Effect of bioclimatic area on the essential oil composition and antibacterial activity of Rosmarinus officinalis L.

Food Control 30 (2013) 463e468

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Effect of bioclimatic area on the essential oil composition and antibacterial activity of Rosmarinus officinalis L. María J. Jordán a, *, Vanesa Lax a, María C. Rota b, Susana Lorán b, José A. Sotomayor a a

Murcia Institute of Agri-Food Research and Development (IMIDA), c/ Mayor s/n, 30150 La Alberca, Murcia, Spain Department of Animal Production and Food Science, Food Hygiene, Inspection, Control and Microbiology Unit, Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 April 2012 Received in revised form 11 July 2012 Accepted 17 July 2012

The essential oil yield, volatile profile and antimicrobial activity of individual Rosmarinus officinalis L. shrubs growing wild in the different bioclimatic areas of the province of Murcia (Spain) were studied. A low thermicity index favoured the production of essential oil; however, no differentiation related to a specific chemotype depended on the geographical origin. In individual plants, the effect of the order of abundance among the components that define the rosemary essential oil chemotype (eucalyptol, camphor, a-pinene), on the antimicrobial activity was also determined. All the chemotypes showed strong antibacterial activity against four food-borne pathogens. Determination of the diameter of inhibition in Salmonella typhimurium pointed to a positive contribution effect of eucalyptol and a-pinene. A high proportion of a-pinene increases the effectiveness of the oil against Staphylococcus aureus, while the presence of eucalyptol, as the most abundant compound, considerably decreases the efficiency of rosemary oil. In contrast, the efficacy of these oils against Listeria monocytogenes and Escherichia coli was not affected by this condition. As regards the minimum inhibitory (MIC) and bactericide (MBC) concentrations, the strong activities exhibited by these essentials oils (<0.5 mL/mL) did not allow the chemotypes and antibacterial activities to be differentiated. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Essential oil Rosmarinus officinalis MIC MBC Chemotype Food-borne

1. Introduction Because of its essential oil and phenol biological properties, Rosmarinus officinalis L. is the most used aromatic and medicinal plant worldwide (Rozman & Jersèk, 2009). The latest research related with rosemary essential oil has mainly focused on its antibacterial (Jiang et al., 2011; Okoh, Sadimenko, & Afolayan, 2010), antifungal (Soylu, Kurt, & Soylu, 2010), insecticidal (Zoubiri & Baaliouamer, 2011), anticancer (Degner, Papoutsis, & Romagnolo, 2009) and antioxidant properties (Özcan & Arslan, 2011; Zaouali, Bouzaine, & Boussaid, 2010). According to Moreno, Scheyer, Romano, and Vojnov (2006), rosemary essential oil from wild populations exhibits high variation in its antimicrobial and antioxidant activities. These disparities are associated with the varying chemical composition of the essential oils. Factors including place of origin (Jamshidi, Afzal, & Afzali, 2009), environmental and agronomic conditions (Moghtader &

* Corresponding author. Tel.: þ34 968 366 790; fax: þ34 968 366 792. E-mail address: [email protected] (M.J. Jordán). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.07.029

Afzali, 2009), the time of harvest (Celiktas et al., 2007), the stage of development of the plants (Ruberto & Barata, 2000) and the method of extraction (Lopez, Sanchez, Batle, & Nerin, 2005; Okoh et al., 2010; Ramirez et al., 2006) influence the effectiveness of these properties. According to Napoli, Curcuruto, and Ruberto (2010), rosemary essential oil can be classified into three chemotypes from a chemical point of view: cineoliferum (high content in 1,8-cineol); camphoriferum (camphor > 20%); and verbenoniferum (verbenone > 15%). The chemical composition and seasonal variations in rosemary oil from southern Spain were reported by Salido, Altarejos, Nogueras, Sánchez, and Luque (2003). For these authors, all the samples studied belonged to the chemotype a-pinene/1,8-cineole/camphor. Later on, Varela et al. (2009), reported the chemical polymorphism of the essential oil from 87 Spanish wild rosemary populations. These authors defined different chemotypes depending on the geographical area. In the northeast of Spain, the major components identified in the volatile profile of the essential oils were camphor/1,8-cineole/ a-pinene; on the Mediterranean coast (southeast Spain), the chemotype was defined by 1,8-cineole/a-pinene/camphor; and in the

