Characterization of aromatic compounds and antimicrobial properties of four spice essential oils from family Lamiaceae

Essential oil Garden thyme Rosemary Spearmint Sweet basil Antibacterial Abstract Introduction. Representatives of the Lamiaceae family are widely used in the food industry as they are characterized by a high content of aromatic compounds. Materials and methods. It was evaluated the chemical composition and antibacterial activity of essential oils of garden thyme (Thymus vulgaris L.), rosemary (Rosmarinus officinalis L.), spearmint (Mentha spicata L.), and sweet basil (Ocimum basilicum L.). Results and discussion. The percentage ratio of volatile components obtained by GC-MS analysis of essential oil from garden thyme contains: thymol (37.90%) and γ-terpinene (19.44%). It has been determined eucalyptol (19.89%) and camphor (16.86%) in the essential oil of rosemary and carvone (50.23%) and limonene (13.90%) in spearmint oil, respectively. The differences in the quantitative and qualitative composition of essential oils and their aromatic components in relation to the previous researches may be probably due to different environmental and genetic factors, different chemotypes and the nutritional status of the plants as well as other factors that can influence the oil composition. Escherichia coli was the most susceptible bacterium strain. The essential oils of spearmint and rosemary possessed the most pronounced antibacterial activities against Escherichia coli (with inhibition zone: 32.00 mm and 30.00 mm). Conclusions. The results obtained provide a basis for a thorough examination of the chemical composition and antimicrobial properties of various representatives of Lamiaceae family with a view to a wide usage in food technology. Article history:


Introduction
Sweet basil (Ocimum basilicum L.), is a culinary herb of major importance. Most culinary and ornamental basils are cultivars of the species O. basilicum, but other species are also grown and there are many hybrids between species [38]. Sweet basil can be dried or used fresh, similar to many other pot-herbs. It is best used fresh, as dried it will lose its flavor. Sweet basil leaves containing essential oil of distinctive aroma can be used both fresh and dried to spice up various kinds of meals. Apart of culinary use, sweet basil has been traditionally applied as a medicinal herb [33]. The presence of essential oil and its composition determine the specific aroma of plants and the flavor of the condiment [33]. Although essential oils in different basil cultivars are variable, prevalent components are monotherpenes and phenylpropanoids [28]. Fresh sweet basil leaves is used as an ingredient in various dishes and food preparations, especially in the Mediterranean cuisine. Due to its antimicrobial [9,37] and insecticidal [8] activities and very pleasant aroma, sweet basil essential oil is widely used in the food.
Its usage in culinary practice is based on the possibility of fresh and dried leaves to emphasize meat and vegetable flavors, giving them a pronounced aromatic flavor. Most often aromatic components in fresh spices are obtained by low temperature extraction with vegetable oils used in culinary practice. The resulting aromatic oil substances are successfully used, such as salad dressings, spices for meat and vegetable soups, poultry and game dishes.
Due to the wide use of the Lamiacea spices in the food technology and the scarce information in Bulgaria about their aromatic composition and antimicrobial properties against foodborne pathogens, it have been involved the need for their further examination.
The aim of the present study is to characterize the aromatic components in the essential oils of four spices of the family Lamiaceae and antimicrobial properties against pathogenic bacteria, causing foodborne illnesses, with potential possibilities for their application in food technology.

Plant Material
Garden thyme (Thymus vulgaris L.), rosemary (Rosmarinus officinalis L.), spearmint (Mentha spicata L.), sweet basil (Ocimum basilicum L.) were purchased from the merchant local market in Plovdiv. Samples were identified by an expert in Agricultural University of Plovdiv, Bulgaria.
The moisture content of raw materials was determined by drying to constant weight at 105°С [34].

Essential oils
The essential oils were obtained with modification of hydrodistillation for 150 min in the laboratory glass apparatus according to the British Pharmacopoeia [6].
The oils were dried over anhydrous sulfate and stored in tightly closed dark vials at 4 °C until analysis.
The GC-MS analysis was carried out with an Agilent 5975C MSD system coupled to an Agilent 7890A gas chromatograph (Agilent Technologies Inc., Santa Clara, CA). Agilent J&W HP-5MS column (0.25 µm, 30 m x 0.25 mm) was used with helium as a carrier gas (1.0 mL min -1 ). The operational conditions were: oven temperature 35 °C/3 min, 5 °C/min to 250 °C for 3 min, total run time 49 min; injector temperature 260 °C; ionization voltage 70 eV; ion source temperature 230 °C; transfer line temperature 280 °C; solvent delay 4.25 min and mass range 50 -550 Da. The MS was operated in scan mode. One μL of the sample was injected into the GC/MS system at split ratio 30:1. The GC analysis was carried out using an Agilent 7890A GC system; FID temperature 270 °C. In order to obtain the same elution order with GC/MS, simultaneous triplicate injections were done by using the same column and the same operational conditions. The identification of compounds was made by comparing their mass spectra with those from mass spectra libraries [1] and by comparing the literature and estimated Kovat's (retention) indices that were determined using mixtures of homologous series of normal alkanes from C8 to C40 in hexane, under the conditions described above. The percentage ratio of volatile components was computed using the normalization method of the GC/FID peak areas.
Determination of antibacterial activity As test microorganisms were used strains of pathogenic bacteria, reported as causing foodborne infections, intoxications and toxicoinfections. Antibacterial activity of essential oils was tested against Gram-positive bacteria -Listeria monocytogenes NCTC  The media were inoculated with 24-hour suspension of the bacterial species.
Melted and cooled to 45 o C selective media were inoculated with the tested microorganisms and next equally dispensed into Petry dishes. After setting of the media, sterile rings (Ø 6 mm) were placed on, and the amount of each sample (0.05 mL) was put into the rings. Petry dishes were incubated at 37 °C for 24 or 48h according to the bacterial spices, and then the distinct zone of growth inhibition (mm) around the rings was measured. The used inoculums have resulted as an actual concentration cells of L. monocytogenes, S. aureus, E. coli, S. enterica into the responding selective medium about 3 × 10 5 CFU/mL. The total plate count was estimated by the conventional plate-counting technique using appropriate dilution.

