Abstract: The present study, was conducted to evaluate in vitro antibacterial properties of Essential Oil (EO) from endemic plants, thyme (Thymus vulgaris) and mint (Mentha piperita) ageist Escherichia coli O157:H7. Antibacterial screening was done by Disk Diffusion (DD), Minimal Inhibitory Concentration (MIC) and Minimum Bactericide Concentration (MBC) methods. About 3.9 And 7.8 μg mL^-1concentrations of thyme EO were required in order to achieving the MIC and MBC. These indexes for mint EO was 15.6 and 31.2 μg mL^-1in E. coli O157:H7. In DD assay (500 μg disk^-1 concentration) thyme and mint were shown 35±1.4 and 13±1.5 mm inhibition zone. The chemical composition of hydro distilled EOs of thyme and mint was analyzed by Gas Chromatography-Mass Spectrometry (GC/MS). A total of 36 compounds of thyme and 23 compounds of mint, representing 93.62 and 90.69% of the oils were identified: Thymol (18.12%), Carvacrol (12.11%), p-Cymene (15.12%) in thyme and α-Terpinene (20.11%), Pipertitinone oxaide (17.10%) and trans-Carveol (19.48%) in mint, were the main components of the oils. Results here show that the EOs of thyme and mint possess antibacterial activity and therefore, it could be used as a natural preservative ingredient in food and/or pharmaceutical industries against E.coli O157:H7.

INTRODUCTION

In spite of modern improvements in slaughter hygiene and food production techniques, food safety is an increasingly important public health issue. It has been estimated that as many as 30% of people in industrialized countries suffer from a food borne disease each year and in 2000 at least 2 million people died from diarrhoeal disease worldwide (Burt, 2004). Among the reported outbreaks in the United States during 1993-1997 periods for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (75%) and the largest percentage of cases (86%) (Oussalah et al., 2007). There is still a need for new methods of reducing or eliminating food borne pathogens, possibly in combination with existing methods (Burt, 2004; Lee et al., 2005). All diarrhoeagenic strains of E. coli were initially termed Enteropathogenic E. coli (EPEC) but as their pathogenic mechanisms were known, they were grouped into seven classes (Duarte et al., 2007). Infections with Shiga Toxin-producing E. coli (STEC) serotype O157:H7 have been associated with a variety of sources, such as minced beef, dairy products, surface water and drinking water (Reinders et al., 2001). E. coli O157: H7 was recognized first in 1982 as a human pathogen during the investigation of 2 outbreaks of bloody diarrhoea in Oregon and Michigan. The relatively recent emergence of E.coli O157:H7 as a food borne pathogen has a significant impact on the food industry (Simsek et al., 2007).

Spices are used as culinary ingredients for taste and tang (Simsek et al., 2007; Seydim and Sarikus, 2006). They are also, used in oriental medicine (Eteghad et al., 2008). Many spices are used for curing minor illnesses like colds and stomach upsets. Research on this problem aimed to understand the natural defence mechanisms of plants and seeds against microorganisms (Simsek et al., 2007). Thyme, a member of the Lamiaceae family, is an aromatic and medicinal plant of increasing economic importance for North America, Europe and North Africa (Bisset and Wichtl, 2001). At present time, this plant is cultivated in large scale in Iran. Thyme volatile phenolic oil has been reported to be among the top 10 EOs, showing antibacterial, antimycotic, antioxidative, natural food preservative and mammalian age delaying properties (Badi et al., 2004). Mint is an herb used extensively in Indian cuisine and also for curing several common ailments. Mint extract did not show any antibacterial activity, though EOs of some Mentha species have been reported to have antibacterial activity (Kanatt et al., 2008; Marino et al., 2001).

The aim of present research, is to study in vitro antibacterial activities of the EOs of thyme and mint against E.coli O157:H7 and to determine the chemical composition of their EOs by GC/MS.

MATERIALS AND METHODS

Plant material: Collective samples of the aerial parts from thyme and mint growing wild in northwest-Iran were collected during the vegetative, the flowering and the post-flowering phases (June and July) from three different localities: Moghan, Misho and Hasanbiglu (in East-Azerbaijan province) and authenticated at Herbarium of Tabriz Faculty of Pharmacy, Tabriz, Iran. The material was dried in the dark at room temperature before extraction.