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south, the principal components were 1,8-cineole/camphor/apinene. Jordán, Lax, Martínez, Aouissat, and Sotomayor (2011), described the intraspecific chemical variability detected among plants belonging to a reduced geographical area in south-eastern Spain, identifying three major chemotypes that may predominate: 1,8-cineol-a-pinene-camphor (representing an 18.42% of the total plants analysed); camphor-1,8-cineole-a-pinene (17.76%); and 1,8-cineole-camphor-a-pinene (13.82%). As stated above, many biological activities e correlated with the presence of terpenes as major volatile components e have been attributed to rosemary essential oil. Zaouali et al. (2010) reported that variations in the antibacterial activity of Tunisian R. officinalis (var. typicus and var. troglodytorum) depended on the quantitative variation of essential oil compounds. Previous studies by our group have demonstrated the chemical variability that can be found between individual wild rosemary plants belonging to the same population (Jordán et al., 2011). This variability should be considered as an advantage that nature provides, enabling us to correlate the chemical composition and the biological activities described for individual plants. Bearing in mind that a high percentage of the worldwide production of rosemary oils, phenol extracts and dry leaves come from wild populations; the analysis of individual plants allows those with the highest biological activities to be identified so that they can be established as homogeneous commercial crops. Taking into account these considerations, the main goal of the present work was to evaluate how variations in the relative concentrations of the components that define the chemotype affect the antimicrobial activities of essential oils against a selection of four food-borne pathogens of significant importance for food hygiene. To the best of our knowledge, analysis of this condition using rosemary essential oil belonging to the same variety has not been carried out before. 2. Material and methods 2.1. Plant material

Directorate for the Quality of Medicines, 2002, pp. 183e184). The oil obtained was separated from water and dried over anhydrous sodium sulphate and kept in amber vials at 4  C until chromatographic analysis. Yield percentage was calculated as volume (mL) of essential oil per 100 g of plant dry matter. 2.3. Gas chromatographyemass spectrometry (GCeMS) analysis Samples (0.1 mL) were subjected to analysis by GCeMS. A 6890 N gas chromatograph (GC) (Palo Alto, CA, U.S.A.) equipped with a 30 m  0.25 mm i.d. HP-5 (5% cross-linked phenyl-methyl siloxane) column with 0.25 mm film thickness, and a DB-Wax (30 m  0.32 mm i.d.) and 1.0 mm film thickness was used. Both stationary phases were supplied by Agilent Technologies (Palo Alto, CA). Helium was used as the carrier gas (constant pressure, b-ionone eluting at 27.6 min for HP-5MS column and 41.38 min for DB-Wax column), and the split ratio was set to 100:1. The GC was linked to an Agilent model 5972 inert mass spectrometry detector. For both stationary phases, the initial oven temperature was set at 60  C, then increased at 2.5  C/min to 155  C, and finally raised to 250  C at a rate of 10  C/min; the injection port and the transfer line to the mass selective detector were kept at 250 and 280  C, respectively. The mass spectrometer was operated in electron impact ionization mode with an ionizing energy of 70 eV, scanning from m/z 50 to 500 at 3.21 scan/s. The quadrupole temperature was 150  C, and the electron multiplier voltage was maintained at 1300 V (Jordan, Martınez, Martınez, Monino, & Sotomayor, 2009). The individual peaks were identified by retention times and retention indices (relative to C6eC17 n-alkanes), compared with those of known compounds, and by comparison of mass spectra using the NBS75K library (U.S. National Bureau of Standards, 2002) and spectra obtained from the standard. Percentage compositions of samples were calculated according to the area of the chromatographic peaks using the total ion current. 2.4. Antimicrobial activity

A total of 150 samples of R. officinalis (identified by the authors) were collected from 31 wild populations belonging to different bioclimatic areas of the province of Murcia between July and August 2009. The bioclimatic zones are described in Table 1, following the classification made by Rivas-Martinez (1987). Cuttings from new shoots of individual plants were collected at the phenological stage of fruit maturation. Before essential oil extraction, the plant material was dried in a forced-air drier at 35  C for 48 h, until it reached a constant weight. 2.2. Essential oil extraction Aerial parts of individual plants were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus (European Table 1 Description of the rosemary populations’ bioclimatic areas ecological traits under study. Bioclimatic zone

