Meat Science 96 (2014) 1355–1360
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Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat Danka Bukvički a,c, Dejan Stojković b,⁎, Marina Soković b, Lucia Vannini c, Chiara Montanari c, Boris Pejin d, Aleksandar Savić d, Milan Veljić a, Slavica Grujić a, 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 ‘Siniša Stankovic’, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia University of Bologna, Department of Agricultural and Food Sciences, Via Fanin 46, 40127 Bologna, Italy d University of Belgrade, Institute for Multidisciplinary Research, Department of Life Science, Kneza Višeslava 1, 11030, Belgrade, Serbia b c
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 17 November 2013 Accepted 24 November 2013 Keywords: Satureja horvatii Antimicrobial activity Pork meat Listeria monocytogenes Antiradical activity Sensorial evaluation
a b s t r a c t The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The genus Satureja L. includes about 200 species of herbs and shrubs, often aromatic, with a centre of distribution in the Mediterranean Basin. In the area of the central and western Balkans, nine species of this genus have been registered (Lakusic, Ristic, Slavkovska, Stankovic, & Milenkovic, 2008). Many of these species from genus Satureja are endemic from the Orjen–Lovćen mountain massive (Montenegro), with a high content of essential oil, up to 4% and are widely applied in ethnomedicine and ethnobotany. Significant proportions of Satureja species are plants that have an important role in the pharmaceutical industry. The essential oils isolated from various Satureja species have shown antibacterial, fungicidal, antiviral and antioxidant activities (Lakusic et al., 2008). Due to antimicrobial effects attributed to the composition of these essential oils, they are widely used in various types of phytotherapies and cosmetics (Momtaz & Abdollai, 2010; Redžić, Tuka, & Pajević, 2006). A wide range of preservatives or antimicrobial treatments is used to extend the shelf-life of a product by inhibiting microbial growth. However, an increasingly negative consumer consideration of food additives, ⁎ Corresponding author at: Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000, Belgrade, Serbia. Tel.: +43 381 11 2078419; fax: +43 381 11 276143. E-mail address: [email protected] (D. Stojković). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.11.024
perceived as non-natural, has spurred an interest in finding natural alternatives to the traditional solutions (Smith-Palmer, Stewart, & Fyfe, 2001). Although essential oils were originally added to change or improve taste, their antimicrobial activity makes them an attractive choice for substituting chemical preservatives (Burt, 2004; Hyldgaard, Mygind, & Meyer, 2012; Lanciotti et al., 2004; Raybaudi-Massilia, MosquedaMelgar, Soliva-Fortuny, & Martín-Belloso, 2009). Many food products are perishable and require protection from microbial spoilage during preparation, storage and distribution to give them an acceptable shelflife and organoleptic characteristics. Because of recent trends in food marketing, the need for an extended shelf-life for these products has increased together with the application of less severe technological treatments (Rasooli, 2007). In addition, the risks due the presence and the growth of pathogenic microrganisms or bacteria producing toxins can be increased by these trends. As an example, contamination of some foods with Listeria monocytogenes is almost inevitable due to its ubiquitous nature in the environment. Oxidative reactions can also affect the shelf-life and overall quality of a food. In particular, oxidative damages by free radicals are implicated in the etiology of many diseases, cancer and cardiovascular diseases being the most common. Antioxidants have been widely used as food additives to prevent oxidative degradation by free radicals (Razali, Mat-Junit, Abdul-Muthalib, Subramaniam, & Abdul-Aziz, 2012). Chemical antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene are widely used as inhibitors of lipid peroxidation.
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Several side effects of these additives have been reported, including a carcinogenic effect (Ames, 1983; Baardseth, 1989). In the present study, the chemical composition of S. horvatii oil was analyzed by GC-MS. The effect of S. horvatii oil against different food spoiling yeasts and bacteria was studied through in vitro antimicrobial assays. Moreover, the potential use of the plant essential oil as a natural preservative in pork against L. monocytogenes and as an antioxidant (more specifically, antihydroxyl radical agent) was assessed.
2.5. In vitro antimicrobial assay
The aerial parts of the plant were dried at room temperature and hydrodistilled (100 g) for 2 h, using a Clevenger-type apparatus. The oil yield was 3.2%. After hydrodistillation, water was removed by decantation and the essential oil obtained was stored at 4 °C and protected against light.
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 Concentration (MIC) determination was performed by serial dilution 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 μL/well) with inoculum of the target microbial species previously adjusted 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 oil showing complete inhibition of visible growth were defined as MICs. The absence of visible growth was determined under a binocular microscope. 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 minimum concentrations of the tested molecule not allowing any microbial growth when 10 μL of the cultures taken from the wells with no visible growth after incubation is plated into solid medium (YPD and TSB for yeasts and bacteria, respectively). Streptomycin and cyclohexamide were used as positive controls.
2.3. Gas chromatographic mass spectrometry (GC-MS) analysis of Satureja horvatii oil
2.6. Fluorescence measurements
2. Materials and methods 2.1. Plant material Plant material of S. horvatii Šilic was collected from Orjen Mt. (Montenegro) in July 2010. A specimen has been deposited in the Herbarium at the Institute of Botany and Botanical Garden “Jevremovac“, University of Belgrade (BEOU). The material was dried at room temperature (25 °C) with constant aeration for ten days. 2.2. Distillation of essential oil
Gas chromatography-mass spectrometry (GC-MS) analysis was 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 μm film diameter) was used. The temperature program started from 50 °C, then was programmed at 3 °C/min to 240 °C, which was maintained for 1 min. 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 4 min after injection. One microliter of the sample was injected directly into the column with a split ratio of 1:100. Component separation was achieved as described above. 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 pure standards (Sigma–Aldrich, Milan, Italy) analyzed under the same conditions. 2.4. Yeast, bacteria strains and culture conditions Yeasts (Saccharomyces cerevisiae 635, Zygosacharomyces bailii 45, Aureobasidium pullulans L6F, Pichia membranaefaciens OC 71, Pichia membranaefaciens OC 70, Pichia anomala CBS 5759 and Pichia anomala DBVPG 3003) obtained from the strain collection of the Department of Agricultural and Food Sciences of the University of Bologna (Italy) were used to evaluate the effect of essential oil. The 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) broth at 27 °C for 48 h, while bacterial strains were grown in Tryptic Soy Broth (TSB) at 37 °C for 24 h. Microorganisms were used immediately to make appropriate cell dilutions in media for further experiments.
Fluorescence spectra in time domain (kinetics mode) were collected using a Fluorolog-3 spectrofluorimeter (Jobin Yvon Horiba, Paris, France) equipped with a 450 W xenon lamp and a photomultiplier tube. Measurements were performed in a well stirred and tempered quartz cuvette (1 cm optical path length, 1.5 mL volume, 25 °C). The slits on the excitation and emission beams were fixed at 2 nm. Wider slits can be used if white noise is present. Total time of measurement was 600 s, while the integration time was set to 0.1 s. Excitation/ emission wavelengths were 500/520 nm for hydroxyl radical measurements (Gomes, Frenandes, & Lima, 2005). APF (2-[6-(4′-amino)phenoxy-3H-xanten-3-on-9-yl] benzoic acid) was dissolved directly in a cuvette during the Fenton reaction (0.5 μL, undiluted). Standard Fenton reaction was used for generation of hydroxyl radicals (•OH) (0.5 mM FeSO4 and 1 mM H2O2, and 0.5 μL APF in a cuvette of 1.5 mL). This was used as the blank. Anti hydroxyl radical was determined in the same reaction system, with addition of 0.1 μL of essential oil. All chemicals (analytical grade or higher) were used as received from Sigma–Aldrich without further purification. All solutions were prepared with deionized water of resistivity not less than 18.2 MΩ cm.
