Variability of Satureja cuneifolia Ten. essential oils and their antimicrobial activity depending on the stage of development

Eur Food Res Technol (2004) 218:367 371 DOI 10.1007/s00217-003-0871-4 ORIGINAL PAPER SkočibuÐić Mirjana Bezić Nada Dunkić Valerija Variability of Satureja cuneifolia Ten. essential oils and their antimicrobial activity depending on the stage of development Received: 9 October 2003 / Revised: 4 December 2003 / Published online: 21 January 2004 Springer-Verlag 2004 Abstract The essential oils obtained from Satureja cuneifolia Ten. harvested in the central part of Dalmatia at three ontogenetic stages were evaluated for their chemical composition and antimicrobial activity against food pathogens. The GC/MS analyses allowed 32 compounds to be determined; the main constituents of the essential oils were linalool (18.2 17.2%), carvacrol (16.0 5.0%), p-cymene (14.8 4.0%), a-pinene (12.0 5.8%) and limonene (11.0 1.8%). The compounds linalool and borneol appeared to be relatively constant but carvacrol, limonene and a-pinene showed variability during the growth cycles. The oils had a broad-spectrum antimicrobial activity against food pathogens in broth microdilution bioassays. Maximum activity was observed against the yeast Candida albicans, the Gram-negative bacteria Escherichia coli, Salmonella typhimurium and Proteus mirabilis and the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus. The essential oils showed good antibacterial effects against E. coli with a minimum inhibitory concentration of 0.06% and a minimum bactericidal concentration of 0.12% during the flowering stage. These inhibitory effects are interesting in relation to the prevention of microbial contamination in many foods and, therefore, essential oils of S. cuneifolia could be used as substitutes for synthetic antimicrobial compounds. Keywords Satureja cuneifolia Antimicrobial Food pathogens Essential oil Linalool S. Mirjana () ) B. Nada D. Valerija Department of Biology, Faculty of Natural Science, and Mathematics and Education, University of Split, Teslina 12, 21000 Split, Croatia e-mail: mirskoc@pmfst.hr Tel.: +385-21-385133 Fax: +385-21-385431 Introduction The increasing global incidence of food poisoning cases originating from food contaminated by pathogens has great social and economic costs and causes major concern, both to the general public and to the food industry [1]. The epidemiology of food-borne diseases is rapidly changing. The increased interest in biopreservation of food systems has recently led to the development of new natural antimicrobial compounds having different origins. Most plants produce antimicrobial secondary metabolites, either as part of their normal programme of growth and development or in response to pathogen attack or stress. A novel way to reduce the proliferation of microorganisms is the use of essential oils. Spices and their derivatives, such as essential oils and oleoresins, are used with the primary purpose of flavouring foods and beverages, although it has long been known that some spices have an antimicrobial activity [2, 3, 4]. Lamiaceae is a plant family within which we can find several species with potential for preservation from microbial spoilage, primarily due to their essential oils, which are a mixture of a great number of volatile constituents [5, 6, 7, 8, 9]. The genus Satureja belongs to the family Lamiaceae, subfamily Nepetoidae, and tribe Mentheae [10]. The essential oil isolated from various species of Satureja has certain biological properties such as antibacterial [11, 12], antiviral [13], fungicidal [11, 2, 14] and antioxidant [15] activity. Satureja cuneifolia is a perennial shrub sprouting every spring with new twigs full of leaves. In the Mediterranean it grows in littoral and island areas up to 1,200 m above sea level. The essential oil of this plant is applied in flavouring condiments, relishes, soups, sausages, canned meats and spicy table sauces. The composition of the oils and their antimicrobial effects depend on the plant species, regional conditions and growth stages [11, 16]. No reports on the composition of essential oil from the wild-growing population in Croatia and its antimicrobial activity have been found. The present work examines the