Thermicity index

Rainfall (mm/year)

Lower Thermo-Mediterranean Upper Thermo-Mediterranean Lower Meso-Mediterranean Upper Meso-Mediterranean Supra-Mediterranean

>411 351e410 301e350 211e300 61e210

200e350 350e600 >600

Thermicity index ¼ 10[(T þ M þ m)] (1) (T, average annual temperature; M, average temperature from the highest temperature in the coldest month; m, average temperature from the lowest temperature in the coldest month. Rivas-Martínez (1983).

2.4.1. Microbial strains The antimicrobial activity of 25 essential oil samples (selected on the basis of their chemotype) was tested against Listeria monocytogenes serovar 4b (CECT 935), Staphylococcus aureus (CECT 240), Salmonella serotype typhimurium (CECT 443) and Escherichia coli O157:H7 (CECT 4267). All the strains were obtained from the Spanish Collection of Types Cultures and the cultures were kept frozen at 80  C in cryovials. Bacterial strains were grown in Trypticase Soy Broth (Merck, Darmstadt, Germany) supplemented with 0.6% of yeast extract (Merck, Darmstadt, Germany) (TSB-YE) and incubated at 37  C. 2.4.2. Disk diffusion method The essential oils were screened for antimicrobial activity using the agar diffusion technique, according to the method described by Rota, Carramiñana, Burillo, and Herrera (2004). Broth subcultures were prepared by inoculating, with one single colony from a plate, a test tube containing 5 mL of sterile TSBeYE. After inoculation, the tubes were incubated overnight at 37  C. The suspension of bacteria being tested (containing about 106 CFU/mL) was spread on Tryptic Soy Agar, with 0.6% of Yeast Extract added (TSAeYE) using sterile swabs. A filter paper disk (Whatman N 1, 6 mm diameter) containing 15 mL of each essential oil were applied to the surface of the agar plates. The plates were incubated overnight at 37  C and the inhibition zone diameters around each of the disks (diameter of inhibition zone plus diameter of the disk) were measured in millimetres. Streptomycin (0.025 g/L) was used as a positive control.

M.J. Jordán et al. / Food Control 30 (2013) 463e468

2.4.3. Determination of minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) The inhibitory and bactericidal activities of the rosemary essential oils (EOs) were determined by the tube dilution method (Rota et al., 2004). Broth subcultures were prepared by inoculating a flask containing 50 mL of sterile TSBeYE with well-isolated, single bacterial colonies from overnight plates. After inoculation, the flasks were incubated overnight at 37  C. Suitable amounts of EOs were added to stationary phase cultures previously diluted to a final concentration around 106 CFU/mL in TSBeYE with 3% ethanol added as a solvent. The highest concentration of EO tested was 30 mL/mL and the lowest concentration tested was 0.5 mL/mL. Negative controls, with TSBeYE plus 3% ethanol and 30% (vol/vol) of EOs, and positive controls containing TSBeYE with microorganism at a final concentration of around 106 CFU/mL plus 3% ethanol were also prepared. The MIC was read after 24 h incubation at 37  C in a shaken thermostatic bath (Bunsen, mod. BTG, Madrid, Spain). The MIC was taken as the lowest concentration of EOs at which bacteria failed to grow, so no visible changes were detected in the broth medium. In order to evaluate MBC, 100 mL of each case, in which microbial growth was not observed, was plated in Tryptic Soy Agar supplemented with 0.6% Yeast Extract (TSAeYE). Plates were incubated at 37  C for 24 h. The MBC was the lowest concentration at which bacteria failed to grow in TSBeYE and the subsequent transfer to TSAeYE plates (Janssen, Scheffer, & Svendsen, 1987). The evaluation of MIC and MBC was carried out in triplicate.

