LWT - Food Science and Technology xxx (2015) 1e6 Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt Potential application of Micromeria dalmatica essential oil as a protective agent in a food system Danka Bukvicki a, c, *, Dejan Stojkovic b, Marina Sokovic b, Milos Nikolic b, Lucia Vannini c, d, Chiara Montanari d, Petar D Marin a a University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, 11000 Belgrade, Serbia University of Belgrade, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia University of Bologna, Department of Agricultural and Food Sciences, Viale Fanin 46, 40127 Bologna, Italy d Interdepartmental Center for Industrial Agri-food Research, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy b c a r t i c l e i n f o a b s t r a c t Article history: Received November 2014 Received in revised form 17 February 2015 Accepted 13 March 2015 Available online xxx Chemical composition of Micromeria dalmatica essential oil (EO) by gas chromatographyemass spectrometry solid phase microextraction (GC/MS-SPME) analysis revealed that the dominant compounds were piperitenone (41.46%), pulegone (19.02%), piperitenone oxide (14.49%), D-limonene (6.23%) and pmenthone (5.06%) The minimum inhibitory concentration (MIC) ranged from 0.03 to 2.32 mg/mL for bacteria, and from 0.62 to 2.49 mg/mL for yeast strains, while the minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11e5.57 mg/mL for bacteria and yeasts, respectively Growth inhibition concentration (GIC50) that caused 50% of growth delay of Salmonella Typhimurium in pork meat system was calculated to be 0.048 mg/mL Experimental results suggest that M dalmatica EO possess high antimicrobial efficacy against food spoilage microorganisms The present study has certainly set up an attractive platform for commercial applications of EO as natural preservative in food, such as pork meat © 2015 Elsevier Ltd All rights reserved Keywords: Micromeria dalmatica GCeMS/SPME Antimicrobial activity Salmonella Typhimurium Pork meat Introduction The genus Micromeria includes flowering plants belonging to the family Lamiaceae with its distribution from MacaronesianeMediterranean region to southern Africa, India and China (Radulovic & Blagojevi c, 2012) Micromeria species are perennial herbs or dwarf shrubs growing mostly in the Mediterranean regions on rocky habitat They are considered as medicinal plants since they have been used against heart disorders, headache, infections and the colds and are known to have sedative, anesthetic, antiseptic, abortifacient, antirheumatic and central nervous system stimulant properties (Ali-Shtayeh, Yaghmour, Faidi, Khalid, & AlNuri, 1998) Their leaves are also used as a tea and for food prepa€ ration because of their mint flavor (Kirimer, Ozek, Bas¸er & Harmandar, 1993) Micromeria dalmatica Benth (Syn Clinopodium * Corresponding author University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, 11000 Belgrade, Serbia Tel.: ỵ381 64 206 5264; fax: ỵ38 111 324 3603 E-mail addresses: dankabukvicki@bio.bg.ac.rs, dankabukvicki@gmail.com (D Bukvicki) €uchler & Heubl; Satureja dalmatica (Benth.) dalmaticum (Benth.) Bra Nyman) is an endemic species of Balkan Peninsula Recently, extensive research on this species, mainly resulting in chemical characterization of its essential oil (EO), has been performed (Karousou, Hanlidou, & Lazari, 2012; Kostadinova et al., 2007; Slavkovska et al., 2005) Unfortunately, there are few quantitative data (minimal inhibitory concentration or minimal bactericidal concentration) related to the antimicrobial activity of essential oil against foodborne pathogens with few articles available to date (Marinkovi c, Marin, Kne zevi c-Vukcevi c, Sokovic, & Brkic, 2002; Savikin et al., 2010) Despite technological development and innovative food production techniques, food safety remains a very important public health issue The effect of EOs and plant extracts on the growth of fungi and bacteria, and their use in food as flavor and fragrance agents, makes them very useful for the preservation of foodstuffs and continues to be studied by scientists (Stojkovic et al., 2013) In that manner, the assurance of inventory and the shelf life of meat products represent an important challenge for the meat industries The EOs of Satureja horvatii and oregano have been shown to be more or less effective against spoilage microbiota in meat products http://dx.doi.org/10.1016/j.lwt.2015.03.053 0023-6438/© 2015 Elsevier