2.6.1. Signal processing Kinetic profile of APF probe represents linear function of time:
Aðt Þ ¼ at þ A0
ð1Þ
A(t) — amplitude of fluorescence emission in given moment of time, a — parameter which determines the slope of line, A0 — starting intensity of fluorescence emission. In the presence of (•OH) radical, fluorescence emission intensity increases with time due to –O–degradation of the fluorescence probe (Setsukinai, Urano, Kakinuma, Majima, & Nagano, 2003).
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Coefficient (COH) which determines (•OH) radical production was calculated according to: C OH ¼
a A0
ð2Þ
2.7. In situ antibacterial preservation of pork meat 2.7.1. Pork meat and preparation of pork meat medium Minced pork meat was purchased from a local supermarket. Pieces (10 g) were added to 90 mL of distilled water and blended to make a homogenous suspension. The pork meat medium was then pasteurized by autoclaving at 70 °C, for 20 min. After cooling, the pH of the pork meat solution was 6.7. Serial dilutions of the pork meat solution were made and cultured on Műller–Hinton (MH) and Malt agar (MA) plates, kept at 37 °C and 25 °C, respectively, to investigate possible bacterial or fungal contaminants after autoclaving. 2.7.2. In situ antibacterial assay in pork meat medium S. horvatii essential oil was added to the pork meat medium to achieve concentrations in the range of 0.16–20 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 for antimicrobial food preserving properties was used as previously described (Stojković, Zivković, et al., 2013) and results were expressed as percentage inhibition (Reis et al., 2012; Stojković, Petrović, et al., 2013; Stojković, Reis, et al., 2013). The pork meat medium was inoculated with Listeria monocytogenes grown overnight at 37 °C in TSB medium. Cell suspension was adjusted with sterile saline solution (to avoid mixing of pork meat medium and TSB medium) to obtain approximately 1 × 106 cells per 100 μL of pork meat medium. Experimental microplates were divided into two groups: one group was kept at 25 °C and the other at 4 °C. Both groups contained equal amounts of essential oil. The inhibition percentages at 25 °C (1) and 4 °C (2) were calculated by optical density measured by an ELISA plate reader (Tecan Austria, GmbH-Austria, Eppendorf-AG, Germany) using the following equations: %Inhibition ¼
ðODsample−OD0sampleÞ −ðODblank−OD0blankÞ ðODgrowth −OD0growthÞ 100 ð3Þ
ðODsample−OD0sampleÞ −ðODblank−OD0blankÞ %Inhibition ¼ ðODgrowth −OD0growthÞ 100−Tinhibition ð4Þ where OD0sample and ODsample corresponded to the absorbance at 612 nm of the strain grown in the presence of the extract before and after incubation, respectively; OD0blank and ODblank corresponded to the broth medium with the essential oil dissolved before and after incubation, respectively; and OD0growth* and ODgrowth* to the strain grown in the absence of the essential oil before and after incubation at 25 °C, respectively. Inhibition corresponded to inhibition of L. monocytogenes at 4 °C, measured according to the formula (5): ODTgrowth 100 TInhibition ¼ 100− ðODT0growthÞ
ð5Þ
where ODT0growth and ODTgrowth are the absorbance values relative to the growth of L. monocytogenes at 4 °C in liquid medium, before and after incubation, respectively.
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2.7.3. Sensory evaluation of antioxidant properties To evaluate possible antioxidant preservation activity of the phenolics (no change in color and flavor) the pork meat was sprayed with essential oil at 10 mg/10 cm2 of pork meat. The panelists were asked to evaluate change in color and flavor of the meat after 4 days of storage with or without essential oil, on a scale from 5 to 1; indicating decreasing taste. A general taste score was calculated as the average of all grades. Acceptance was evaluated using a 5-point scale, according to a previous report, where 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like. Results were expressed as average grades given by 10 panelists (Stojković, Petrović, et al., 2013). 3. Results and discussion 3.1. Chemical composition of Satureja horvatii essential oil Qualitative and quantitative analysis of the S. horvatii essential oil is listed in Table 1. Thirty-four components were identified, 54.42% monoterpenes, 44.20% oxygenated monoterpenes, 0.30% sesquiterpenes and 0.94% alcohols, which together accounted for 99.88% of the total
Table 1 Chemical composition of Satureja horvatii essential oil. Compounds
RI
%
Monoterpenes α-thujene α-pinene camphene β-pinene β-thujene thuja-2,4(10)-diene 3-carene myrcene α-phellandrene α-terpinene D-limonene β-phellandrene β-trans-o-cymene γ-terpinene p-cymene terpinolene menthone pulegone Total:
1025 1035 1074 1113 1124 1136 1143 1163 1189 1195 1204 1226 1247 1262 1289 1290 1480 1654
1.02 4.26 2.40 0.51 0.03 0.25 0.17 1.63 0.45 4.02 1.36 0.58 0.06 4.05 33.14 0.33 0.03 0.15 54.44
Oxygenated monoterpenes cineole b dehydro-1,8-N cis-β-terpineol α-terpineol (-)-borneol carvacrol thymol thymol, methyl ether Total:
1198 1427 1685 1691 2204 2173 1590
0.04 0.27 0.18 1.40 1.12 26.11 15.08 44.20
Sesquiterpenes α-cubebene β-bourbonene Aromadendrene alloaromadendrene γ-muurolene δ-cadinene Total:
1472 1510 1600 1641 1682 1750
0.05 0.07 0.12 0.01 0.01 0.04 0.30
1446 1535 1548
0.76 0.17 0.01 0.94 99.88
Alcohols 1-okten-3-ol linalol 1-nonen-3-ol Total: Total identified:
RI = Retention Index on CP WAX 52 CB capillary column.
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detected constituents. The major constituents of the oil were pcymene (33.14%), thymol (26.11%), thymol methyl ether (15.08%). %), γ-terpinene (4.05%), α-pinene (4.26%) and α-terpinene (4.02%). Lakusic et al. (2008), reported that the main constituent of S. horvatii oil was thymol. The chemical composition of various Satureja species essential oils has been reported (Adiguzel, Ozer, Kilic, & Cetin, 2007; Cavar, Maksimovic, Šolic, Jerkovic-Mujkic, & Bešta, 2008; Matasyoha, Kiplimoa, Karubiub, & Hailstorks, 2007) and the results are comparable to the present findings. Chemical differences could be due to genetic and environmental factors, and stage of plant development (Redžić et al., 2006).