3. Results and discussion 3.1. Essential oil yield and chemical composition According to the results shown in Fig. 1, the ecological traits of the particular area in which the rosemary shrubs are grown affect the essential oil yielded by these shrubs. Thus, statistically significant differences were detected between the essential oils obtained from plants growing in the lower thermo Mediterranean area (1.74  0.38%) with respect to the oil levels extracted from plants of the upper meso- and supra-Mediterranean areas respectively (2.44  1.26 and 2.58  0.75%). This phenomenon could be

Essential oils yield (%)

3 2,7 2,4 2,1

465

attributed to certain environmental factors that might stimulate the essential oil production by the rosemary plants. Different studies can be found in the scientific literature regarding rosemary essential oil yield and how it changes depending on the plant geographical origin. For example, Angioni et al. (2004) reported differences at the essential oil yield of the Sardinian R. officinalis L., collected from different natural stations. For these authors, yields obtained from the northern and eastern samples were, on average, 2-fold higher than those of the southern and central samples, although they did not relate these variations to any edaphoclimatic condition in particular. Later, Zaouali et al. (2010) described the essential oil yielded by R. officinalis var. typicus and var. troglodytorum endemic to Tunisia, growing wild in different bioclimates. In this case, the essential oil yield for the typicus variety was higher in upper semiarid zones than that obtained from sub-humid regions. From these results it is clear that rosemary essential oil is influenced by the habitat in which the plants grow. However, the main edaphoclimatic factor that modifies this parameter is not clear. According to our results, and taking into account that no edaphic differences existed between the bioclimatic zones studied, we suggest that the climate of the zone plays an important role at the essential oil level production. In the province of Murcia, and contrary to that published by Zaouali et al. (2010) considering the same variety, rosemary essential oil yield increases in bioclimatic zones with lower thermicity indexes. As regards the essential oil volatile profile, chromatographic analysis allowed the identification of 32 major components, which represent 96.9e97.5% of the volatile components identified in rosemary essential oil (Table 2). Although, at a quantitative level, the essential oil chemical composition undergoes variations over the different bioclimatic areas studied, the relative concentration of the three components that define the rosemary essential oil chemotype (1,8-cineol, camphor and a-pinene) did not show statistically significant differences between the zones studied. Thus, no differentiation related to a specific chemotype can be defined depending on the bioclimatic area. These results are in agreement with those published by Zaouali et al. (2010), who affirmed that variations in the chemical composition of rosemary essential oil from Tunisia should be attributed to varieties rather than bioclimatic conditions. However, studies conducted by Tigrine-Kordjani et al. (2007) into the chemical composition of 32 rosemary samples collected at the same time from 12 different sites in the north of Algeria highlighted a strong correlation between the volatile chemical compositions of the samples and their origins. These differences could be attributed to the different methods used for essential oil extraction (microwave-accelerated distillation) and for the isolation of the volatile components before the chromatographic analysis (headspace solid phase microextraction), applied by these authors.

1,8

3.2. Antimicrobial activity

1,5

To study the antimicrobial activity of the essential oils, from the 150 plants previously analysed, a total of 25 individual plants with similar chemical compositions, but different relations between the major components defining the volatile profile of the essential oils, were selected (Table 3). The chemotypes described correspond eucalyptol-camphor-a-pinene, eucalyptol-a-pinene-camphor, camphor-eucalyptol-a-pinene, camphor-a-pinene-eucalyptol and a-pinene-eucalyptol-camphor. The antimicrobial activity was tested against two food-borne pathogens of Gram negative strains (S. typhimurium and E. coli) and two Gram positive strains (L. monocytogenes and S. aureus). Preliminary screening of the in vitro antimicrobial activity was developed using the filter paper disc agar diffusion technique

5.S.m

4.U.m.m

3.L.m.m

2. U.t.m

1.L.t. m

1,2

Bioclimatic Areas 1.L.t.m, Lower Thermo-mediterranean, 2 U.t.m, Upper Thermo-mediterranean,

3 L.m.m. Lower Meso-mediterranean, 4 U.m.m Upper Meso-mediterranean, 5 S.m, Supra-Mediterranean Fig. 1. Rosemary essential oil yield at the different bioclimates zones in the region of Murcia.