Ltd All rights reserved Please cite this article in press as: Bukvicki, D., et al., Potential application of Micromeria dalmatica essential oil as a protective agent in a food system, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.053 D Bukvicki et al / LWT - Food Science and Technology xxx (2015) 1e6 (Bukvicki et al., 2014; Stojkovic et al., 2013) The use of oregano EO in a vacuum packed and pasteurized minced (ground) pork product resulted in a delay in meat spoilage (Ismaiel & Pierson, 1990) The delay of spoilage and improvement of organoleptic qualities of packed meat is highly interesting from a commercial point of view Nevertheless, to the best of our knowledge, no previous reports on the use of M dalmatica EO in preservation of foodstuffs are available in literature Therefore, the aim of this study was to investigate chemical profile of M dalmatica EO wild growing in Montenegro Furthermore, in vitro antimicrobial activity was evaluated against food spoilage bacteria and yeasts Finally, the practical application of the EO as a natural preservative in a meat product was investigated by using Salmonella Typhimurium as a contaminant microorganism This strategy will certainly represent a good way in reducing the use of synthetic compounds in food preservation Material and methods 2.1 Plant material M dalmatica Benth was collected from Lovcen Mt (Montenegro) in July 2010 A voucher specimen has been deposited in the Herbarium at the Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade (BEOU) Material was dried at room temperature 2.2 Distillation of essential oil The aerial parts of the plant were dried at room temperature and hydrodistilled (100 g) for h, using a Clevenger-type apparatus The oil yield was 1.36% After hydrodistillation, water was removed by decantation and the essential oil obtained was stored at C and protected against light to avoid alteration in its composition 2.3 Solid phase microextraction (SPME) gas chromatographyemass spectrometry (GCeMS) analysis of M dalmatica oil A divinylbenzene-poly(dimethylsiloxane)-coated stable flex fiber (65 mm) and a manual SPME holder (Supelco Inc., Bellefonte, PA, USA) were used in this study after preconditioning according to the manufacturer's instruction manual Samples were put into sealed vials and then equilibrated for 10 at room temperature The SPME fiber was exposed to each sample for 10 by manually penetrating the septum, and, finally, the fiber was inserted into the injection port of the GC for 10 sample desorption Gas chromatographyemass spectrometry (GCeMS) analyses were carried out on an Agilent 7890 gas chromatograph (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 5975 mass selective detector operating in electron impact mode (ionization voltage, 70 eV) A CP-Wax 52 CB capillary column (50 m length, 0.32 mm inner diameter, 1.2 mm film diameter) was used The temperature program started from 50 C, then programmed at C/ to 240 C, which was maintained for Injector, interface, and ion source temperatures were 250 C, 250 C, and 230 C, respectively Injections were performed in split mode and helium (1 mL/min) was used as the carrier gas The mass selective detector was operated in the scan mode between 20 and 400 m/z Data acquisition started after injection The identification of the molecules was based on comparison of mass spectra of compounds both with those contained in available databases (NIST version 2005) and with those of some pure standards, i.e a-pinene, bpinene, D-limonene, p-menthone, a-terpineol and 1-octen-3-ol (SigmaeAldrich, Milan, Italy) analyzed under the same conditions Data are expressed as percentage of each individual peak area (expressed as AU) and the total peak area 2.4 Yeast, bacterial strains and culture conditions Different yeast strains (Saccharomyces cerevisiae 635, Zygosaccharomyces bailii 45, Aureobasidium pullulans L6F, Pichia membranaefaciens OC 71, P membranaefaciens OC 70, Pichia anomala CBS 5759 and P anomala DBVPG 3003) were obtained from the microbial culture collection of the Department of Agricultural and Food Sciences, Alma Mater Studiorum e University of Bologna (Italy) and used to evaluate the effect of the essential oil Different bacterial strains (Listeria monocytogenes NCTC 7973, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 35210 and Salmonella Typhimurium ATCC 13311) were obtained from the strain collection of the Department of Plant Physiology, Laboratory of