Isolation of the essential oils: The air-dried and ground herbal part of the plant collected, was submitted for 4 h to water-distillation using a British-type Clevenger apparatus. The obtained EOs were dried over anhydrous sodium sulphate and extracts yield were recorded:

and then stored at 4°C in sealed glass vials until tested and analyzed.

Gas chromatography-mass spectrometry: A mass spectrometer with an ion trap detector in full scan mode under electron impact ionization (70 eV) was used. The chromatographic column used for the analysis was HP-5 capillary column (30 m x 0.25 mm, film thickness 0.25 μm). The carrier gas used was helium, at a flow rate of 1 mL min^-1. The injections were performed in split less mode at 230°C. One micro liter EO solution in hexane (HPLC grade) was injected and analyzed with the column held initially at 60°C for 2 min and then increased to 260°C with a 5°C min^-1 heating ramp and subsequently kept at 260°C for 13 min. The relative percentages amounts of the separated compounds were calculated from total ion chromatograms by a computerized integrator.

Preparation of bacterial strains: E.coli O157:H7 was obtained from the culture collection of the Department of Food Hygiene and Quality Control, Azad University (Tabriz, Iran). The organism was maintained on tryptic soy agar (TSA, PH 7.0: Difco) slants at 4°C. Cultures were activated by transferring loop inoculating into 10 mL of tryptic soy broth (TSB, PH 7.0: Difco) at 37°C for 20 h. Following 2 consecutive 2 0 h culture transfers, a 50 mL TSB was inoculated with culture and incubated at 37°C for 20 h. The bacteria was grown overnight at 37°C in sorbitol McConkey Agar (Merck) and inoculum for the assays was prepared by diluting cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5 and confirmed by spectrophotometrical reading at 620 nm. Cell suspensions were finally diluted to 10⁸ cfu mL^-1 for being used in the antibacterial assays.

Antibacterial screening: The method of DD was employed for the determination of antibacterial activity of the EOs. The MIC of the samples against the test microorganism was determined by the broth micro dilution method (Sharififar et al., 2007). All growth-negative wells were spread on agar media separately for MBC test. All tests were performed in triplicate for each plant.

Disc diffusion assay: The EOs were dissolved in Dimethylsulfoxide (DMSO) (Merk, Schauchardt OHG, Germany) to 5 concentration (3.1, 6.2, 12.5, 25 and 50 mg mL^-1) and sterilized by filtration with 0.45 μm Millipore filters. Antibacterial tests were then carried out by DD method, using 100 μL of inculum, spread on nutrient agar. The sterile discs (Whatman No. 1, 6 mm in diameter) were impregnated with 10 μL of each concen-tration of EOs (31.2, 62.5, 125, 250 and 500 μg disc^-1) and placed on the inoculated agar. Negative controls were prepared using DMSO. The inoculated plates were incubated at 37°C for 24 h. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism.

Minimal inhibitory concentration and minimum bactericide concentration test: MIC tests were carried out according to Duarte et al. (2007), using Muller-Hinton Broth on a tissue culture test plate (96 wells) for each EO. The stock solutions of the EOs were diluted and transferred into the first well and serial dilutions were performed, so that concentrations in 0.9, 1.9, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250 and 500 μg mL^-1 were obtained. The inoculum was added to all wells and the plates were incubated at 37°C for 24 h. MIC was defined as the lowest concentration of oil that inhibited visible growth.

MBCs of tests were determined by spreading 100 μL of each clear well on Sorbitol MacConkey agar (SMAC) fallowing incubated at 37°C for 24 h.

Statistical analysis of data: Results from the experiments were analyzed by using the SPSS 13.5 statistical package (SPSS Ltd., Woking, UK). All experiments were accomplished in triplicate and the results reported are averages.

Table 1:	MICs, MBCs (μg mL -1) and Disk diffusion (millimeters) results against E. coli O157:H7
aInclude disk (6 mm in diameter). bEO concentration (μg disc-1); cNot affect

Table 2:	Chemical composition of thyme and mint EOs
RI: Kovats Retention Indices. aOther components were found at <0.1%

RESULTS

According to the results given in Table 1, the EOs of thyme and mint had great antibacterial activity against E. coli O157: H7. Results obtained from DD method, followed by measurements of MICs and MBCs.

The identified components of the EOs, as well as the percentages and Retention Index (RI) of each component are listed in Table 2 GC, GC/MS analysis revealed 36 compounds of thyme and 23 compounds of mint oils, respectively these compounds represent over 90% of the oils. Also, extracts yield were 1.43% for thyme and 1.87% for mint.