368 composition of these essential oils and their antimicrobial activity against food pathogens, depending on the stage of plant development. Materials and methods Plant material Plant material of Satureja cuneifolia Ten. was collected during 1999 from the Kozjak Mountain (Croatia, near the city of Split) prior to flowering (leaves and stalks, July), in the course of flowering (flowering tops, leaves and stalks, September) and after flowering (leaves and stalks, November). Voucher specimens are deposited in the herbarium at the Faculty of Natural Science, Mathematics and Education of the University of Split. Isolation of the essential oils Air-drying of the plant was performed in a shady place at room temperature for 10 days. Leaves were used for the analysis of essential oil composition. Dried plant material (100 g), consisting of flowered tops, was subjected to hydrodistillation for 3 h using a modified Unger-type apparatus. The body of the apparatus consisted of two concentric tubes. The inner tube was graduated and filled with water and a known amount of n-pentane, whose role was to retain the essential oil and to separate it from the water. The outer tube was filled with running water. The essential oil obtained was dried over anhydrous sodium sulphate and 2 l was used for GC/MS measurements. Analysis of essential oil The analyses of the volatile compounds were carried out on a Hewlett-Packard GC/MS system (GC 5890 Series II; MSD 5971A). The fused-silica HP-20 M polyethylene glycol column (50 m 0.2 mm i.d., 0.2 m film thickness) was directly coupled to the mass spectrometer. The carrier gas was helium (1 ml/min). The temperature programme used was 4 min isothermal at 70 C, then 70 180 C at a rate of 4 C/min, then held isothermal for 10 min. The injection port temperature was 250 C. The ionisation of the sample components was performed in the EI mode (70 ev). The linear retention indices for all the compounds were determined by co-injection of the sample with a solution containing the homologous series of C 8 C 22 n-alkanes [17]. The individual constituents were identified by comparison of their retention indices with those of known compounds retrieved from the literature [18], and also by comparing their mass spectra with either those of the known compounds or with the Wiley mass spectral database. Biological material The antimicrobial activity of the essential oil was evaluated at different vegetative stages (before, during and after flowering) using a panel which included laboratory control strains obtained from the American Type Culture Collection (Rockville, MD, USA): Gram-positive bacteria Bacillus cereus (ATCC 11778), Bacillus subtilis (ATCC 26633), Enterococcus faecium (ATCC 29212), Listeria monocytogenes (ATCC 700302) and Staphylococcus aureus (ATCC 25923); Gram-negative bacteria Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 25933) and Salmonella typhimurium (ATCC 19430); and fungal organisms Aspergillus niger (ATCC 9142), Aspergillus fumigatus (ATCC 9142), Candida albicans (ATCC 10231), Candida rugosa (ATCC 10571) and Saccharomyces cerevisiae (ATCC 561). In vitro antimicrobial bioassay In order to quantify the antimicrobial activity of S. cuneifolia oils, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) were determined using a broth microdilution method [19, 20]. The MIC was the lowest concentration, which resulted in a significant decrease (>90%) in inoculum viability, while the MBC/ MFC were the concentrations corresponding to 0.1% or less of viability of the initial inoculum. Determination of MIC was performed by a serial dilution technique using 96-well microtitre plates. The essential oils investigated were dissolved in 0.5% dimethyl sulphoxide (DMSO) to give solutions in malt medium broth with microbial suspensions to a final concentration range of 0.06 4.0% (w/v). The microbial suspensions of selected strains were prepared by inoculating Mueller Hinton broth (MHB) and potato dextrose broth (PDB) with one or two pieces of culture from a blood agar plate and then incubating for 18 h at 37 C in a rotary shaker. Freshly grown bacterial suspensions in double strength MHB (Merck) and yeast suspensions of Candida albicans in yeast medium were standardized to 1 10 8 CFU/ml. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline. The spore suspension was adjusted with sterile saline to a concentration of approximately 2.5 10 3 CFU/ml. In the tests, triphenyltetrazolium chloride (TTC) (Aldrich Chemical Company Inc., Milwaukee, WI, USA) was also added to the culture medium at a concentration of 0.05% as a growth indicator. After aerobic incubation at 37 C for 24 h (bacteria) and 25 C for 48 h (yeast and filamentous fungal strains), the first well without turbidity was determined as the MIC. The MBC/MFC were determined by serial subcultivation of 10 l in microtitre plates containing 100 l of broth per well and further incubation for 18 h at 37 or 25 C. Each test was performed in triplicate and the results analysed. DMSO (0.5%) and TTC (0.05%) without oil were also used as a negative control and, in this case, no antimicrobial activity was observed. Results and discussion Our work gives comparative GC/MS analyses of the S. cuneifolia essential oils obtained at three different stages of the plant life cycle. Essential oils were isolated by hydrodistillation and the yield of oils ranged from 0.2 to 0.5% w/w (SD=0.15) (Table 1). The yield was highest in July at the beginning of summer. The analyses allowed identification of 32 compounds and the major fraction was monoterpene. Table 1 shows that the main component was linalool (17.2 18.2%, SD=0.51) which appeared to be constant during the plant growth cycle. All samples showed relatively good content of carvacrol (5.0 16.3%, SD=6.01) and p-cymene (1.8 14.8%, SD=6.96). Carvacrol, limonene (4.7 11.0%, SD=4.70) and a-pinene (5.8 12.0%, SD=3.13) showed variable concentration. In the investigated plant, carvacrol, p-cymene and b- caryophyllene showed the highest concentration during the flowering stage (16.3, 14.8 and 9.3%, respectively). The content of terpinen-4-ol (2.8<5.1<6.4%, SD=1.82) increased with the maturation of the plant, to the contrary of b-cubebene (9.1>3.5>1.7%, SD=3.86). Relatively constant concentrations were observed for borneol (6.8 7.6%, SD=0.42) and d-cadinene (1.3 1.7%, SD=0.23) during the three phenological stages investigated. The essential oils also contained small percentages of myrcene (0.3 2.8%, SD=1.31), spathulenol (1.9 1.3%, SD=0.31),

369 Table 1 Composition (%) of Satureja cuneifolia Ten. essential oils in three different stages of life cycle No. Component RI Flowering period Mode of Before During After SD identification 1 a-pinene 1038 8.1 5.8 12.0 3.13 GC, MS 2 b-pinene 1102 1.5 3.4 / GC, MS 3 Myrcene 1149 2.8 0.3 0.9 1.31 GC, MS 4 Limonene 1183 4.7 1.8 11.0 4.70 GC, MS 5 cis-b-ocimene 1218 4.2 / GC, MS 6 g-terpinene 1231 4.1 / GC, MS 7 p-cymene 1247 1.8 14.8 4.0 6.96 GC, MS, RC 8 aallo-oocimene 1351 2.6 / GC, MS 9 1-Octen-3-ol 1411 0.7 / GC, MS, RC 10 Sabinene hydrate 1423 0.5 / GC, MS 11 a-copaene 1466 0.7 / GC, MS 12 Camphor 1472 1.4 1.9 / GC, MS 13 b-bourbonene 1496 2.2 3.2 / GC, MS 14 Linalool 1507 18.2 17.2 17.9 0.51 GC, MS, RC 15 Terpinen-4-ol 1559 2.8 5.1 6.4 1.82 GC, MS 16 Calaren 1564 1.1 / GC, MS 17 b-caryophyllene 1578 5.2 9.3 2.4 3.47 GC, MS 18 Neral 1633 2.0 2.8 / GC, MS 19 a-terpineol 1646 1.9 1.9 / GC, MS 20 Borneol 1653 6.8 7.6 7.4 0.42 GC, MS, RC 21 b-cubebene 1680 9.1 3.5 1.7 3.86 GC, MS 22 b-bisabolene 1692 0.6 / GC, MS 23 Geranial 1680 0.9 1.2 / GC, MS, RC 24 d-cadinene 1729 1.7 1.7 1.3 0.23 GC, MS 25 Myrtenol 1733 