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Table 2 Rosemary essential oil volatile profile at the different bioclimatic areas. Components

tricyclene a-thujene a-pinene camphene sabinene b-pinene 4-octanone myrcene 3-octanol a-phellandrene D-3-carene a-terpinene r-cymene limonene eucaliptol g-terpinene terpinolene linalool a-campholenal camphor borneol terpinen-4-ol r-cymen-8-ol a-terpineol verbenone cuminaldehyde bornyl acetate ylangene (E)-b-caryophyllene a-caryophyllene a-amorphene caryophyllene oxide Total identification (%)

RI

Bioclimatic zones

(1)

(2)

1. L.t.m

958 960 965 973 987 989 995 998 1004 1008 1012 1017 1024 1028 1030 1053 1080 1095 1118 1141 1162 1173 1183 1188 1206 nd 1288 1370 1412 1441 1466 1553

nd 1064 1071 1082 1106 nd 1116 1120 nd 1133 1139 1146 1255 1164 1171 1194 1230 1246 nd 1301 1327 1341 1352 1358 1380 1415 1468 1552 1595 1625 1650 1739

0.4 0.1 15.6 8.4 tr 1.1 0.1 1.6 tr 0.3 0.2 0.6 2.9 2.9 22.1 0.7 0.7 0.7 0.3 18.5 7.1 1.0 0.2 2.8 5.4 tr 0.7 0.1 0.4 0.2 0.1 0.2 97.5

   

0.07ab 0.13 3.76a 1.23b

 0.44ab  0.09a  0.98a                

0.33 0.36 0.23a 1.55a 1.16 5.08 0.34a 0.18 0.33 0.09 4.82ab 2.45c 0.45ab 0.03 0.83 1.45

     

0.57 0.07 0.29a 0.12a 0.09 0.09a

2. U.t.m 0.4 tr 19.2 8.0 tr 1.1 0.3 3.7 tr 0.5 0.4 0.8 2.5 3.9 19.1 0.8 0.9 1.2 0.2 15.0 6.6 1.2 0.2 2.8 5.0 0.1 1.1 0.1 0.8 0.2 0.1 0.2 97.7

3. L.m.m.

 0.09b  4.59b  1.02b  0.44a  0.5a  2.36b                       

0.45 0.76 0.23ab 0.91a 1.70 5.48 0.20ab 0.24 0.68 0.1 4.86a 1.88c 0.15b 0.31 0.64 2.56 0.09 1.93 0.06 0.46ab 0.23a 0.02 0.16ab

0.3 0.1 14.6 7.7 tr 1.2 0.4 2.7 tr 0.5 0.7 0.8 2.3 3.3 19.7 0.9 1.2 1.1 0.3 20.2 5.8 1.2 0.2 2.8 4.8 0.1 0.8 0.1 1.2 0.3 0.1 0.2 96.9

   

0.16ab 0.11 3.65a 1.7b

 0.64ab  0.64a  2.26ab                       

0.55 1.17 0.42ab 1.11a 1.87 6.45 0.41abc 1.53 0.82 0.15 7.19ab 1.92bc 0.16b 0.55 0.58 3.02 0.05 0.88 0.09 1.17bc 0.27a 0.05 0.13a

4. U.m.m 0.3 0.1 16.2 7.4 tr 1.6 0.4 3.3 tr 0.7 0.4 0.9 2.3 3.4 21.1 1.1 1.0 1.2 0.2 17.7 5.2 1.2 0.1 2.8 5.1 0.1 1.0 0.1 1.0 0.3 0.1 0.2 97.1

   

0.09ab 0.09 4.21a 1.54ab

 0.81b  0.57a  2.10b                       

0.97 0.90 0.39b 1.10a 2.04 6.99 0.46c 36 0.68 0.17 7.34a 1.40ab 0.18ab 0.04 0.61 2.47 0.09 0.79 0.08 0.76ab 0.19a 0.04 0.09a

5. S.m 0.3 0.1 14.1 6.5 tr 2.2 0.9 3.4 0.1 0.5 0.7 0.7 1.5 3.1 18.9 1.0 1.1 0.9 0.3 24.0 4.1 1.0 0.1 2.7 4.0 0.1 1.2 0.1 1.6 0.6 0.1 0.3 97.5

   

0.11a 0.04 4.35a 1.57a

                          

0.83c 0.95b 2.14b 0.13 0.42 1.27 0.21ab 1.16b 1.13 6.01 0.30bc 0.41 0.46 0.42 9.96b 1.82a 0.21a 0.07 0.64 2.80 0.08 0.92 0.09 0.90c 0.50b 0.05 0.23b

(1), (2) Retention indices calculated using, respectively, (1) an apolar column (HP-5) and (2) a polar columna (DB-Wax); volatile compound proportions were calculated from the chromatograms obtained on the HP-5 column. Values followed by the different letters shared significant differences at 5% (Duncan test). nd, not detected. tr, trace (<0.01).