3.2. Antimicrobial activity The MIC and MBC/MYC of S. horvatii essential oil were determined against various bacterial (L. monocytogenes, S. aureus, E. coli and S. typhimurium) and yeast (S. cerevisiae, Z. bailii, A. pullulans, P. membranaefaciens and P. anomala) strains. These MIC and MBC/ MYC values are shown in Table 2 in comparison with values related to two antibiotics, streptomycin and cycloheximide, used as references compounds. According to the MICs and MBCs, the Gram positive bacteria were more resistant than the Gram negative ones. For the bacterial strains, the MIC varied from 0.03 to 0.57 mg/mL (Table 2). The MIC values of oil for Gram positive bacteria (L. monocytogenes and S. aureus) were significantly higher than those for the Gram negative bacteria (S. typhimurium and E. coli). The MBC of oil for the bacterial strains varied from 0.07 to 1.15 mg/mL and showed a similar pattern, i.e. L. monocytogenes (1.15 mg/mL) N S.aureus (0.29 mg/mL) N E. coli (0.07 mg/mL) = S. tiphymurium (0.07 mg/mL). In general, S. horvatii essential oil showed significant antimicrobial activity, where bacterial strains were more resistant than the yeast strains. These results are in agreement with a report on the sensitivity of Gram negative bacteria to the essential oil of Thymus vulgaris (Imelouane et al., 2009). The authors reported that Gram positive bacteria (S. aureus, MIC = 1.33 mg/mL, Streptococcus sp., MIC = 2.67 mg/mL) were more resistant to the essential oil of T. vulgaris than Gram negative ones (E. coli MIC = 0.33 mg/mL). Streptomycin expressed inhibitory effects at 0.05–0.2 mg/mL and bactericidal activity at 0.2–0.4 mg/mL. It can be seen that the essential oil possessed (in some cases) a higher antibacterial effect than streptomycin against the Gram negative bacteria (MIC = 0.03 mg/mL). The MIC of the oil for the yeast strains varied from 0.56 to 2.23 mg/mL (Table 2). The lowest MIC (0.56 mg/mL) was shown by P. membranaefaciens OC71 and highest MIC (2.23 mg/mL) was shown by Z. bailii, A. pullulans. A similar pattern was found for the MFC of the oil against the yeast strains. The commercial antimycotic
cycloheximide was used as a control and had MICs (b 0.5 mg/mL) lower than that of the essential oil against the yeasts tested. Antimicrobial activity of a plant essential oil depends on its chemical composition. The presence of significant amounts of oxygenated monoterpenes (44.2 %), mainly represented by thymol and thymol methyl ether compounds, indicated a high antimicrobial potential of Satureja oil (Lakusic et al., 2008). The effect of some essential oil components on the cell membrane integrity of Gram positive and Gram negative bacteria has been reported. The most elucidated action concerns the antimicrobial components of oregano and thyme essential oils, i.e. carvacrol and thymol. It is recognized that their action results in the release of the lipopolysaccharides (LPS) from Gram negative bacteria with consequent cell membrane permeability increase and ATP loss (Faleiro, 2011). Carvacrol and thymol are able to disintegrate the outer membrane of Gram negative bacteria, releasing LPS and increasing the permeability of the cytoplasmic membrane to ATP (Gill & Holley, 2006; Lambert, Skandamis, Coote, & Nychas, 2001; Ultee, Bennik, & Moezelaar, 2002). The primary mode of antibacterial action of thymol is not fully known, but is believed to involve outer and inner membrane disruption, and interaction with membrane proteins and intracellular targets (Hyldgaard et al., 2012). The antimicrobial action of phenolic compounds, such as thymol and carvacrol, is expected to cause structural and functional damage to the cytoplasmic membrane (Sikkema, DeBont, & Poolman, 1995). Interestingly, thymol induced cell lysis and only altered the cell structure of proliferating S. cerevisiae cells, indicating that the effect of thymol depends on cell proliferation (Bennis, Chami, Chami, Bouchikhi, & Remmal, 2004). Contrary to this, Rao, Zhang, Muend, and Rao (2010), proposed that thymol activates specific signaling pathways in yeasts. Delgado, Fernández, Palop, and Periago (2004), reported that the use of thymol and p-cymene may be applied simultaneously for preservation of minimally processed foods. 3.3. Antihydroxyl radical property of essential oil Essential oil of S. horvatii has strong antihydroxyl radical activity (Fig. 1). In fact fluorescence emission intensity, which is positively correlated to radical production, dropped to 5% of the initial intensity immediately after addition of essential oil in the Fenton reaction system. After 1 min, radical production slowed to 1% of the initial production, and after 3 min hydroxyl radical production ceased. As the main component of S. horvatii oil is p-cymene, antiradical activity to hydroxyl radicals was expected, because monoterpene
Table 2 Antimicrobial activity (mg/mL) of Satureja horvatii essential oil. Microorganisms
S. horvatii oil 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 NCTC7973 Staph. aureus ATCC 6538 E. coli ATCC 35210 S. Typhimurium ATCC 13311
1.11 2.23 2.23 0.56 1.11 1.11 1.11 0.57 0.15 0.03 0.03
± ± ± ± ± ± ± ± ± ± ±
0.03 0.01 0.02 0.01 0.02 0.02 0.03 0.03 0.01 0.02 0.03
Control MYC/MBC
Strep. MIC/MBC
2.23 5.57 2.23 1.11 2.23 2.23 5.57 1.15 0.29 0.07 0.07
– – – – – – – 0.05 0.05 0.20 0.10
± ± ± ± ± ± ± ± ± ± ±
0.02 0.03 0.02 0.03 0.02 0.03 0.02 0.01 0.02 0.03 0.02
± ± ± ±
Cyclo. MIC
b0.05 ± 0.02 b0.05 ± 0.01 b0.05 ± 0.03 b0.05 ± 0.02 b0.05 ± 0.01 b0.05 ±0.01 b0.05 ± 0.03 0.03/0.10 ± 0.01 0.03/0.10 ± 0.01 0.01/0.40 ± 0.01 0.02/0.20 ± 0.02
MIC: Minimal Inhibitory Concentration; MBC: Minimal Bactericidal Concentration; MYC: Minimal Yeast-cidal Concentration; Strep.: Streptomycin; Cyclo.: Cycloheximide, mean ± standard deviation.
Fig. 1. Antiradical activity of S. horvatii essential oil, examined using fluorescence probe APF. Analysis was performed according to Eq. (5), in fixed size time Windows of 30 s.
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hydrocarbons are known as possible reactants with hydroxyl radicals (Winer, Lloyd, Darnall, & Pitts, 1976). The second most abundant component, thymol has shown a protective effect to hydroxyl radical and lipid peroxidation (Aeschbach et al., 1994).
