Abstract: The antibacterial effect of different concentrations (0.01 to 15%) of thyme (Thymus vulgaris), peppermint (Mentha piperita L.) caraway seed (Carum carvi), fennel (Foeniculum vulgar), tarragon (Artmesia dracunculus) and pennyroyal (Mentha pullegium) essential oils on the Staphylococcus aureus and Escherichia coli was studied in nutrient broth medium. The MIC values of peppermint, fennel, thyme, pennyroyal and caraway essential oils against Escherichia coli were 0.5±0.03, 1±0.03, 0.3±0.01, 0.7±0.03 and 0.6±0.02% and in contrast, for Staphylococcus aureus were 0.4±0.01, 2±0.13, 0.1±0.01, 0.5±0.02 and 0.5±0.02%, respectively. The MBC values of peppermint, fennel, thyme, pennyroyal and caraway essential oils for Escherichia coli were 0.7±0.02, 2±0.05, 0.5±0.02, 1±0.02 and 0.8±0.02 and for Staphylococcus aureus were 0.5±0.02, 4±0.26, 0.3±0.02, 0.7±0.02 and 0.6±0.01, respectively. Statistical evaluation of the results indicated that the essential oils of thyme (Thymus vulgaris) showed the broadest spectrum of action (p<0.05). Essential oils of peppermint (Mentha piperita), caraway seed (Carum carvi), pennyroyal (Menthae pullegium) and fennel (Foeniculum vulgar) had moderate effect against tested microorganisms and in contrast, tarragon essential oil were less effective against tested microorganisms. In conclusion, essential oils of edible plants could be a potential source for inhibitory substances for some foodborne pathogens. Natural substances that extracted from plants have applications in controlling pathogens in foods.
INTRODUCTION
The food industry has tended to reduce the use of chemical preservatives in their products due to increasing pressure from consumers or legal authorities, to either completely remove or to adopt more herbal alternatives for the maintenance or extension of product shelf life (Nychas, 1995). Medicinal plants may offer a new source of antibacterial agents for use. In many parts of the world medicinal plants are used for antibacterial, antifungal and antiviral activities (Essawi and Srour, 2000; Nevas et al., 2004; Pina-Vaz et al., 2004; Proestos et al., 2005). Medicinal plants contain numerous biologically activity compounds, many of which have been shown to have antibacterial properties.
There is considerable interest in the possible use of these compounds as food additives, to delay the onset of food spoilage or to prevent the growth of foodborne pathogens. Among these pathogens Escherichia coli and Staphylococcus aureus are of great importance.
Although the antibacterial activity of Essential Oils (EOs) from spices has been recently reviewed (Nychas, 1995), their mechanism of action against the microorganisms has not been studied in great detail. Only a few studies have focused on the mechanism by which spices or their essential oils inhibit microorganisms. Since essential oils consist of terpenes (phenolics in nature), it would seem reasonable that their mode of action might be related to those of other phenolic compounds. According to Conner and Beuchat (1984a and b) the antimicrobial action of essential oils may be due to impairment of a variety of enzyme systems including those involved in energy production and structural component synthesis.
The major antimicrobial components of spices and their essential oils are, for example, eugenol in cloves, allicin in garlic, cinnamic aldehyde and eugenol in cinnamon, carvacrol and thymol in oregano and thyme and vanillin in vanilla beans. The essential oil shows antioxidant (Dhuley, 1999), antibacterial (Nevas et al., 2004; Friedman et al., 2004) antifungal (Chami et al., 2004; Wang et al., 2005) and some other therapeutic activities. However, there are often large differences in the reported antimicrobial activity of oils from the same plant.
The antimicrobial activity of some essential oil components against foodborne pathogens, including mycotoxin-producing fungi, has also been tested (Kim et al., 1995; Thompson, 1996; Cox et al., 2000; Delaquis et al., 2002; Benkeblia, 2004). More recently, plant extracts have been developed and proposed for use in foods as natural antioxidants (Curvelier et al., 1996; Basaga et al., 1997; Dhuley, 1999) and/or antimicrobials (Del Campo et al., 2000; Hsieh, 2001; Mejlholm and Dalgaard, 2002; Burt and Reinders, 2003).