(Table 4). According to the classification made by Rota, Herrera, Martínez, Sotomayor, and Jordán (2008), all the chemotypes tested showed strong activity (inhibition zone  20 mm) against S. typhimurium, S. aureus, L. monocytogenes, and E. coli except for the chemotypes eucalyptol-camphor-a-pinene and

camphor-a-pinene-eucalyptol, which showed moderate activity (inhibition zone < 20e12 mm) against E. coli. The relative concentrations of the volatile components that define the essential oil chemotype affects the antibacterial activity of these oils against S. typhimurium and S. aureus, although this

Table 3 Volatile profile of rosemary essential oils selected by chemotype. Components

Chemotypes E-aP-C

E-C-aP

C-E-aP

C-aP-E

aP-E-C

a-pinene

17.7  0.93 8.0  0.09 2.1  0.68 2.2  0.93 1.1  0.98 1.0  0.38 2.4  0.47 2.8  0.82 23.4  0.18 1.2  0.38 1.0  0.02 1.1  0.02 13.4  0.75 5.3  1.07 1.3  0.15 2.7  0.17 5.0  1.34 0.6  0.19 0.7  0.42 0.5  0.46 0.2  0.03

13.3  1.11 6.0  2.13 2.4  0.57 1.8  0.94 0.2  0.04 0.7  0.12 2.1  1.26 1.9  0.15 26.4  1.25 0.9  0.25 0.7  0.17 0.4  0.10 21.7  0.66 4.4  1.29 1.1  0.23 3.3  0.26 4.1  1.31 1.1  0.70 1.2  0.89 0.1  0.06 0.3  0.17

8.9  1.33 5.8  1.31 1.8  0.65 3.2  1.93 0.3  0.04 0.5  0.13 0.9  0.16 3.7  0.91 13.6  2.51 0.8  0.17 0.9  0.15 1.0  0.45 39.7  1.89 4.6  0.74 0.9  0.30 2.3  0.27 2.8  0.77 1.4  1.13 2.0  1.48 0.7  0.54 0.3  0.12

17.1  0.92 9.3  0.61 1.2  0.38 4.6  2.53 0.3  0.07 0.8  0.35 2.2  0.78 4.6  1.19 13.3  3.56 1.2  0.66 0.9  0.20 0.9  0.8 25.7  5.32 6.5  1.30 1.1  0.11 1.9  0.34 2.6  1.65 2.2  1.0 1.0  0.49 0.6  0.45 0.1  0.06

22.3  0.76 7.8  0.66 0.9  0.22 3.1  0.11 0.6  0.34 0.9  0.22 3.7  0.15 3.5  0.37 20.7  0.91 0.9  0.10 0.9  0.07 0.8  0.28 13.2  0.55 5.3  2.09 1.3  0.11 2.9  0.17 4.6  1.29 0.6  0.47 0.6  0.38 0.2  0.14 0.1  0.02

camphene b-pinene myrcene a-phellandrene a-terpinene p-cymene limonene eucalyptol g-terpinene terpinolene linalool camphor borneol terpinen-4-ol a-terpineol verbenone bornyl acetate (E)-b-caryophyllene a-caryophyllene caryophyllene oxide

Chemotypes classification in relation with the chemical compounds found in rosemary samples. E ¼ eucalyptol; C ¼ camphor; aP ¼ a-pinene. Bold values signify relative concentration of the three major components that define the essential oil chemotype.