0.3 / GC, MS 26 Nerol 1752 0.8 / GC, MS 27 Geraniol 1796 0.4 1.0 / GC, MS 28 Caryophyllene oxide 1927 0.4 1.4 1.8 0.72 GC, MS 29 Viridiflorol 2023 0.3 / GC, MS 30 Spathulenol 2061 1.3 1.5 1.9 0.31 GC, MS 31 Thymol 2115 1.8 1.6 0.8 0.53 GC, MS, RC 32 Carvacrol 2140 5.0 16.3 7.1 6.01 GC, MS, RC Oil yield (%) 0.5 0.4 0.2 0.15 RI retention indices (Kovats index) on HP-20 M column, GC identification by comparison of retention indices, MS identification on the basis of the mass spectra Wiley (MS) only, RC identification by comparison of their mass spectra of reference compounds, SD standard deviation, / not calculated SD, trace <0.1% thymol (0.8 1.8%, SD=0.53), caryophyllene oxide (0.4 1.8% SD=0.72) and d-cadinene. Tümen et al. [21] showed that the occurrence of carvacrol-rich (26 72%) and thymol-rich (22 58%) oils in S. cuneifolia may not be regarded as a genuine chemotype situation in Thymus vulgaris and some Satureja species. Müller-Riebau et al. [22] indicated that the phenolic constituents, carvacrol and thymol, of S. thymbra essential oils were low in the early phenological stages and increased gradually with plant development. It is interesting that Croatian S. cuneifolia is relatively rich in linalool, carvacrol, p- cymene, a-pinene, limonene and low percentages of thymol. The in vitro activity of S. cuneifolia essential oils was evaluated by a broth microdilution method at different vegetative stages using a panel of microorganisms, which included laboratory control strains. The results of the MIC and MBC or MFC are shown in Table 2. The data indicate that the oils exhibit varying levels of antimicrobial activity against the investigated food pathogens. The inhibitory properties of the oils were observed within a range of concentrations from 0.06 to 4.0% (w/v). In liquid medium the essential oils were active against all the test strains with the exception of P. aeruginosa. The Gramnegative P. aeruginosa seemed to be the most resistant to the lowest dilution (4%) of the investigated oils, compared to the other strains used in this study. Maximum activity was observed against the yeast C. albicans, the Gram-negative bacteria E. coli, S. typhimurium and P. mirabilis and the Gram-positive bacteria S. aureus and B. cereus. The Gram-negative E. coli displayed varying degrees of susceptibility to the investigated oils. The oils showed the highest antimicrobial effect with MIC of 0.06% and MBC of 0.12% against E. coli during the flowering stage, with carvacrol, linalool and p-cymene in high concentration (Table 1). Before and after the flowering period the MIC for E. coli was 0.25% and the MBC was equal or higher (0.25 and 0.5%) than the MIC. The investigated oils demonstrated good antibacterial effect against S. typhimurium during the flowering stage (MIC/MBC, 0.12%), but before and after the flowering period the activity was lower (MIC/MBC, 0.25%). Satureja cuneifolia oils showed good in vitro antibacterial effects against the five Gram-positive bacteria, with the MBC in a range between 0.12 and 1%. The MIC for P. mirabilis was 0.25, 0.12 and 0.5% and the MBC was 0.5,

370 Table 2 Antimicrobial activity of Satureja cuneifolia Ten. essential oils in three different stages of life cycle. Percentages (% w/v) of oils in liquid cultures broth dilution method Microorganisms Flowering period Before During After MIC a MBC/MFC b MIC a MBC/MFC b MIC a MBC/MFC b Gram-positive bacteria Bacillus subtilis 0.5 0.5 0.25 0.25 0.25 0.5 Enterococcus faecium 0.5 1.0 0.25 0.5 0.25 0.5 Listeria monocytogenes 1.0 1.0 0.5 0.5 0.5 1.0 Staphylococcus aureus 0.25 0.5 0.12 0.25 0.25 0.25 Gram-negative bacteria Escherichia coli 0.25 0.25 0.06 0.12 0.25 0.5 Proteus mirabilis 0.25 0.5 0.12 0.5 0.5 1.0 Pseudomonas aeruginosa >4 >4 >4 >4 >4 >4 Salmonella typhimurium 0.25 0.25 0.12 0.12 0.25 0.25 Fungi Aspergillus fumigatus 1.0 1.0 0.5 1.0 0.5 1.0 Candida albicans 0.12 0.12 0.06 0.06 0.06 0.12 Saccharomyces cerevisiae 0.5 0.5 0.12 0.12 0.12 0.5 a