DISCUSSION

The oils were characterized with prominent (>10%) concentrations of Thymol, p-Cymene and Carvacrol in thyme and α-Terpinene, Pipertitinone oxide and trans-Carveol in mint. The thyme oil was also distinctive in its high concentrations (>5%) of α-Pinene and Geraniol, while, β-Caryophyllene, Isomenthone were two compounds in mint oil in these concentrations. The presence of Tricyclene, Camphene, β-Pinene, α-Terpinene, 1, 8-Cineole, p-Cymene, Limonene, γ-Terpinene, Terpinolene, γ-Terpineol, Geranyl acetate and β-Caryophyllene was detected in both oils in different concentrations.

To the best of our knowledge, there are many reports on the chemical composition of the oils isolated from thyme and mint (Dimitrijevi et al., 2007; Martinez et al., 2005; Jordan et al., 2006; Oussalah et al., 2007; Rota et al., 2007; Kitajima et al., 2004; Yadegarinia et al., 2006; Gherman et al., 2000; Tassou et al., 2000; Burt, 2004). Most of these reports indicate that Thymol, p-Cymene and Carvacrol in thyme and α-Terpinene, Pipertitinone oxide and trans-Carveol in mint are the main and/or characteristic constituents of these plant oils.

Based on the results of chemical composition of the EOs, we can conclude that the antibacterial nature of the EOs studied is apparently related to Carvacrol and Thymol in thyme and α-Terpinene, Isomenthone, Piperitone and Carvone in mint (Burt, 2004; Tassou et al., 2000). This claim is further supported by our findings; indicating high contents of Thymol, Carvacrol and α-Terpinene (Table 2).

The inherent activity of EOs can be expected to relate to the chemical configuration of the components, the proportions in which they are present and interactions between them. An additive effect is observed when the combined effect is equal to the sum of the individual effects. Antagonism is observed when the effect of one or both compounds is less when they are applied together than when individually applied (Burt, 2004). For example in mint, minor compounds such as Carvone and p-Cymene have synergistic antibacterial effects along major compounds such α-Terpinene.

According to Rota et al. (2007), ≤0.2 μL mL^-1 concentration of thyme EO was required in order to achieve the MIC and MBC in E. coli O157:H7. In another study that was prepared with Oussalah et al. (2007), EO was required in order to achieve the MIC in same bacteria was 50 μL mL^-1. In other study, MIC for Enteroinvasive E. coli (EIEC) and STEC, 400 and 300 μg mL^-1 was recorded (Duarte et al., 2007). In same condition, MIC and MBC for mint EO 20 and 22 μL mL^-1was reported (Moreira et al., 2005). The previous studies, showed that Iranian mint EO had antibacterial effect against E. coli in 2 μL mL^-1 Concentration (MIC) (Yadegarinia et al., 2006).

In current study, the results of MIC and MBC for each plant EOs against test pathogen (E. coli O157:H7) are written in Table 1, they are different from previous findings.

In studies done with DD method we seen wide range of concentration in Eos usages. According to Martinez et al. (2005), thyme EO inhibits zone for E. coli was 18.4 mm (6 μL disk^-1). In another study that was prepared by Sagdic (2003) inhibits zone for E. coli O157:H7, 12 mm was recorded. In same condition, 17 mm inhibit zone was reported for mint EO (Moreira et al., 2005). According to Kanatt et al. (2008) mint extract (0.1%) did not have any antibacterial activity. But Yadegarinia et al. (2006) shown that Iranian mint EO can certain 31.33 mm inhibit zone in E.coli. By attention to Table 1, our results are different from previous findings in DD method and same concentration.

The composition of EOs from a particular species of plant can differ between harvesting seasons and between geographical sources. So, EO components have been shown to exhibit antimicrobial activity to different extents (Burt, 2004) in different plants.

By notice to results, thyme and mint essences cultured in area can be used as a natural antibacterial compounds, preservative ingredient in food and/or pharmaceutical industries in said concentrations against E. coli O157:H7. Also inhibition in same concentration in thyme is grater than mint.

ACKNOWLEDGEMENT

The authors are thankful to Dr. Vakil Zadeh and his cooperators (Research office of Tabriz Islamic Azad University, Tabriz, Iran) for spiritual auspices.