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Table 4 Acceptability scores given by panelist for pork meat alone and sprayed with essential oil, after 4 days of storage (10 mg/10 cm2 of meat).
color flavor
3.4. In situ antimicrobial activity in pork meat The antimicrobial activity of S. horvatii essential oil against L. monocytogenes in pork meat medium was better under refrigerated conditions, 4 °C (Table 3). With the two highest tested concentrations (20 mg/mL and 10 mg/mL) 100% inhibition was achieved in 24 h regardless of the incubation temperature. Inhibition percentage was relatively constant at concentrations of 0.67 to 5 mg/mL during storage at 4 °C, in the range 93.45–96.62%. No inhibition was recorded at the lowest concentrations used (0.16 and 0.32 mg/mL). At room temperature S. horvatii essential oil was active in the concentration range 0.62–5.0 mg/mL, similarly to 4 °C. However, the inhibition percentage was dose dependent and its activity was almost halved at the lowest active concentration. Furthermore, inhibition slightly increased during storage. The best effect was for the highest concentrations used (20 and 10 mg/mL) at 25 °C. Application of low amounts of naturally occurring antimicrobial agents as ingredients should be explored, in order to control spoilage (Stojković, Petrović, et al., 2013; Stojković, Reis, et al., 2013; Stojković, Zivković, et al., 2013). The present work provides a basis for developing effective naturally occurring antimicrobial agents to extend the shelf-life of pork meat and provides strong evidence that the essential oil of S. horvatii might be efficient in the control of Listeria monocytogenes in pork meat. Eugenol and coriander, clove, oregano and thyme oils were found to be effective at 5 – 20 μL/g in inhibiting L. monocytogenes, A. hydrophila and autochthonous spoilage flora in meat products, sometimes causing a marked initial reduction in the number of recoverable cells (Skandamis & Nychas, 2001; Tsigarida, Skandamis, & Nychas, 2001), whilst mustard, cilantro, mint and sage oils were less effective or ineffective (Gill, Delaquis, Russo, & Holley, 2002; Lemay et al., 2002). One must consider the essential oil not only as a preservative, but also as a flavour component, especially as many herb and spice flavoured cheeses and other products already exist. Alternatively they could be incorporated into products which already have a strong flavour, that could mask the presence of the essential oil. However, the antimicrobial activity of any plant essential oil is likely to be a result of the interactions between different components, which can be additive, antagonistic or synergistic (Lanciotti et al., 2004; Smith-Palmer et al., 2001).
Pork meat
Pork meat + EO
2.2 1.7
4.1 3.7
The results are expressed as the average of all grades. 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like. EO — essential oil of S. horvatii.
3.5. Sensorial evaluation of flavor and color of meat sprayed with essential oil The results of the sensory evaluation are presented in Table 4. In meat non-treated with essential oil, after 4 days, due to oxidative changes the panelists mostly disliked the color and flavor and would refuse to eat such meat. As for the pork meat sprayed with essential oil, changes in color and flavor was recorded by panelists, but compared to non-treated samples the acceptability scores were better. Since the essential is a good antioxidant it may be of use in the meat industry, for more than just its antimicrobial properties. Meat in which oxidation reactions have occurred is brown in color; the flavor is rancid and stale and such meat would likely be rejected by the consumer (Greene & Price, 1975). Changes in meat color are due to oxidation of red oxymyoglobin to metmyoglobin (MMG), which gives rise to an unattractive brown color (Velasco & Williams, 2011). 4. Conclusion S. horvatii essential oil is a rich source of compounds such as pcymene and thymol and shows antimicrobial potential against a range of food spoilage microorganisms. The oil is much more active against bacterial than yeast strains. Thus, essential oils of S. horvatii may be useful for preservation and/or extension of the shelf life of raw or processed meat products. Acknowledgments This research was supported by a grant from the Ministry of Education and Science of Serbia (Project No. 173029, 173032 and 173040), Department of Agricultural and Food Sciences, Alma Mater Studiorum,
Table 3 Antibacterial activity of S. horvatii essential oil in pork meat medium, against the foodborne Listeria monocytogenes (mean ± SD). Concentration (mg/mL)
Temp. (°C)
Percentage of inhibition of L. monocytogenes in meat 0h
Satureja horvatii essential oil
20.00 10.00 5.00 2.50 1.25 0.62 0.31 0.16
+25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
24 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 97.43 96.58 87.25 96.24 81.39 93.45 42.61 93.65 0.00 0.00 0.00 0.00
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 .2.23 1.46 1.51 2.36 1.17 1.05 2.08 1.15 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 98.52 95.12 91.33 98.18 81.75 93.82 45.37 94.32 0.00 0.00 0.00 0.00
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 1.38 2.54 1.13 3.64 3.65 1.02 2.21 2.72 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 98.82 96.62 93.14 96.53 87.93 95.24 48.96 95.13 0.00 0.00 0.00 0.00
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 1.14 1.32 1.02 1.73 1.77 1.44 2.54 1.02 0.00 0.00 0.00 0.00
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University of Bologna, Italy and Erasmus Mundus fellowship under EMECW to Danka Bukvicki. The authors wish to thank Professor Maria Elisabetta Guerzoni for her valuable suggestions and help during experiments.
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Matasyoha, J. C., Kiplimoa, J. J., Karubiub, N. M., & Hailstorks, T. P. (2007). Chemical composition and antimicrobial activity of the essential oil of Satureja biflora (Lamiaceae). Bulletin of the Chemical Society Ethiopia, 21, 249–254. Momtaz, S., & Abdollai, M. (2010). An update on pharmacology of Satureja species, from antioxidant, antimicrobial, antidiabetes and anti-hyperlipidemic to reproductive stimulation. International Journal of Pharmacology, 6, 346–353. NCCLS (National Committee for Clinical Laboratory Standards) (1999). Performance standards for antimicrobial susceptibility testing; 9th International Supplement, Wayne Pa. M100-S9. Rao, A., Zhang, Y., Muend, S., & Rao, R. (2010). Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway. Antimicrobial Agents Chemotherapy, 54, 5062–5069. Rasooli, I. (2007). Food preservation – A biopreservative approach. Food, 1(2), 111–136 (Global Science Books). Raybaudi-Massilia, R. M., Mosqueda-Melgar, J., Soliva-Fortuny, R., & Martín-Belloso, O. (2009). Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. Comprehensive Reviews in Food Science and Food Safety, 8, 157–180. Razali, N., Mat-Junit, S., Abdul-Muthalib, A. F., Subramaniam, S., & Abdul-Aziz, A. (2012). Effects of various solvents on the extraction of antioxidant phenolics from the leaves, seeds, veins and skins of Tamarindus indica L. Food Chemistry, 131, 441–448. Redžić, S., Tuka, M., & Pajević, A. (2006). Research into microscopic structure and essential oils of endemic medicinal plant species Satureja subspicata Bartl. Ex Vis. (Lamiaceae). Bosnian Journal of Basic Medical Sciences, 6, 25–31. Reis, F. S., Stojkovic, D., Sokovic, M., Glamoclija, J., Ciric, A., Barros, L., & Ferreira, I. C. F. R. (2012). Chemical characterization of Agaricus bohusii, antioxidant potential and antifungal preserving properties when incorporated in cream cheese. Food Reserch International, 48, 620–626. Setsukinai, K., Urano, Y., Kakinuma, K., Majima, H. J., & Nagano, T. (2003). Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. The Journal of Biological Chemistry, 278, 3170–3175. Sikkema, J., DeBont, J. A.M., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbioogical Review, 59, 201–222. Skandamis, P. N., & Nychas, G. J. E. (2001). Effect of oregano essential oil on microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres. Journal of Applied Microbiology, 91, 1011–1022. Smith-Palmer, A., Stewart, J., & Fyfe, L. (2001). The potential application of plant essential oils as natural food preservatives in soft cheese. Food Microbiology, 18, 463–470. Stojković, D., Petrović, J., Soković, M., Glamočlija, J., Kukić-Marković, J., & Petrović, S. (2013c). In situ antioxidant and antimicrobial activities of naturally occurring caffeic acid, p-coumaric acid and rutin, using food systems. Journal of the Science of Food and Agriculture, http://dx.doi.org/10.1002/jsfa.6156. Stojković, D., Reis, F. S., Ferreira, I. C. F. R., Barros, L., Glamočlija, J., Ćirić, A., Nikolić, M., Stević, T., Giveli, A., & Soković, M. (2013b). Tirmania pinoyi, desert truffle from Libya: chemical composition, in vitro antioxidant and antibacterial activities and in situ control of Staphylococcus aureus in chicken soup. Food Research International, 53, 56–62. Stojković, D., Zivković, J., Soković, M., Glamočlija, J., Ferreira, I. C. F. R., Janković, T., & Maksimović, Z. (2013a). Antibacterial activity of Veronica montana L. extract and of protocatechuic acid incorporated in a food system. Food and Chemical Toxicology, 55, 209–213. Tsigarida, E., Skandamis, P., & Nychas, G. J. E. (2001). Behaviour of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified atmosphere packaging conditions with or without the presence of oregano essential oil at 5 °C. Journal of Applied Microbiology, 89, 901–909. Ultee, A., Bennik, M. H., & Moezelaar, R. (2002). The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology, 68, 1561–1568. Velasco, V., & Williams, P. (2011). Improving meat quality through natural antioxidants. Chilean Journal of Agricultural Research, 71, 313–322. Winer, A.M., Lloyd, A.C., Darnall, R. K., & Pitts, N. J. (1976). Relative rate constants for the reaction of the hydroxyl radical with selected ketones, chloroethenes, and monoterpene hydrocarbons. The Journal of Physical Chemistry, 80, 1635–1639.
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat Danka Bukvički a,c, Dejan Stojković b,⁎, Marina Soković b, Lucia Vannini c, Chiara Montanari c, Boris Pejin d, Aleksandar Savić d, Milan Veljić a, Slavica Grujić a, 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 ‘Siniša Stankovic’, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia University of Bologna, Department of Agricultural and Food Sciences, Via Fanin 46, 40127 Bologna, Italy d University of Belgrade, Institute for Multidisciplinary Research, Department of Life Science, Kneza Višeslava 1, 11030, Belgrade, Serbia b c
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 17 November 2013 Accepted 24 November 2013 Keywords: Satureja horvatii Antimicrobial activity Pork meat Listeria monocytogenes Antiradical activity Sensorial evaluation
a b s t r a c t The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The genus Satureja L. includes about 200 species of herbs and shrubs, often aromatic, with a centre of distribution in the Mediterranean Basin. In the area of the central and western Balkans, nine species of this genus have been registered (Lakusic, Ristic, Slavkovska, Stankovic, & Milenkovic, 2008). Many of these species from genus Satureja are endemic from the Orjen–Lovćen mountain massive (Montenegro), with a high content of essential oil, up to 4% and are widely applied in ethnomedicine and ethnobotany. Significant proportions of Satureja species are plants that have an important role in the pharmaceutical industry. The essential oils isolated from various Satureja species have shown antibacterial, fungicidal, antiviral and antioxidant activities (Lakusic et al., 2008). Due to antimicrobial effects attributed to the composition of these essential oils, they are widely used in various types of phytotherapies and cosmetics (Momtaz & Abdollai, 2010; Redžić, Tuka, & Pajević, 2006). A wide range of preservatives or antimicrobial treatments is used to extend the shelf-life of a product by inhibiting microbial growth. However, an increasingly negative consumer consideration of food additives, ⁎ Corresponding author at: Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000, Belgrade, Serbia. Tel.: +43 381 11 2078419; fax: +43 381 11 276143. E-mail address: [email protected] (D. Stojković). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.11.024
perceived as non-natural, has spurred an interest in finding natural alternatives to the traditional solutions (Smith-Palmer, Stewart, & Fyfe, 2001). Although essential oils were originally added to change or improve taste, their antimicrobial activity makes them an attractive choice for substituting chemical preservatives (Burt, 2004; Hyldgaard, Mygind, & Meyer, 2012; Lanciotti et al., 2004; Raybaudi-Massilia, MosquedaMelgar, Soliva-Fortuny, & Martín-Belloso, 2009). Many food products are perishable and require protection from microbial spoilage during preparation, storage and distribution to give them an acceptable shelflife and organoleptic characteristics. Because of recent trends in food marketing, the need for an extended shelf-life for these products has increased together with the application of less severe technological treatments (Rasooli, 2007). In addition, the risks due the presence and the growth of pathogenic microrganisms or bacteria producing toxins can be increased by these trends. As an example, contamination of some foods with Listeria monocytogenes is almost inevitable due to its ubiquitous nature in the environment. Oxidative reactions can also affect the shelf-life and overall quality of a food. In particular, oxidative damages by free radicals are implicated in the etiology of many diseases, cancer and cardiovascular diseases being the most common. Antioxidants have been widely used as food additives to prevent oxidative degradation by free radicals (Razali, Mat-Junit, Abdul-Muthalib, Subramaniam, & Abdul-Aziz, 2012). Chemical antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene are widely used as inhibitors of lipid peroxidation.
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Several side effects of these additives have been reported, including a carcinogenic effect (Ames, 1983; Baardseth, 1989). In the present study, the chemical composition of S. horvatii oil was analyzed by GC-MS. The effect of S. horvatii oil against different food spoiling yeasts and bacteria was studied through in vitro antimicrobial assays. Moreover, the potential use of the plant essential oil as a natural preservative in pork against L. monocytogenes and as an antioxidant (more specifically, antihydroxyl radical agent) was assessed.
2.5. In vitro antimicrobial assay
The aerial parts of the plant were dried at room temperature and hydrodistilled (100 g) for 2 h, using a Clevenger-type apparatus. The oil yield was 3.2%. After hydrodistillation, water was removed by decantation and the essential oil obtained was stored at 4 °C and protected against light.
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 Concentration (MIC) determination was performed by serial dilution 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 μL/well) with inoculum of the target microbial species previously adjusted 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 oil showing complete inhibition of visible growth were defined as MICs. The absence of visible growth was determined under a binocular microscope. 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 minimum concentrations of the tested molecule not allowing any microbial growth when 10 μL of the cultures taken from the wells with no visible growth after incubation is plated into solid medium (YPD and TSB for yeasts and bacteria, respectively). Streptomycin and cyclohexamide were used as positive controls.