Tests of antimicrobial activity can be classified as diffusion, dilution or bioautographic methods (Rios et al., 1988; Koutsoumanis et al., 1998). The principles and practice of these test methods are explained in the literature (Barry, 1976; Davidson and Parish, 1989) but it appears that no standardised test has been developed for evaluating the antibacterial activity of possible preservatives against food related microorganisms, although the need for such has been indicated (Davidson and Parish, 1989). The NCCLS method for antibacterial susceptibility testing, which is principally aimed at the testing of antibiotics has been modified for testing essential oils (NCCLS, 2000). The Minimum Inhibitory Concentration (MIC) is cited by most researchers as a measure of the antibacterial performance of EOs. The definition of the MIC differs between publications and this is another obstacle to comparison between studies. In some cases, the Minimum Bactericidal Concentration (MBC) or the bacteriostatic concentration is stated, both terms agreeing closely with the MIC.
The aim of the present study was to evaluate the antibacterial activity of several Iranian essential oils against the Escherichia coli and Staphylococcus aureus in broth media. It is well known that the activity of antibacterial varies according to the pathogen strain and may be influenced by the storage temperature of foods.
MATERIALS AND METHODS
Essential oils: The commercial and scientific names of the investigated plants are given in the Table 1. All essential oils used in this study were kindly provided by Dr. H. Aruei, Department of Horticulture, Ferdowsi University of Mashhad. The extractions were carried out by the method described by Helander et al. (1998), basically, plant materials (100 g) were cut into small pieces and placed in a flask (2 L) with double distilled water (1.5 L). A steam distillation continuous extraction head was attached to the flask. After steam distillation (3 h), the oils were isolated and dried over anhydrous sodium sulphate.
Strains, preparation of inocula: Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used in this study both kindly supplied by Mashhad University of Medical Sciences, Mashhad and Iran Medical Laboratory. Each culture was activated by transferring a loopful from brain heart infusion (BHI, Oxoid, CM0375) slants into nutrient broth (Oxoid, CM0001) followed by incubation at 37°C for 24 h. The optical density of each active culture was adjusted to 0.1 at 625 nm using fresh broth to give a standard inoculum of ca. 106 Colony Forming Units (CFU) per ml. Bacterial counts were confirmed by plating out on Standard plate count agar (Oxoid, CM0325), plates incubated at 37°C for 24 h.
Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) test: Dilutions of each plant essential oil to be tested were prepared in 1.0 mL volumes of sterile brain heart infusion broth to give a range of concentrations from 0.01 to 15%. After preparation of suspensions of test microorganisms (ca.106 organisms per ml), one drop of suspension (0.02 mL) was added to the extract broth dilutions. After 24 h incubation at 37°C, the tubes were then examined for growth. MIC was defined as the lowest concentration of essential oil, showing no visible bacterial growth after incubation time (Hammer et al., 1999; NCCLS, 2000; Delaquis et al., 2002). MBC was defined as the lowest concentration of essential oil at which no visible bacterial growth is observed after subculturing into fresh broth (Onawunmi, 1989 NCCLS, 2000). 0.1 mL from tubes that showed no visible growth added and spread on the nutrient agar medium (Oxoid, CM0003) and incubated for 24 h at 37°C for determination of MBC of essential oils. Oxacillin and norfloxacin (Sigma) served as positive controls for Staphylococcus aureus and Escherichia coli, respectively. Methanol (or water) were included in every experiment as negative controls.
Statistical analysis of data: All experiments were conducted in triplicate
and tests were duplicated (two extractions). Experiment was conducted twice
and data were averaged and analysed statistically using SPSS software (Version
10.0.5). The results are expressed as mean values±SE.
| Table 1: | Essential oils of plants investigated for antibacterial activity |
| Table 2: | The in vitro antibacterial activity of essential oils from plants against bacteria |
RESULTS AND DISCUSSION
Because of Staphylococcus aureus and Escherichia coli are important bacteria among foodborne pathogens, they were selected for antibacterial assay of the essential oils (Table 2). The Minimum Inhibitory Concentration (MIC) values of peppermint, fennel, thyme, pennyroyal and caraway essential oils for Escherichia coli were 0.5±0.03, 1±0.03, 0.3±0.01, 0.7±0.03 and 0.6±0.02% and in contrast, for Staphylococcus aureus were 0.4±0.01, 2±0.13, 0.1±0.01, 0.5±0.02 and 0.5±0.02%, respectively. The Minimum Bactericidal Concentration (MBC) of peppermint, fennel, thyme, pennyroyal and caraway essential oils for Escherichia coli were 0.7±0.02, 2±0.05, 0.5±0.02, 1±0.02 and 0.8±0.02 and for Staphylococcus aureus were 0.5±0.02, 4±0.26, 0.3±0.02, 0.7±0.02 and 0.6±0.01, respectively (Table 2). Statistical evaluation of the results showed that thyme, caraway, peppermint and pennyroyal essential oils proved to be more effective in inhibiting the bacterial growth in vitro than fennel and tarragon essential oils (p<0.001).
