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Composition and Antimicrobial Activity of Some Essential Oils from Serbia

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Composition and Antimicrobial Activity of Some Essential Oils from Serbia FOOD ANALYSIS LJUBIŠA ŠARIĆ1*, IVANA ČABARKAPA1, BOJANA ŠARIĆ1, DRAGANA PLAVŠIĆ1, JOVANKA LEVIĆ1, SAVA PAVKOV2, BOJANA KOKIĆ1 *Corresponding author 1. University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara 1, Novi Sad, 21000, Serbia 2. Institute for Medicinal Plant Research, Dr. Josif Pančić, Tadeuša Košćuška 1, Belgrade, 11000, Serbia Ljubiša Šarić Composition and antimicrobial activity of some essential oils from Serbia KEYWORDS: essential oils, chemical composition, antimicrobial activity The aim of this work was to determine chemical composition and antimicrobial properties of essential oils of AbstractRosmarinus officinalis L., Salvia officinalis L, Lavandula angustifolia L. and Mentha piperita L. originated from Serbia. The main components of rosemary essential oil were camphor (17.66 %), 1,8 cineole (16.11 %), verbenone (13.84 %), α-pinene (12.45 %) and borneol (9.22 %). Sage essential oil contained 28.64 % of camphor 21.90 % of 1,8 cineole and 19.92 % of α-thujone (16.92 %). Camphor (17.14 %), 1,8 cineole (12.74 %) and linalyl acetate (9.62 %) were the most abundant compounds of lavander essential oil. Concentrations of menthol and menthone as the main compounds of mint essential oil were 38.82 % and 26.12 %, respectively. All essential oils showed antimicrobial activity against all tested bacteria in the MIC range of 0.16 – 5.00 mg/ml and MBC range of 0.63 – 5.00 mg/ml. INTRODUCTION and diseases. In the popular medicine, it is used to treat nausea, flatulence, vomiting, indigestion, stomach Increased demand for natural and safe food, without cramps, menstrual cramps and parasitosis. It is also chemical preservatives, provokes many researches to recognized for its carminative, stimulant, antispasmodic, investigate the antimicrobial effects of natural antiseptic, anti-inflammatory, antibacterial and antifungal compounds. Aromatic plants are generally considered as activities (28). Antimicrobial activity of EOs mainly the most important source of natural antimicrobial agents depends on their chemical composition (27), while the (3). Numerous studies have confirmed the antimicrobial variation in chemical composition of EOs from the same activity of essential oils (EOs) in model food systems as species can be caused by different growing factors such well as in real food (24), including the EOs of Rosmarinus as climatic, seasonal and geographic conditions (13). officinalis L., Salvia officinalis L, Lavandula angustifolia L. Despite the fact that chemical composition and and Mentha piperita L. (10, 17, 19, 29). Rosemary antimicrobial properties of EOs of rosemary, sage, (Rosmarinus officinalis L.) is an evergreen shrub originated lavender and mint are well documented (6, 14, 16, 9, 1, in the Mediterranean area. Nowadays it is cultivated 10, 17, 19, 29), the literature sources on composition and almost everywhere in the world, primarly for its aromatic antimicrobial potential of these EOs obtained from plants leaves. Rosemary is often widely used as a common grown in Serbia are very limited. household spice, and also as a fragrant aromatic The objective of this research was to determine chemical flavoring agent in several commercially available composition and to evaluate the antimicrobial activity of products, such as vegetables, prepared meats, baked essential oils of rosemary, sage, lavender and mint against goods, etc. (25). Althought sage (Salvia officinalis L.) selected food borne pathogens from Enterobacteriaceae could be found near the cost of Mediterranean sea as an and Staphylococcaceae family. autochtonous plant it is also cultivated in many countries. The extracts and EOs of sage are used as spices and for healing of different diseases. Sage EOs exibit antioxidant, MATERIAL AND METHODS anti-inflammatory, antispasmodic, antimicrobial and stimulant properties (21). Lavender (Lavandula Plant material angustifolia L.) is well known species from genus The leaves of mint (Mentha piperita), lavender (Lavandula Lavandula (Laminaceae). Lavender is one of the most angustifolia), sage (Salvia officinalis) and rosemary useful medicinal plants. Several therapeutic effects of (Rosemary officinalis) were collected in August 2012 at lavender, such as sedative, spasmolytic, antiviral and the experimental field of the Institute for Medicinal Plant antibacterial activities have been reported (2). Mint Research “Dr Josif Pančić”, Belgrade, Serbia. The plant (Mentha piperita L.), commonly called peppermint, is a material was dried at room temperature and milled in the well-known herbal remedy used for a variety of symptoms form of powder with granulation of up to 3 mm. 40 Agro FOOD Industry Hi Tech - vol 