Mycology, Institute for Biological Research “Sinisa Stankovic”, Belgrade, Serbia Yeast strains were grown in Yeast extract Peptone Dextrose (YPD) at 27 C for 48 h, while bacterial strains were grown in Tryptic Soy Broth (TSB) at 37 C for 24 h After harvesting, microbial cells were suspended in sterile saline solution and immediately used 2.5 In vitro antimicrobial assay In order to investigate the antimicrobial activity of the essential oil, the modified micro-dilution technique was used (Daouk, Dagher, & Sattout, 1995; NCCLS, 1999) Minimum inhibitory concentrations (MICs) determination was performed by a serial dilution technique by using 96-well microtitre plates (Sarstedt, Milan, Italy) The tested oil was added to the TSB medium for bacteria and YPD medium for yeasts and then filled into 96-wells microplates (100 mL/well) with inoculum (100 mL/well) of the target microbial species previously adjusted with sterile saline solution to a concentration of approximately 1.0  106 CFU/mL The microplates were incubated for 24 h at 37 C for bacteria and 48 h at 27 C for yeasts A sterile medium incubated under the same condition was used as a blank, while the medium inoculated with the target microorganisms (without the oil) was used as a positive control of growth The lowest concentrations of the EO showing complete inhibition of visible growth were defined as MICs All determinations were performed in triplicate Also the Minimum Bactericidal Concentration (MBC) and Minimum Yeast-cidal Concentration (MYC) were determined Generally, MBC/MYC values are defined as the minimal concentrations of the tested molecule not allowing the microbial growth when 10 mL of the cultures taken from the wells with no visible growth are plated into solid medium and incubated at the optimal temperature; MBCs and MYCs indicate 99.5% killing of the original inoculum Streptomycin was used to control the sensitivity of the tested bacteria, whereas cycloheximide was used as a control against the fungi 2.5.1 Growth curve of Salmonella Typhimurium Since Salmonella Typhimurium was chosen for further in situ analysis, the above mentioned strain was used to monitor its growth at MIC and MBC concentrations of the essential oil Growth of Salmonella Typhimurium without essential oil was used as a control For that purposes ELISA plate reader was used (Tecan Austria, GmbH-Austria, Eppendorf-AG, Germany) and absorbance was measured at 610 nm The measurements were carried out on h, h, h, h, 10 h, 14 h, 18 h and 24 h (Piccirillo, Demiray, Silva Ferreira, Pintado, & Castro, 2013) Please cite this article in press as: Bukvicki, D., et al., Potential application of Micromeria dalmatica essential oil as a protective agent in a food system, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.053 D Bukvicki et al / LWT - Food Science and Technology xxx (2015) 1e6 2.6 In situ antibacterial preservation of pork meat 2.6.1 Preparation of pork meat medium Milled pork meat was purchased from a local store Pieces of pork meat (10 g) were added to 90 mL of distilled water and homogenized The pork meat solution was then pasteurized by autoclaving at 70 C for 20 After cooling, pork meat medium was filtered through sterile Whatman no filter paper in order to exclude denatured proteins and their possible influence on spectrophotometric readings used in further experiments pH value of the pork meat solution was adjusted to 6.7 Serial dilutions of the pork meat solution were made and cultured on Mueller Hinton and Malt Agar plates, kept at 37 C and 25 C, respectively, in order to investigate possible bacterial or fungal contaminants after autoclaving 2.6.2 In situ antibacterial assay in pork meat medium Different concentrations of the M dalmatica essential oil were added to the pork meat medium to achieve final concentrations in the range of 0.04e0.30 mg/mL The controls contained pork meat medium, but not the essential oil The flasks were homogenized for 30 s to ensure mixing of the essential oil compounds with pork meat medium The modified method to test the ability of antimicrobials to preserve food properties was used as previously described by authors, and results were expressed as percentage of inhibition (Bukvi cki et al., 2014; Stojkovi c et al., 2013) Briefly, the pork meat medium was inoculated with ~106 cells of S Typhimurium that had been prepared by growing the bacterium overnight at 37 C in TSB medium Cells suspension was adjusted with sterile saline