2.3. Gas chromatographic mass spectrometry (GC-MS) analysis of Satureja horvatii oil
2.6. Fluorescence measurements
2. Materials and methods 2.1. Plant material Plant material of S. horvatii Šilic was collected from Orjen Mt. (Montenegro) in July 2010. A specimen has been deposited in the Herbarium at the Institute of Botany and Botanical Garden “Jevremovac“, University of Belgrade (BEOU). The material was dried at room temperature (25 °C) with constant aeration for ten days. 2.2. Distillation of essential oil
Gas chromatography-mass spectrometry (GC-MS) analysis was 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 μm film diameter) was used. The temperature program started from 50 °C, then was programmed at 3 °C/min to 240 °C, which was maintained for 1 min. 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 4 min after injection. One microliter of the sample was injected directly into the column with a split ratio of 1:100. Component separation was achieved as described above. 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 pure standards (Sigma–Aldrich, Milan, Italy) analyzed under the same conditions. 2.4. Yeast, bacteria strains and culture conditions Yeasts (Saccharomyces cerevisiae 635, Zygosacharomyces bailii 45, Aureobasidium pullulans L6F, Pichia membranaefaciens OC 71, Pichia membranaefaciens OC 70, Pichia anomala CBS 5759 and Pichia anomala DBVPG 3003) obtained from the strain collection of the Department of Agricultural and Food Sciences of the University of Bologna (Italy) were used to evaluate the effect of essential oil. The 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) broth at 27 °C for 48 h, while bacterial strains were grown in Tryptic Soy Broth (TSB) at 37 °C for 24 h. Microorganisms were used immediately to make appropriate cell dilutions in media for further experiments.
Fluorescence spectra in time domain (kinetics mode) were collected using a Fluorolog-3 spectrofluorimeter (Jobin Yvon Horiba, Paris, France) equipped with a 450 W xenon lamp and a photomultiplier tube. Measurements were performed in a well stirred and tempered quartz cuvette (1 cm optical path length, 1.5 mL volume, 25 °C). The slits on the excitation and emission beams were fixed at 2 nm. Wider slits can be used if white noise is present. Total time of measurement was 600 s, while the integration time was set to 0.1 s. Excitation/ emission wavelengths were 500/520 nm for hydroxyl radical measurements (Gomes, Frenandes, & Lima, 2005). APF (2-[6-(4′-amino)phenoxy-3H-xanten-3-on-9-yl] benzoic acid) was dissolved directly in a cuvette during the Fenton reaction (0.5 μL, undiluted). Standard Fenton reaction was used for generation of hydroxyl radicals (•OH) (0.5 mM FeSO4 and 1 mM H2O2, and 0.5 μL APF in a cuvette of 1.5 mL). This was used as the blank. Anti hydroxyl radical was determined in the same reaction system, with addition of 0.1 μL of essential oil. All chemicals (analytical grade or higher) were used as received from Sigma–Aldrich without further purification. All solutions were prepared with deionized water of resistivity not less than 18.2 MΩ cm.
2.6.1. Signal processing Kinetic profile of APF probe represents linear function of time:
Aðt Þ ¼ at þ A0
ð1Þ
A(t) — amplitude of fluorescence emission in given moment of time, a — parameter which determines the slope of line, A0 — starting intensity of fluorescence emission. In the presence of (•OH) radical, fluorescence emission intensity increases with time due to –O–degradation of the fluorescence probe (Setsukinai, Urano, Kakinuma, Majima, & Nagano, 2003).
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Coefficient (COH) which determines (•OH) radical production was calculated according to: C OH ¼
a A0
ð2Þ
2.7. In situ antibacterial preservation of pork meat 2.7.1. Pork meat and preparation of pork meat medium Minced pork meat was purchased from a local supermarket. Pieces (10 g) were added to 90 mL of distilled water and blended to make a homogenous suspension. The pork meat medium was then pasteurized by autoclaving at 70 °C, for 20 min. After cooling, the pH of the pork meat solution was 6.7. Serial dilutions of the pork meat solution were made and cultured on Műller–Hinton (MH) and Malt agar (MA) plates, kept at 37 °C and 25 °C, respectively, to investigate possible bacterial or fungal contaminants after autoclaving. 2.7.2. In situ antibacterial assay in pork meat medium S. horvatii essential oil was added to the pork meat medium to achieve concentrations in the range of 0.16–20 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 for antimicrobial food preserving properties was used as previously described (Stojković, Zivković, et al., 2013) and results were expressed as percentage inhibition (Reis et al., 2012; Stojković, Petrović, et al., 2013; Stojković, Reis, et al., 2013). The pork meat medium was inoculated with Listeria monocytogenes grown overnight at 37 °C in TSB medium. Cell suspension was adjusted with sterile saline solution (to avoid mixing of pork meat medium and TSB medium) to obtain approximately 1 × 106 cells per 100 μL of pork meat medium. Experimental microplates were divided into two groups: one group was kept at 25 °C and the other at 4 °C. Both groups contained equal amounts of essential oil. The inhibition percentages at 25 °C (1) and 4 °C (2) were calculated by optical density measured by an ELISA plate reader (Tecan Austria, GmbH-Austria, Eppendorf-AG, Germany) using the following equations: %Inhibition ¼
ðODsample−OD0sampleÞ −ðODblank−OD0blankÞ ðODgrowth −OD0growthÞ 100 ð3Þ
ðODsample−OD0sampleÞ −ðODblank−OD0blankÞ %Inhibition ¼ ðODgrowth −OD0growthÞ 100−Tinhibition ð4Þ where OD0sample and ODsample corresponded to the absorbance at 612 nm of the strain grown in the presence of the extract before and after incubation, respectively; OD0blank and ODblank corresponded to the broth medium with the essential oil dissolved before and after incubation, respectively; and OD0growth* and ODgrowth* to the strain grown in the absence of the essential oil before and after incubation at 25 °C, respectively. Inhibition corresponded to inhibition of L. monocytogenes at 4 °C, measured according to the formula (5): ODTgrowth 100 TInhibition ¼ 100− ðODT0growthÞ
ð5Þ
where ODT0growth and ODTgrowth are the absorbance values relative to the growth of L. monocytogenes at 4 °C in liquid medium, before and after incubation, respectively.