Minimum inhibitory concentration (MIC) b Minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) 0.5 and 1.0% in the three phenological stages, respectively. The oils exhibited the highest inhibitory effect against S. aureus with MIC/MBC of 0.12 to 0.25% during the flowering stage. The MIC (0.25%) of this oil against S. aureus appeared to be constant before and the after flowering period. During the flowering stage oils were effective against B. cereus with a range of 0.12 to 0.25%, but they had lowest activity against B. subtilis (MIC/ MBC, 0.25%) and E. faecium (MIC/MBC, 0.25 to 0.5%). The oil isolated during flowering was at least two to four dilutions more active than oils in the other vegetative stages against B. subtilis, B. cereus and E. faecium strains. The oils exhibited modest activities against important food pathogens such as L. monocytogenes, with MIC/ MBC of 1% before flowering and lowest at 0.5% during the flowering period. The essential oils of S. cuneifolia were effective against all fungal strains tested in the study. The oils exhibited the greatest antifungal effects against the yeast C. albicans during the flowering period, with MIC/MBC of 0.06%, but before and after flowering the activity was lower (MIC/MBC, 0.12%; MIC, 0.06%; MBC, 0.012%). The antifungal effects of the oils were observed in a range from 0.12 to 0.5% against S. cerevisiae. The oils exhibited an antifungal effect with MIC/MFC of 0.12% during flowering, but the lowest antifungal effects were observed before the flowering stage (MIC/MFC, 0.5%). The filamentous fungus A. fumigatus exhibited modest sensitivity to the essential oils of all the vegetative stages. The MIC/MBC values for the flowering stage were 0.5 and 1.0% and before flowering the MIC/MFC values were higher (1%). In liquid medium, S. cuneifolia essential oil inhibited the growth of almost all strains tested at 1% concentration. The oils showed the greatest antimicrobial effects against the investigated food pathogens during the flowering period. The antimicrobial effects were attributed to the presence of some volatile components in the oil. The major components of the investigated S. cuneifolia essential oils were identified as linalool, carvacrol, p- cymene, a-pinene, limonene and b-caryophyllene. Furthermore, the antimicrobial activities of different Satureja species were shown in other studies [23]. Linalool has previously been reported as having antibacterial and antifungal activity [24]. Numerous studies indicate that carvacrol and thymol are bactericidal [8, 25] and fungitoxic to pathogenic and spoilage microorganisms [26]. For example, Kim et al. [27] found that a 1.5% solution of carvacrol was necessary to kill Salmonella enterica serovar typhimurium on fish cubes, a level which is significantly higher than the concentration (0.1%) needed to kill the microorganisms in liquid medium. In fact, the MICs with carvacrol and thymol in a pure culture system are 3 and 1 mm for E. coli 0157:H7 and S. enterica serovar typhimurium, respectively [4]. It has frequently been reported that Gram-positive bacteria are more susceptible to essential oils than Gramnegative bacteria [28]. The tolerance of Gram-negative bacteria to essential oils has been ascribed to the presence of a hydrophilic outer membrane that blocks the penetration of hydrophobic essential oils into the target cell membrane. In contrast, data in this study on the antibacterial effects showed that the Gram-negative bacteria E. coli and S. typhimurium were more susceptible than all the Gram-positive strains investigated. Similar results have been reported by Kim et al. [4], who found that L. monocytogenes (Gram-positive) was more resistant to the inhibitory effects of 11 essential oils than the Gramnegative bacteria. However, it has to be considered that minor components, as well as possible interaction between the other active compounds, could also affect the microbiological properties of essential oils.