The investigations of Valero and Salmeron (2003) showed that the essential oil of cinnamon, oregano and thyme were most effective on controlling of B. cereus Essawi and Srour (2000) reported that Salvia officinalis, Teucrium polium, Majorana syriaca, Thymus origanium, Thymus vulgaris, Commiphora opobalsamum Foeniculum vulgare and Rosmarinus officinalis exhibited an antibacterial effect against some of both Gram-positive and Gram-negative bacteria.
In our present study, it is deduced that the Essential oils of thyme (Thymus vulgaris) significantly showed the broadest spectrum of action (p<0.05). Essential oils of peppermint (Mentha piperita), caraway seed (Carum carvi), pennyroyal (Menthae pullegium) and fennel (Foeniculum vulgare) had moderate effect against tested microorganisms and in contrast, tarragon essential oil were less effective against tested microorganisms.
Determination of MIC and MBC of tested essential oils against tested bacteria showed that Staphylococcus aureus were significantly more susceptible to the essential oils than Escherichia coli. In accordance with previous findings, it seems that the antibacterial action of the essential oils is more pronounced on Gram-positive than on Gram-negative bacteria and these findings correlate with the observations of previous screenings of medicinal plants for antimicrobial activity, where most of the active plants showed activity against Gram-positive strains only (few are active against Gram-negative bacteria) (Kelmanson, 2000; Ali et al., 2001). The resistance of Gram-negative bacteria towards antibacterial substances is related to lipopolysaccharides in their outer membrane (Gao et al., 1999).
In literature, it has been indicated that the antibacterial activity is due to different chemical agents in the extract, including essential oils (especially thymol), flavonoids and triterpenoids and other compounds of phenolic nature or free hydroxyl group, which are classified as active antimicrobial compounds (Rojas et al., 1992; Essawi and Srour, 2000). Camporese et al. (2003) reported that various concentrations of the plants species extracts showed activity to some extent against Escherichia coli and Pseudomonas aeruginosa, while Aristolochia trilobata leaves and bark Syngonium podophyllum leaves and bark were active also against Staphylococcus aureus. The highest activity was shown against the Gram-positive bacterium Staphylococcus aureus. A recent study demonstrated an antibacterial activity of rosmarinic acid against Escherichia coli and Staphylococcus aureus (Teng et al., 2000). Sagdic et al. (2002) reported that between extracts of seven spices, thyme and oregano showed higher activity than others on the growth of Escherichia coli 0157:H7 strain.
CONCLUSIONS
In conclusion, edible plants could be a potential source for inhibitory substances for some foodborne pathogens. Natural substances that extracted from plants (specially, medicinal, aromatic and spice plants) have applications in controlling pathogens in foods (Davidson, 1997; Bowles and Juneja, 1998). Since the medicinal plants studied appear to have a broad antimicrobial activity spectrum, they could be useful in antiseptic and disinfectant formulations as well as in chemotherapy.
The essential oils of thyme (Thymus vulgaris) showed the broadest spectrum of action against Staphylococcus aureus and Escherichia coli (p<0.05). Results reported in this study showed that essential oils of peppermint (Mentha piperita), caraway seed (Carum carvi), pennyroyal (Menthae pullegium) and fennel (Foeniculum vulgare) had moderate effect and in contrast, tarragon essential oil were less effective against tested microorganisms (p<0.001). Recently, some of these herbal drugs showed also a topical anti-inflammatory, activity that, together with the antibacterial properties supports their traditional use as wound healing in Central America. Furthermore, beside the confirmation of the popular use, the obtained results demonstrate that these herbal drugs could represent a new source of antimicrobial agents, less expensive than the imported drugs (Nychas, 1995).
The ability of edible plant extracts in inhibiting pathogenic bacteria in food matrices and the impact of plant materials on organoleptic properties of food require further studies.
ACKNOWLEDGMENTS
I would like to express my appreciation and thanks to Dr. H. Aruei for providing us with essential oils, Dr. J. Mehrzad and Dr. K. Ghazvini, Mashhad University of Medical Sciences for technical help and Iran Medical Laboratory, for providing us with the microorganisms.