solution to approximately  106 cells per 100 mL of pork meat medium The inhibition percentage at C was calculated by optical density measured by ELISA plate reader (Tecan Austria, GmbH-Austria, Ependorf-AG, Germany) after days of storage, by using the following equation: ðODsample À OD0sampleÞ %Inhibition ¼ ðODgrowth* À OD0growth*Þ À ðODblank À OD0blankÞ Â 100 where OD0sample and ODsample correspond to the absorbances at 612 nm of the strain grown in the presence of the essential oil before and after incubation, respectively; OD0blank and ODblank correspond to the broth medium with dissolved the essential oil before and after incubation, respectively; and OD0growth* and ODgrowth* to the strain grown in the absence of the essential oil before and after incubation, respectively The results were further presented as growth inhibition concentrations (GIC) that cause 50% retardation of bacterial growth The GIC50 was calculated as follows 1 e Inhibition percentage was calculated; 2 e A linear regression was made using the concentrations and respective % of inhibition that are immediately before and after the 50% of inhibition; 3 e Using the equation curve the value of 50% of inhibition was obtained The concentrations were calculated after days following the incubation period 2.7 Sensory evaluation Sensory analysis of pork meat and pork meat with added M dalmatica EO was conducted for samples at the end of storage by a sensory panel composed of 25 untrained staff from the laboratory The pork meat medium was prepared as stated in the Section 2.6.1 The panelists were asked to read informant consent prior to sensorial evaluation Overall acceptance of the samples was evaluated using a 5-point scale, according to a previous report, where: ¼ extremely dislike, ¼ dislike, ¼ neither like nor dislike, ¼ like; ¼ extremely like (Stojkovi c et al., 2013) The panelists were asked to evaluate overall acceptance of pork meat and pork meat enriched with essential oil, as well as change in color and flavor of pork meat after days of storage at 25 C, on a scale from to indicating decreasing taste Overall acceptance of the samples was visually analyzed by panelists, due to the possibility of foodborne contaminants and potentially harmful oxidation products of the meat Results and discussion 3.1 Chemical composition of M dalmatica essential oil In Fig the GC/MS-SPME profile of M dalmatica essential oil is shown, while the retention indexes (RI) and chemical composition of the EO are presented in Table The most abundant components found in the essential oil of M dalmatica were piperitenone, pulegone, piperitenone oxide, D-limonene and p-menthone which accounted for 41.46%, 19.02%, 14.49%, 6.23%, and 5.06%, respectively Previous studies on chemical composition of oil of this species have demonstrated that at least two different chemotypes, with different percentages of their main compounds, were present (Radulovi c & Blagojevi c, 2012) Identified monoterpenes such as pulegone (29.6%), menthone (11.7%) and piperitenone (10.8%) have been found as the most represented essential constituents of the oil These data are in agreement with previous literature reporting that the oil of M dalmatica is rich in pulegone (26.7%) and piper itenone (21.8%) in addition to piperitenone oxide (25.4%) (Savikin et al., 2010) Main reasons for the significant differences in percentage contents of Micromeria essential oil could be due to different genetic and environmental factors 3.2 In vitro antimicrobial activity The effects of antibacterial activity of M dalmatica essential oil (Table 2) resulted very high against bacteria (MIC varied from 0.03 to 2.32 mg/mL) Streptomycin showed lower activity against S aureus (MIC ¼ 0.05 mg/mL) than the essential oil (MIC ¼ 0.03 mg/ mL), while MIC activity of the antibiotic for E coli (0.2 mg/mL) and Salmonella Typhimurium (0.10 mg/mL) were similar to the MIC values of M dalmatica oil (0.29 and 0.15 mg/mL, respectively) On the contrary, the strain of L monocytogenes showed the least sensitiveness to the tested EO The MIC of the yeasts ranged between 0.62 and 2.49 mg/mL P membranaefaciens OC 70, P membranaefaciens OC 71, P anomala DBVPG 3003 and P anomala CBS 5759 (0.62 mg/mL) showed higher MIC values than A pullulans and S cerevisiae (1.24 mg/mL) Z bailii (2.49 mg/mL) was the most resistant yeast tested Growth curves of Salmonella Typhimurium in laboratory media (control) and in the presence of MIC and MBC