371 Conclusion In this study essential oils obtained from Satureja cuneifolia Ten. at three phenological stages were evaluated for their chemical composition and antimicrobial activity against food pathogens. The results indicated that there is a seasonal variation of the main components such as linalool, carvacrol, p-cymene, a-pinene and limonene. We suggest that oil isolated during the flowering period has a high concentration of biologically active components required for strong antimicrobial effects against the yeast Candida albicans and important food pathogens (Escherichia coli, Salmonella typhimurium). These results may constitute a significant connection between activity and chemical composition for the future development of S. cuneifolia essential oil as an antimicrobial agent to be used as a potential preservative in food products, to protect them from microbial spoilage. Acknowledgements The Ministry of Science and Technology of Republic of Croatia supported this work, through grant No. 0177140. References 1. Bennett A (1997) Salmonella. Seminar abstracts: food-borne pathogens a review for the practical microbiologist and food technologist. CCFRA, Gloucestershire, UK 2. Deans SG, Svoboda KP, Gundoidza M, Brechany EY (1992) Acta Hortic 306:229 232 3. Helander IK, Alakomi HL, Latva-Kala K, Mattila-Sandholm T, Pol IE, Smid J, Von Wright (1998) J Agric Food Chem 46:3590 3595 4. Kim JM, Maurice R, Wei Ch (1995) J Agric Food Chem 43:2839 2845 5. Beuchat LR (1994) Antimicrobial properties of spices and their essential oils. In: Dillon VM, Board RG (eds) Natural antimicrobial systems and food preservation. CAB, Wallingford, pp 167 179 6. Charai M, Mosaddak M, Faid M (1996) J Essent Oil Res 8:657 664 7. Dorman HJ, Deans SG (2000) J Appl Microbiol 88:308 316 8. Hammer KA, Carson CF, Riley TV (1999) J Appl Microbiol 86:985 990 9. Juven BJ, Kanner J, Schved F, Weisslowicz H (1994) J Appl Bacteriol 76:626 631 10. Cantino PD, Harley RM, Wagstaff SJ (1992) Genera of Labiatae status and classification. In: Harley RM, Reynolds T (eds) Advances in Labiatae science. Royal Botanic Gardens, Kew, pp 511 522 11. Bezić N, SkočibuÐić M, Dunkić V (1999) Acta Bot Croat 58:99 104 12. Smith-Palmer A, Stewart J, Fyfe L (1998) Lett Appl Microbiol 26:118 122 13. Yamasaki K, Nakano M, Kawahata T, Mori H, Otake T, Ueba N, Oishi I, Inami R, Yamane M, Nakamura M, Murata H, Nakanishi T (1998) Biol Pharm Bull 21:829 833 14. Paster N, Menasherov M, Ravid U, Juven B (1995) J Food Prot 58:81 85 15. Esquivel MM, Ribeiro MA, Bernardo-Gil MG (1999) J Supercrit Fluids 14:129 138 16. MiloÐ M, Radonić A, Bezić N, DunkićV (2001) Flav Fragr J 16:157 160 17. Van Den Dool H, Kratz PD (1963) J Chromatogr 11:463 471 18. Adams RP (1995) Identification of essential oil components by gas chromatography and mass spectroscopy. Allured, Carol Stream 19. Mislivec PB, Beuchat LR, Causin MA (1992) Yeast and molds. In: Vanderzant C, Splittstoesser DF (eds) Compendium of methods for the microbiological examination of food, 3rd edn. American Public Health Association, Washington, pp 239 249 20. Swanson KMJ, Busta FF, Peterson EH, Johanson MG (1992) Colony count methods. In: Vanderzant C, Splittstoesser DF (eds) Compendium of methods for microbiological examination of food, 3rd edn. American Public Health Association, Washington, pp 75 95 21. Tümen G, Kirimer N, Ermin N, Baser KHC (1998) Planta Med 64:81 83 22. Müller-Riebau FJ, Berger BM, Yegen O, Cakir C (1997) J Agric Food Chem 45:4821 4825 23. Müller-Riebau FJ, Berger BM, Yegen O (1995) J Agric Food Chem 43:2262 2266 24. Mazzanti G, Battinelli L, Salvatore G (1998) Flav Fragr J 13:289 294 25. Ultee AE, Kets PW, Smid, EJ (1999) Appl Environ Microbiol 65:4606 4610 26. Thompson DP (1996) J Food Prot 59:412 415 27. Kim JM, Marshall MR, Cornell JA, Preston JF III, Wei CI (1995) J Food Sci 60:1364 1374 28. Mann CM, Cox SD, Markham JL (2000) Lett Appl Microbiol 30:294 297