Plant Essential Oils Antimicrobial Properties Against Bacteria

Experiments were carried out in the laboratory to examine the efficacy of plant essential oils such as thyme, lemon, tea tree, peppermint basil, and clove. The agar diffusion test was used to investigate the effects of essential oils on specific bacteria such as E.coli at various MCI percentages. These essential oils significantly inhibited bacterial growth, indicating that they have antimicrobial powers.
Plants contain essential oils that are partially soluble in alcohol and slightly soluble in water. Plant essential oils are made up of a complex combination of aldehydes, ketones, esters, and terpenes. The primary method for the extraction of essential oils from plants is by means of steam distillation although some can be achieved by cold, dry or vacuum distillation.
Plant essential oils have a long history of usage by humanity. Examples of plant essential oils include tea tree oil that has been widely used in Australia by the Aborigines to treat wounds and man other ailments that are minor. The essential oils of plants have become a big and familiar topic in scientific research both in pharmaceutical and food industries. These scientific research interests have led to the emergence of bacterial strains that have developed resistance to antibiotics. Such bacteria include S. aureus that is resistant to methicilin and Enterococcus faectum that has extensively developed resistance to vancomycin.
Food safety remains an increasingly significant issue of public health despite the modern improvements in food safety and production techniques for food (WHO 2002). According to Burt (2004), approximately 30% of people living in industrial countries face ailments that are food bone related each and every year. He also estimates that nearly two million people died in 2002 as a result of such food bone diseases causing diarrhea.
Such occurrences need the intervention of methods that are new in order to help inhibiting such food borne diseases. This can be achieved by combining the existing methods such as refrigeration, hurdle principle, modifying the atmosphere and heating to addition of antimicrobial compounds. It is important that production of safe food that is green and natural is promoted. The use of organic fertilizers such as animal waste in agriculture has at most occasions contributed to the agricultural produce contamination by the pathogens. Such contamination has also been reported for surface water and the grounds. The use of essential oils for various reasons is reported to might have been the cause of the continued spread of bacterial pathogens. Essential oils are historically known to contain antibacterial properties based on their chemical composition.
Biological activity of essential oils depends on their chemical composition, which is determined by the genotype of the plant and certain factors such as agronomic, geographical origin and environmental conditions. Plant essential oils contain variable mixtures of terpenoids that are essential, diterpenes, alcohols, acids, phenolic compounds, lactones or acyclic esters (Rota et al. 2004).
Most species of plants and more so herbal plants produces antimicrobial properties because of the factions of essential oils they contain. Antimicrobial properties in plant essential oils have been reported in plants such as peppermint, cloves, orange, basil, nutmeg and tea tree amongst others. The structure, functional groups and composition of the essential oil acts as the key determinants for the antimicrobial properties of these plant essential oils (Omidbeygi et al. 2007; Yesil Celiktas et al. 2007).
Plant essential oils containing phenolic group compounds have been much associated with antimicrobial properties. Oils with suc compounds as phenol have been classified as Generally Recognized as Safe (GRAS) and have been recommended for use in agriculture as organic fertilizers in order to prevent post harvest development of bacteria that are deemed as contaminants (Singh et al. 2002; Moreira et al. 2005).
The aim of this laboratory experiment was to evaluate the antimicrobial properties of plant essential oils such as peppermint, cloves, orange, basil, nutmeg and tea tree on selected bacteria.
Material and methods
Materials
Bacterial strains and inoculum preparation. Escherichia coli, Staphylococcus aureus, Staphylococcus epiderdimis, Listeria monocytogenes or Pseudomonas aeruginosa. These strains of bacteria were preserved in nutrient broth to prevent them from deteriorating. The cultures were obtained through aerobic incubation in nutrient agar for 24hours in 37oC.
Essential oils: peppermint, clove, orange, basil, nutmeg and tea tree.

Methods
100ul of the bacterial culture was added to each of the three TSA plates and spread over the entire surface. Using a disc dispenser, 6 disks were evenly placed around the side of the plate. To each disc on one plate, 10ul of the neat plant essential oil was added and the process repeated on the second plate using the 10% solutions of the plate essential oils. The plates with essential oils were properly labeled before the oil was added into the disc. The third plate was left as a spare and used to repeat either of the two other plates.
The plates were then incubated for 24 hours at 37oC. The expected result after incubation was that zones of inhibition would appear round the edge of the disks where the oils have inhibited the growth of the test bacteria.
In order to determine the concentration of bacteriostatic and bactericidal concentrations, all the test tubes were inoculated with 20ul of the overnight culture of the bacteria and mixed together. Appropriate volumes of 10% solution of the plant essential oil was added in order to achieve the final concentrations of the oils in the broth of 1, 0.5, 0.25, 0.1, 0.05, 0.01 and 0.005%. control was used and it contained no plant essential oil.
In order to examine the tubes that had evidence of bacterial growth, 100ul of TSB culture media were was plated and incubated for 24 hours at 37oC. It was noted that the bacteriostatic concentration was lowest concentration at which bacteria fail to grow in the broth but are cultured when samples are plated onto agar.
Preparation of the strip
Incubation box, tray and lid were prepared and 5mls of water distributed into the honeycombed wells of the tray in order to create a humid chamber. The strain reference number was recorded on the elongated tab of the tray and then the strip was placed on the tray.Oxidase test was performed on the identical colony by placing a piece of filter paper on a glass slide. The paper was moistened with one drop of water. The chosen colony was rubbed n the moistened filter paper and one drop of oxidase reagent added on to it in order for a deep violet coloration indicating a positive reaction to be noted.