25(1) - January/February 2014 Isolation of essential oils essential oils were added (20 µl) in a 96-well microtitre The essential oils were isolated from dried plant material plates, then 160 µl of MHB were added into the by hydrodistillation according to the standard procedure microplates and finally 20 µl of 1.0 ´ 108 cfu/ml (according reported in the Fifth European Pharmacopeia (7). The to 0.5 Mc Farland turbidity standards) of standardized distillation was performed during 2 hours using Clevenger microorganisms suspensions were inoculated into type apparatus. Oil samples were dried with anhydrous microplates and the test was performed in a volume of 200 sodium sulphate, dissolved in ethanol and analyzed by µl. Plates were incubated at 37 °C for 24 hours. The same GC/FID and GC/MS. tests were performed simultaneously on control samples (MHB + test microorganism) and sterility control (MHB + test Gas chromatography (GC/FID) oil). The MIC was defined as the lowest concentration of GC/FID analysis of the essential oils was carried out on a essential oil which inhibits the visible growth of HP-5890 Series II GC apparatus (Hewlett-Packard, microorganisms. The MBC is defined as the lowest Waldbronn, Germany), equipped with split-splitless injector concentration of the essential oil at which ≥ 99.9 % of and automatic liquid sampler (ALS), attached to HP-5 inoculated microorganisms were killed. MBC of tested column (25 m 0.32 mm, 0.52 µm film thickness) and fitted to essential oils was determined by subculturing the 5 µl of flame ionisation detector (FID). Carrier gas flow rate (H2) test dilution from each well on to a nutrient agar (Himedia, was 1 ml/min, split ratio 1:30, injector temperature was 250 India) plates and incubating under previously-mentioned °C, detector temperature 300°C, while column conditions. According to MBC definition the presence of ≤ temperature was linearly programmed from 40-260 °C (at 2 cfu of the tested microorganism per plate is acceptable. rate of 4 °C /min). Solutions of essential oil samples in All samples were analyzed in triplicate. ethanol (~1 %) were consecutively injected by ALS (1 µl, split mode). Area percent reports, obtained as result of standard processing of chromatograms, were used as RESULTS AND DISCUSSION base for the quantification purposes. Chemical composition of the essential oils Gas chromatography/mass spectrometry (GC/MS) The rasults of qualitative analyses of the tested essential oils The same analytical conditions as those specified for GC/ (Table 1) are in agreement with the results of earlier FID were applied for GC/MS analyses, along with column investigations (1, 6, 9, 14, 16). However, the quantitative HP-5MS (30 mm, 0.25 mm, 0.25 µm film thickness), using analysis of the tested oils showed some differences in their HPG 1800C Series II GCD system (Hewlett-Packard, Palo composition in comparison to the results of above mention Alto, CA, USA). Instead of hydrogen, helium was used as autors which could be explained by the differences in carrier gas. Transfer line was heated at 260°C. Mass climatic, seasonal, geographic conditions, harvest period, spectra were acquired in EI mode (70 eV), in m/z range distillation technique, as well as genotype and the age of the 40-450. The sample solutions in ethanol (~1 %) were plants (13). injected by ALS (200 nl, split mode). The major components of the rosemary EOs were: camphor The components of the essential oils were identified by (17.6 %), 1,8 cineole (16.1 %), verbenone (13.8 %), α-pinene comparison of their mass spectra to those from Wiley 275 (12.45 %) and borneol (9.2 %) (Table 1). Significant amount of and NIST/NBS libraries, using different search engines. The linalool (4.05 %) was also found. Composition of the tested experimental values for retention indices were determined rosemary EOs made them the most similar to camphor by the use of calibrated Automated Mass Spectral chemotype which is wide spread in the European Deconvolution and Identification System software (AMDIS Mediterranean region (9). In comparison with the chemical ver. 2.1.), compared to those from available literature (12), composition of EOs of Mediterranean rosemary (16) those and used as additional tool to approve MS findings. analyzed in this study were characterized by higher content of verbenone. This can be explained by the cooler climate in Antimicrobial activity assessment Serbia than climate in Mediterranean region. Similar Escherichia coli ATCC 25922, Escherichia coli ATCC 10536, differences in verbenone content in rosemary EOs from Escherichia coli ATCC 8739, Salmonella enteritidis ATCC diferent region of Turkey were also reported(4). 13076, Salmonella typhimurium ATCC 14028, Salmonella Chemical composition of sage EO (Table 1) is characterized choleraesuis 10708, Staphylococcus aureus ATCC 25923 by 19 compounds which represent
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