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2.7.3. Sensory evaluation of antioxidant properties To evaluate possible antioxidant preservation activity of the phenolics (no change in color and flavor) the pork meat was sprayed with essential oil at 10 mg/10 cm2 of pork meat. The panelists were asked to evaluate change in color and flavor of the meat after 4 days of storage with or without essential oil, on a scale from 5 to 1; indicating decreasing taste. A general taste score was calculated as the average of all grades. Acceptance was evaluated using a 5-point scale, according to a previous report, where 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like. Results were expressed as average grades given by 10 panelists (Stojković, Petrović, et al., 2013). 3. Results and discussion 3.1. Chemical composition of Satureja horvatii essential oil Qualitative and quantitative analysis of the S. horvatii essential oil is listed in Table 1. Thirty-four components were identified, 54.42% monoterpenes, 44.20% oxygenated monoterpenes, 0.30% sesquiterpenes and 0.94% alcohols, which together accounted for 99.88% of the total
Table 1 Chemical composition of Satureja horvatii essential oil. Compounds
RI
%
Monoterpenes α-thujene α-pinene camphene β-pinene β-thujene thuja-2,4(10)-diene 3-carene myrcene α-phellandrene α-terpinene D-limonene β-phellandrene β-trans-o-cymene γ-terpinene p-cymene terpinolene menthone pulegone Total:
1025 1035 1074 1113 1124 1136 1143 1163 1189 1195 1204 1226 1247 1262 1289 1290 1480 1654
1.02 4.26 2.40 0.51 0.03 0.25 0.17 1.63 0.45 4.02 1.36 0.58 0.06 4.05 33.14 0.33 0.03 0.15 54.44
Oxygenated monoterpenes cineole b dehydro-1,8-N cis-β-terpineol α-terpineol (-)-borneol carvacrol thymol thymol, methyl ether Total:
1198 1427 1685 1691 2204 2173 1590
0.04 0.27 0.18 1.40 1.12 26.11 15.08 44.20
Sesquiterpenes α-cubebene β-bourbonene Aromadendrene alloaromadendrene γ-muurolene δ-cadinene Total:
1472 1510 1600 1641 1682 1750
0.05 0.07 0.12 0.01 0.01 0.04 0.30
1446 1535 1548
0.76 0.17 0.01 0.94 99.88
Alcohols 1-okten-3-ol linalol 1-nonen-3-ol Total: Total identified:
RI = Retention Index on CP WAX 52 CB capillary column.
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detected constituents. The major constituents of the oil were pcymene (33.14%), thymol (26.11%), thymol methyl ether (15.08%). %), γ-terpinene (4.05%), α-pinene (4.26%) and α-terpinene (4.02%). Lakusic et al. (2008), reported that the main constituent of S. horvatii oil was thymol. The chemical composition of various Satureja species essential oils has been reported (Adiguzel, Ozer, Kilic, & Cetin, 2007; Cavar, Maksimovic, Šolic, Jerkovic-Mujkic, & Bešta, 2008; Matasyoha, Kiplimoa, Karubiub, & Hailstorks, 2007) and the results are comparable to the present findings. Chemical differences could be due to genetic and environmental factors, and stage of plant development (Redžić et al., 2006).
3.2. Antimicrobial activity The MIC and MBC/MYC of S. horvatii essential oil were determined against various bacterial (L. monocytogenes, S. aureus, E. coli and S. typhimurium) and yeast (S. cerevisiae, Z. bailii, A. pullulans, P. membranaefaciens and P. anomala) strains. These MIC and MBC/ MYC values are shown in Table 2 in comparison with values related to two antibiotics, streptomycin and cycloheximide, used as references compounds. According to the MICs and MBCs, the Gram positive bacteria were more resistant than the Gram negative ones. For the bacterial strains, the MIC varied from 0.03 to 0.57 mg/mL (Table 2). The MIC values of oil for Gram positive bacteria (L. monocytogenes and S. aureus) were significantly higher than those for the Gram negative bacteria (S. typhimurium and E. coli). The MBC of oil for the bacterial strains varied from 0.07 to 1.15 mg/mL and showed a similar pattern, i.e. L. monocytogenes (1.15 mg/mL) N S.aureus (0.29 mg/mL) N E. coli (0.07 mg/mL) = S. tiphymurium (0.07 mg/mL). In general, S. horvatii essential oil showed significant antimicrobial activity, where bacterial strains were more resistant than the yeast strains. These results are in agreement with a report on the sensitivity of Gram negative bacteria to the essential oil of Thymus vulgaris (Imelouane et al., 2009). The authors reported that Gram positive bacteria (S. aureus, MIC = 1.33 mg/mL, Streptococcus sp., MIC = 2.67 mg/mL) were more resistant to the essential oil of T. vulgaris than Gram negative ones (E. coli MIC = 0.33 mg/mL). Streptomycin expressed inhibitory effects at 0.05–0.2 mg/mL and bactericidal activity at 0.2–0.4 mg/mL. It can be seen that the essential oil possessed (in some cases) a higher antibacterial effect than streptomycin against the Gram negative bacteria (MIC = 0.03 mg/mL). The MIC of the oil for the yeast strains varied from 0.56 to 2.23 mg/mL (Table 2). The lowest MIC (0.56 mg/mL) was shown by P. membranaefaciens OC71 and highest MIC (2.23 mg/mL) was shown by Z. bailii, A. pullulans. A similar pattern was found for the MFC of the oil against the yeast strains. The commercial antimycotic
cycloheximide was used as a control and had MICs (b 0.5 mg/mL) lower than that of the essential oil against the yeasts tested. Antimicrobial activity of a plant essential oil depends on its chemical composition. The presence of significant amounts of oxygenated monoterpenes (44.2 %), mainly represented by thymol and thymol methyl ether compounds, indicated a high antimicrobial potential of Satureja oil (Lakusic et al., 2008). The effect of some essential oil components on the cell membrane integrity of Gram positive and Gram negative bacteria has been reported. The most elucidated action concerns the antimicrobial components of oregano and thyme essential oils, i.e. carvacrol and thymol. It is recognized that their action results in the release of the lipopolysaccharides (LPS) from Gram negative bacteria with consequent cell membrane permeability increase and ATP loss (Faleiro, 2011). Carvacrol and thymol are able to disintegrate the outer membrane of Gram negative bacteria, releasing LPS and increasing the permeability of the cytoplasmic membrane to ATP (Gill & Holley, 2006; Lambert, Skandamis, Coote, & Nychas, 2001; Ultee, Bennik, & Moezelaar, 2002). The primary mode of antibacterial action of thymol is not fully known, but is believed to involve outer and inner membrane disruption, and interaction with membrane proteins and intracellular targets (Hyldgaard et al., 2012). The antimicrobial action of phenolic compounds, such as thymol and carvacrol, is expected to cause structural and functional damage to the cytoplasmic membrane (Sikkema, DeBont, & Poolman, 1995). Interestingly, thymol induced cell lysis and only altered the cell structure of proliferating S. cerevisiae cells, indicating that the effect of thymol depends on cell proliferation (Bennis, Chami, Chami, Bouchikhi, & Remmal, 2004). Contrary to this, Rao, Zhang, Muend, and Rao (2010), proposed that thymol activates specific signaling pathways in yeasts. Delgado, Fernández, Palop, and Periago (2004), reported that the use of thymol and p-cymene may be applied simultaneously for preservation of minimally processed foods. 3.3. Antihydroxyl radical property of essential oil Essential oil of S. horvatii has strong antihydroxyl radical activity (Fig. 1). In fact fluorescence emission intensity, which is positively correlated to radical production, dropped to 5% of the initial intensity immediately after addition of essential oil in the Fenton reaction system. After 1 min, radical production slowed to 1% of the initial production, and after 3 min hydroxyl radical production ceased. As the main component of S. horvatii oil is p-cymene, antiradical activity to hydroxyl radicals was expected, because monoterpene
Table 2 Antimicrobial activity (mg/mL) of Satureja horvatii essential oil. Microorganisms
S. horvatii oil 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 NCTC7973 Staph. aureus ATCC 6538 E. coli ATCC 35210 S. Typhimurium ATCC 13311
1.11 2.23 2.23 0.56 1.11 1.11 1.11 0.57 0.15 0.03 0.03
± ± ± ± ± ± ± ± ± ± ±
0.03 0.01 0.02 0.01 0.02 0.02 0.03 0.03 0.01 0.02 0.03
Control MYC/MBC
Strep. MIC/MBC
2.23 5.57 2.23 1.11 2.23 2.23 5.57 1.15 0.29 0.07 0.07
– – – – – – – 0.05 0.05 0.20 0.10
± ± ± ± ± ± ± ± ± ± ±
0.02 0.03 0.02 0.03 0.02 0.03 0.02 0.01 0.02 0.03 0.02
± ± ± ±
Cyclo. MIC
b0.05 ± 0.02 b0.05 ± 0.01 b0.05 ± 0.03 b0.05 ± 0.02 b0.05 ± 0.01 b0.05 ±0.01 b0.05 ± 0.03 0.03/0.10 ± 0.01 0.03/0.10 ± 0.01 0.01/0.40 ± 0.01 0.02/0.20 ± 0.02
MIC: Minimal Inhibitory Concentration; MBC: Minimal Bactericidal Concentration; MYC: Minimal Yeast-cidal Concentration; Strep.: Streptomycin; Cyclo.: Cycloheximide, mean ± standard deviation.