concentrations of essential oil are presented in Fig Absorbance values increased with time of incubation in the control sample, while they decreased in the presence of essential oil On the basis of the data on volatile composition (Table 1), it can be hypothesized that oxygenated monoterpenes, which are the dominant components of the EO accounting for 63.91%, are responsible for its antimicrobial activity Compounds such as piperitone, piperitone oxide (PO), piperitenone, cis-piperitenone epoxide and piperitenone oxide (PEO), as well as pulegone are the main aromatic constituents of the EOs of several plants including Mentha suaveolens (Oumzil et al., 2002) and Satureja parvifolia (Phil.) Epling (Luna et al., 2008), and their bioactivity has been proven in vitro against several Gram-positive and Gram-negative bacteria In Please cite this article in press as: Bukvicki, D., et al., Potential application of Micromeria dalmatica essential oil as a protective agent in a food system, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.053 D Bukvicki et al / LWT - Food Science and Technology xxx (2015) 1e6 Fig GC/MS-SMPE profile of M dalmatica essential oil (1: 1R-a-Pinene; 2: b-Pinene; 3: Myrcene; 4: D-Limonene; 5: o-Cymene; 6: 1-Octen-3-ol; 7: p-Menthone; 8: Pulegone; 9: aTerpineol; 10: Piperitone epoxide; 11: Piperitone; 12: Piperitenone; 13: Piperitenone oxide) Table Chemical composition of Micromeria dalmatica essential oil Compounds Rt (min) RI %a Moneterpenes 1R-a-Pinene b-Pinene Myrcene D-Limonene o-Cymene p-Menthone Pulegone a-Terpineol 8.242 11.305 13.308 14.999 18.142 28.093 34.517 36.045 1027 1113 1168 1212 1277 1474 1652 1688 0.87 1.23 0.48 6.23 1.69 5.06 19.02 0.13 Alcohols 1-Octen-3-ol 25.540 1438 0.20 Oxygenated monoterpenes Piperitone epoxide Piperitone Piperitenone Piperitenone oxide 36.975 37.729 44.932 46.008 1710 1728 1918 1936 2.07 5.89 41.46 14.49 Total identified % 98.82 RI ¼ retention index on CPWAX 52 CB capillary column a Data are expressed as percentage of the total peak area particular, pulegone was the most effective one followed by PEO and PO (Oumzil et al., 2002) Moreover, according to previous findings, pulegone is responsible for the high antibacterial activity against bacteria S aureus, Staphylococcus epidermidis, Bacillus subtilis, E coli, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae with the most efficiency against Staphylococcus species (Sonboli, Mirjalili, Hadian, Ebrahimi, & Yousefzadi, 2006) 3.3 In situ antimicrobial activity in pork meat Recent studies report that Salmonella Typhimurium is the most frequent serovar isolated from the most common meat food products (Kramarenko et al., 2014) Therefore, this serovar was chosen for in situ experiments, which were performed with pork meat medium deliberately inoculated with S Typhimurium stored for days at C From Table it can be noted that growth inhibition percentage raised with increasing concentrations of the essential oil At the lowest concentration used (0.04 mg/mL) inhibition rate was 43.7% When the highest concentration was applied (0.29 mg/mL) a complete inhibition was achieved As previously mentioned, GIC represents the growth inhibition that caused a 50% delay in bacterial growth Guided by experimental procedure, calculation of GIC could be an interesting tool to study bacterial growth and/or inhibition in liquid foods Although the method has practical applications it has some limits regarding the use of initial inoculum which is linked to the limitations of spectrophotometric technique being able to measure the presence of bacteria at a level of 106 CFU/mL or even higher Therefore, the method was developed for validating inhibitory activities of natural preservatives in liquid foods when bacterial growth is 106 CFU/mL or higher, and for the determination of GIC50 value Table Antimicrobial activity (mg/mL) of Micromeria dalmatica essential oil Microorganisms MIC S cerevisiae 635 Z bailii 45 A pullulans L6F P membranaefaciens OC 71 P membranaefaciens OC 70 P anomala CBS 5759 P anomala DBVPG 3003 L monocytogenes NCTC 7973 S aureus ATCC 6538 E coli ATCC 35210 S Typhimurium ATCC 13311 1.24 2.49 1.24 0.62 0.62 0.62 0.62 2.32 0.03 0.29 0.15 MYC/MBC ± ± ± ± ± ± ± ± ± ± ± 0.03 0.01 0.02 0.01 0.02 0.02 0.03 0.03 0.01 0.02 0.03 2.49 6.22 2.49 2.49 2.49 2.49 2.49 4.63 0.03 0.058 0.15 ± ± ± ± ± ± ± ± ± ± ± 0.02 0.03 0.02 0.03 0.02 0.03 0.02 0.01 0.02 0.03 0.02 Strep MIC/MBC Cyclo MIC e e e e e e e 0.05 0.05 0.20 0.10