Preparation of the inoculum
An ampoule of suspension medium was opened with the aid of pipette and a single, well-isolated colony removed from the isolation plate. The inoculum was carefully emulsified in order to achieve a homogenous bacterial suspension.
Inoculation of the strip
The same pipette used for the preparation of the inoculum was used to fill both the tube and cupule of tests with the bacterial suspension. Only the tubes of the other tests were filled and not the cupules. We created anaerobiosis in the tests ADH, LDC, ODC, URE and H2S by overlaying wit mineral oil. The incubation box was closed and incubated for 18 to 24 hours at 37oC.
Reading the strip
After the incubation time was over, the strip was read by referring to he interpretation table. All the spontaneous reactions were recorded on the report sheet.
Identification
By the use of the identification table, the recorded results were compared to the ones given in the table. The patterns of the reactions obtained were coded into numerical profile by the use of Analytical Profile Index. On the report sheet, the tests were separated into groups of three and a number 1, 2, or 4 was indicated for each. By adding the numbers corresponding to POSITIVE reactions within each group, a 7-digit profile number was obtained for the 20 tests of the API 20 Estrip.

Results
Table 2. MIC, Cidal and Static Concentrations
Bacterium Oil MIC Static Cidal E.coli Peppermint 0.05% P.vulgaris Basil 0.25% B.subtilis Clove 0.05% P.auroginosa Clove 0.1% S.aureus Tea tree 0.1% S.aureus Lemon 0.5% S.epidedimis Peppermint 0.1% E.coli Tea tree 0.25% P.vulgaris Clove 0.1% P.auroginosa Thyme 0.1% S.epidedimis Tea tree 0.1% B.subtilis Clove 0.25% S.aureus Lemon 0.5% S.aureus Thyme 0.1% P.vulgaris Thyme 0.1% S.epidedimis Clove 0.1% E.coli Lemon 0.5% E.coli Peppermint 0.01% P.auroginosa Lemon 0.5%
Table showing the antimicrobial activity of essential oils such as: thyme, clove, tea tree, lemon, basil and peppermint against various bacteria.

Discussion
Table 1 summarizes the antimicrobial properties of the five essential oils (thyme, basil, peppermint, tea tree and clove). Bacteria susceptibility to the essential oils, as determined by the agar diffusion method, showed that oils with the highest inhibitory effects produced inhibition zones of 20–25 mm diameter. In the dose response study, the inhibition zone increased with the increasing concentration of essential oil. Low concentrations (10μl) of essential oils inhibited weakly the development of bacteria. However, E. coli, and S. aureus were more sensitive than monocytogenes in the medium containing thymol essential oil. At a high concentration (20 μl/ml), the essential oil extracts exhibited a marked inhibition activity against bacteria, and the inhibition of the essential oil extract of thyme was stronger than that of the others, showing inhibition zones ranging from 23-30 mm. Comparatively, e. coli and S. aureus were less sensitive to the inhibitory activity of the orange and myrtle essential oils than monocytogenes which was more inhibited at the same concentrations of the same essential oils extracts. On the other hand, all bacteria showed low susceptibility to laurel essential oil, with 12–19 mm diameter inhibition zones compared to the same levels of thyme, basil, lemon, and peppermint essential oils.
Conclusion
Thyme, lemon, peppermint, basil, and tea tree essential oils have a potential to inhibit and inactivate certain bacteria in agar and milk medium at different concentrations. The inhibitory effects of essential oils increased with increasing concentration. It is suggested to investigate higher essential oils concentrations than were those used in research, and to study the effects over a longer time period in milk and other available milk products to access the potential of plant species essential oils as preservatives.

References
Burt S. (2004): Essential oils: their antibacterial properties and potential applications in foods – a review. International Journal of Food Microbiology, 94: 223–253.
Omidbeygi M., Barzegar M., Hamidi Z., Naghdibadi H. (2007): Antifungal activity of thyme, summer savory and clove essential oils against aspergillus flavus in liquid medium and tomato paste. Food Control, 18: 1518–1523.
Rota C., Carramiñana J.J., Burillo J., Herrera A. (2004): in vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens. Journal of Food Protection, 67: 1252–1256.
Singh N., Singh R.K., Bhunia A.K., Stroshine R.L. (2002): Efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing escherichia coli O157:H7 on lettuce and baby carrots. Lebensmittel- Wissenschaft und – Technologies, 35: 720–729.
WHO (2002b): World Health Report. 2002. Reducing Risks, Promoting Healthy Life. 30 October 2002. World Health Organization, Geneva.