Results and discussion
Chemical composition of essential oils Garden thyme (Thymus vulgaris L.). The moisture of the plants was determined as 82.75%±0.80. The yield of essential oil was 0.56%±0.00 (in abs. dry mass was 3.26%±0.03). The oil was light yellow liquid with a characteristic, spicy-phenolic odor ( Table 1). The oil is consisted by 35 components, representing 98.89% of the total content. Twelve of them were in concentrations over 1% and the rest 23 constituents were in concentrations under 1%. The major constituents (up 3%) of the oil were as follows: thymol (37.90%), γ-terpinene (19.44%), p-cymene (8.84%), δ-2carene (3.92%), carvacrol (3.60%), and β-caryophyllene (3.37%). The results indicated that Bulgarian essential oil obtained from T. vulgaris is from thymol chemotype [19], which may also have a profound influence on its bioactivity, flavor, and aroma profile. The moisture of the rosemary sample was 64.00%±0.60. The yield of essential oil was 1.09%±0.01 (in abs. dry mass was 3.03%±0.03). The oil was light yellow liquid with a characteristic, refreshing, pleasant odor. The oil is composed by 34 components (Table 1) representing 99.00% of the total content. Sixteen of them were in concentrations over 1% and the rest 18 constituents were in concentrations under 1%. It is obvious that the major constituents (up 3%) of the oil were as follows: 1,8-cineole (19.89%), (+)-camphor (16.86%), α-pinene (13.37%), camphene (7.22%), borneol (5.27%), α-phellandrene (4.27%), αterpineol (4.10%), verbenone (3.69%), and bornyl acetate (3.37%). One of the values of aromatic components in the composition of the essential oil of rosemary is comparable to those obtained by De Mastro et al. [14]. The results indicated that Bulgarian essential oil obtained from R. officinalis is from camphor chemotype [19]. The differences in chemical composition were explained with the different extraction methodologies used.
The moisture of the spearmint sample was 84.60%±0.81. The yield of essential oil was 0.83%±0.00 (in abs. dry mass was 5.41%±0.05). The oil was light yellow liquid with a fresh, caraway-minty odor ( Table 1). The results show that 40 components representing 98.95% of the total content were identified in the oil. Fourteen of them were in concentrations over 1% and the rest 26 constituents were in concentrations under 1%. It is clear that the major constituents (up 3%) of the oil were as follows: (-) carvone (50.23%) and limonene (13.90%), that qualitative and quantitative results are comparable with that identified from Nikšić et al. [31]. Through aromatic compounds isolated from M. spicata in Bulgaria, the carvone/limonene chemotype of the tested sample was established [19].
The differences in the quantitative and qualitative composition of essential oils of garden thyme, rosemary, spearmint and sweet basil and their aromatic components in relation to the previous researches may be probably due to different environmental and genetic factors, different chemotypes and the nutritional status of the plants as well as other factors that can influence the oil composition.

Antibacterial activity
The results of antibacterial testing are presented in Table 2. All essential oils showed good antibacterial potential against tested four strains of foodborne pathogens. E.coli was the most susceptible bacterium strain. The essential oils of spearmint and rosemary possessed the most pronounced antibacterial activities against E. coli (with inhibition zone: 32.00 mm and 30.00 mm). Garden thyme was most effective against E. coli due to the two major constituents as thymol and carvacrol, because of their ability to break the outer membrane of Gram-negative bacteria and increase the permeability of the cytoplasmic membrane. On the other hand, the essential oil of sweet basil showed highest antibacterial property against S. enterica owing to the presence of the major compound linalool.
The weakest potential was observed by the spearmint oil against S. enterica. All these spices, widely used in culinary technology contain compounds that have been shown to possess antibacterial functions. Studies have shown that constituents with a phenolic structure in essential oils, such as eugenol, carvacrol and thymol have the greatest antibacterial activities, followed by aldehydes, ketones, alcohols, ethers and hydrocarbons [39,24]. Our results for garden thyme were in agreement with the findings of El Hattabi et al. [17].
The results of the antibacterial activity of the spearmint essential oil were lower than the findings of Horváth and Koščová [23] reported the highest antibacterial properties against S. aureus CCM 4223 with inhibition zone varied at a range of 35.67 mm. The results obtained in this study are comparable to the findings of Moghaddam et al. [30,37] reported that sweet basil essenatial oil showed inhibition zones against S. aureus (29.20-30.56 mm), and E. coli (17.48-23.58 mm).
Differences in the geographic environment, the cultivar type, age of the plant, different methods of isolation, and seasonality of the samples could be the reasons for the obtained differences in spectrum of antibacterial activity.

Conclusion
In the present study the aromatic composition and antibacterial properties of essential oils of thyme, rosemary, spearmint, and sweet basil were investigated. The results show the presence of alcohols and ketones in the aromatic composition of the essential oil. The identified aromatic compounds exhibit antimicrobial properties against foodborne pathogens. The essential oil of garden thyme possessed the strongest antimicrobial activity against S. aureus and L. monocytogenes. Otherwise, essential oil of sweet basil showed most pronounced antimicrobial properties against S. enterica, while E. coli was most susceptible to the essential oil of spearmint and rosemary. The results obtained provide a basis for a thorough examination of the chemical composition and antimicrobial properties of various representatives of Lamiaceae family with a view to a wide usage in food technology.