₁, T₂, T₄, T₅, T₇ and T₈ were also shown antimicrobial effects. Essential oils of mention chemotypes with this concentration had biocide effects on all tested bacteria.

Comparison of the antibacterial effects of tested oils on bacteria we pointed out that essential oils of chemotypes of basil T₁, T₂, T₄, T₅, T₇ and T₈ showed antibacterial effects with both concentrations. The most active oil was essential oil of chemotype of basil - T₇.

Comparison of the sensitivity of tested bacteria towards essential oils showed that S. aureus and S. enteritidis were the most resistant. On the other hand the most sensitive bacterium was E. coli.

The chemical composition of the essential oil of green chemotypes of basil T7 which showed the highest activity was evaluated by gas chromatographic analysis and results are presented in Tab. 2.

The results obtained by using disc method or agar diffusion indicate that essential oils of eight different Ocimum basilicum chemotypes may inhibit the growth of tested foodborne bacteria, but throughout to variable extent. These inhibition effects depended on qualitative and quantitative composition of their essential oils and also different concentrations of oils showed microbiostatic or microbiocidal effects on tested bacteria.

The essential oils of chemotype of basil T₅ with concentration (1:1) had biocidal effects on E. coli, and S. enteritidis, chemotype T₇ on S. enteritidis and chemotype T₈ on B. subtilis. Essential oils of other chemotypes with this concentration were shown biostatic effects. Higher concentration of essential oils (2:1) of chemotypes T₁,T₂, T₄, T₅, T₇ and T₈ showed biocide effects on all investigated bacteria.

Essential oil of chemotype of basil T₇ with methyl chavicol and linalool as main components has greatest influenced on majority of tested bacteria, but we do not know if these particular components had antimicrobial effects. For production practice we would recommend T₇ chemotype of basil as type of choice.

1. Reuveni R., Fleischer A. and Putievsky E. (1984): Fungistatic Activity of Essential oils from Ocimum basilicum Chemotypes. Phytopath. Z., 110, 20-22.

2. Dube S., Upadhyay P.D. and Tripathi S.C. (1988): Antifungal, physicochemical, and insect-repelling activity of essential oils of Ocimum basilicum. Can. J. Bot. 67, 2085-2087.

3. Kivanc M., Akgul A. (1987): Inhibitory effects of spice Essential oils on Yeast.

4. Hasegawa Y., Tajima K., Toi N. and Sugimura Y. (1997): Characteristic components found in the essential oils of Ocimum basilicum L. Flavour and fragrance journal, Vol. 12, 195-200.

5. Ph. Jug. IV, 1984.