Fig. 1. Antiradical activity of S. horvatii essential oil, examined using fluorescence probe APF. Analysis was performed according to Eq. (5), in fixed size time Windows of 30 s.
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hydrocarbons are known as possible reactants with hydroxyl radicals (Winer, Lloyd, Darnall, & Pitts, 1976). The second most abundant component, thymol has shown a protective effect to hydroxyl radical and lipid peroxidation (Aeschbach et al., 1994).
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Table 4 Acceptability scores given by panelist for pork meat alone and sprayed with essential oil, after 4 days of storage (10 mg/10 cm2 of meat).
color flavor
3.4. In situ antimicrobial activity in pork meat The antimicrobial activity of S. horvatii essential oil against L. monocytogenes in pork meat medium was better under refrigerated conditions, 4 °C (Table 3). With the two highest tested concentrations (20 mg/mL and 10 mg/mL) 100% inhibition was achieved in 24 h regardless of the incubation temperature. Inhibition percentage was relatively constant at concentrations of 0.67 to 5 mg/mL during storage at 4 °C, in the range 93.45–96.62%. No inhibition was recorded at the lowest concentrations used (0.16 and 0.32 mg/mL). At room temperature S. horvatii essential oil was active in the concentration range 0.62–5.0 mg/mL, similarly to 4 °C. However, the inhibition percentage was dose dependent and its activity was almost halved at the lowest active concentration. Furthermore, inhibition slightly increased during storage. The best effect was for the highest concentrations used (20 and 10 mg/mL) at 25 °C. Application of low amounts of naturally occurring antimicrobial agents as ingredients should be explored, in order to control spoilage (Stojković, Petrović, et al., 2013; Stojković, Reis, et al., 2013; Stojković, Zivković, et al., 2013). The present work provides a basis for developing effective naturally occurring antimicrobial agents to extend the shelf-life of pork meat and provides strong evidence that the essential oil of S. horvatii might be efficient in the control of Listeria monocytogenes in pork meat. Eugenol and coriander, clove, oregano and thyme oils were found to be effective at 5 – 20 μL/g in inhibiting L. monocytogenes, A. hydrophila and autochthonous spoilage flora in meat products, sometimes causing a marked initial reduction in the number of recoverable cells (Skandamis & Nychas, 2001; Tsigarida, Skandamis, & Nychas, 2001), whilst mustard, cilantro, mint and sage oils were less effective or ineffective (Gill, Delaquis, Russo, & Holley, 2002; Lemay et al., 2002). One must consider the essential oil not only as a preservative, but also as a flavour component, especially as many herb and spice flavoured cheeses and other products already exist. Alternatively they could be incorporated into products which already have a strong flavour, that could mask the presence of the essential oil. However, the antimicrobial activity of any plant essential oil is likely to be a result of the interactions between different components, which can be additive, antagonistic or synergistic (Lanciotti et al., 2004; Smith-Palmer et al., 2001).
Pork meat
Pork meat + EO
2.2 1.7
4.1 3.7
The results are expressed as the average of all grades. 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like. EO — essential oil of S. horvatii.
3.5. Sensorial evaluation of flavor and color of meat sprayed with essential oil The results of the sensory evaluation are presented in Table 4. In meat non-treated with essential oil, after 4 days, due to oxidative changes the panelists mostly disliked the color and flavor and would refuse to eat such meat. As for the pork meat sprayed with essential oil, changes in color and flavor was recorded by panelists, but compared to non-treated samples the acceptability scores were better. Since the essential is a good antioxidant it may be of use in the meat industry, for more than just its antimicrobial properties. Meat in which oxidation reactions have occurred is brown in color; the flavor is rancid and stale and such meat would likely be rejected by the consumer (Greene & Price, 1975). Changes in meat color are due to oxidation of red oxymyoglobin to metmyoglobin (MMG), which gives rise to an unattractive brown color (Velasco & Williams, 2011). 4. Conclusion S. horvatii essential oil is a rich source of compounds such as pcymene and thymol and shows antimicrobial potential against a range of food spoilage microorganisms. The oil is much more active against bacterial than yeast strains. Thus, essential oils of S. horvatii may be useful for preservation and/or extension of the shelf life of raw or processed meat products. Acknowledgments This research was supported by a grant from the Ministry of Education and Science of Serbia (Project No. 173029, 173032 and 173040), Department of Agricultural and Food Sciences, Alma Mater Studiorum,
Table 3 Antibacterial activity of S. horvatii essential oil in pork meat medium, against the foodborne Listeria monocytogenes (mean ± SD). Concentration (mg/mL)
Temp. (°C)
Percentage of inhibition of L. monocytogenes in meat 0h
Satureja horvatii essential oil
20.00 10.00 5.00 2.50 1.25 0.62 0.31 0.16
+25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4 +25 +4
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
24 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 97.43 96.58 87.25 96.24 81.39 93.45 42.61 93.65 0.00 0.00 0.00 0.00
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 .2.23 1.46 1.51 2.36 1.17 1.05 2.08 1.15 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 98.52 95.12 91.33 98.18 81.75 93.82 45.37 94.32 0.00 0.00 0.00 0.00
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 1.38 2.54 1.13 3.64 3.65 1.02 2.21 2.72 0.00 0.00 0.00 0.00
100.00 100.00 100.00 100.00 98.82 96.62 93.14 96.53 87.93 95.24 48.96 95.13 0.00 0.00 0.00 0.00
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 1.14 1.32 1.02 1.73 1.77 1.44 2.54 1.02 0.00 0.00 0.00 0.00
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University of Bologna, Italy and Erasmus Mundus fellowship under EMECW to Danka Bukvicki. The authors wish to thank Professor Maria Elisabetta Guerzoni for her valuable suggestions and help during experiments.
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