M.J. Jordán et al. / Food Control 30 (2013) 463e468 Table 4 Zone of inhibition growth (mm) of 5 different chemotypes from rosemary essential oil. Chemotype

S. typhimurium

E-aP-C E-C-aP C-E-aP C-aP-E aP-E-C

38.0 26.3 30.3 25.0 26.2

    

8.08c 2.89ab 3.48bc 2.49ab 1.71ab

S. aureus 29.3 26.6 36.4 40.5 44.7

    

7.72ab 5.94ab 8.90bc 6.87bc 2.25c

L. monocytogenes 22.1 20.9 22.8 22.1 22.3

    

2.04 2.52 3.91 3.60 1.50

E. coli 21.2 15.9 21.2 19.8 20.3

    

7.23 6.39 1.35 2.30 1.28

Rosemary’s, vol ¼ 15 mL Essentials oil. E ¼ eucalyptol; C ¼ camphor; aP ¼ a-pinene. Values followed by the different letters shared significant differences at 5% (Duncan test).

behaviour was not observed against L monocytogenes and E. coli. This is in agreement with the affirmation that the antibacterial activity of an oil may be related to the chemical configuration of its components, the proportions in which they are present and the interactions between them (Bajpai, Kwang-Hyun Baek, & Kang, 2012; Delaquis, Stanich, Girard, & Mazza, 2002; Dorman & Deans, 2000; Marino, Bersani, & Comi, 2001). In the case of S. typhimurium, the antibacterial activity of the rosemary essential oil is favoured by the combination of eucalyptol and a-pinene, since the inhibition zones obtained were greater in the presence of oils with a chemotype defined by E-aP-C and C-EaP. Also, it seems that camphor does not play an important role in the inhibition growth of these bacteria. According to Bajpai et al. (2012), it is possible that synergistic interactions exist between the essential oil components and the antibacterial activity detected for some oils against different strains of S. typhimurium. In addition to this, terpenes have the ability to disrupt and penetrate the lipid structure of the cell wall of bacteria, leading to the denaturing of proteins and the destruction of cell membranes, leading to cytoplasmic leakage, cell lysis and eventually cell death (Bajpai et al., 2012), which could explain the effectiveness of the chemotype rich in a-pinene against S. aureus. For this bacterium, the essential oils with camphor as the major component exhibited a moderate action, while, when eucalyptol was the most abundant compound, the effectiveness of rosemary oil against S. aureus decreased considerably. These results are agreement with those published by Zaouali et al., 2010, who also found that rosemary oils rich in eucalyptol exhibited moderate or no action against S. aureus. This affirmation supports the theory that a major proportion of apinene and camphor induces the antibacterial activity against S. aureus. As regards E. coli, any of the essentials oils tested exhibited stronger activity with respect to the other chemotypes. Zaouali et al. (2010) also reported a moderate antibacterial activity for rosemary essential oils from the var typicus (eucalyptol chemotype) and var troglodytorum (camphor chemotype) against this Gram () bacteria. These statements underline the affirmation that the antibacterial activity of rosemary essential oil against E. coli can be attributed to the combination of various minor components present in the oil, which act synergistically with the rest of the volatile compounds present. The antibacterial activity measured against L. monocytogenes was not affected by the relative abundance among the major volatile components present in the rosemary essential oil. All of them showed a strong activity against this bacterium. In contrast, Viuda-Martos et al. (2010) published that rosemary essential oil with a chemotype defined by eucalyptol-camphor-a-pinene did not show antibacterial activity against Listeria sp. Such differences may be attributed to the fact that these authors only used one specimen per species, whereas we studied 150 rosemary plants, providing a more representative pattern of behaviour for the different chemotypes against each bacterium.

467

The bacteriostatic and bactericidal effectiveness of the five rosemary essential oil chemotypes estimated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) respectively are shown in Table 5. On the one hand, these results indicate that all the oils studied exhibited low and similar MIC and MBC values against the tested bacteria, with the exception of E. coli, in which case only the oils with high eucalyptol content showed moderate MIC and MBC values. This bacterium was resistant to the rest of the chemotypes assayed. This agree with Roldán, Díaz, and Duringer (2010), who mention that the content of active substances in the essential oil determines its in vitro and in vivo efficacy, although the susceptibility of a given microorganism to an essential oil depends not only on the properties of the essential oil but also on the microorganism itself. On the other hand, the high degree of effectiveness exhibited by these essentials oils does not allow differentiation between chemotype and antibacterial activity against L. monocytogenes, S. aureus and S. typhimurium. This fact corroborates the high antibacterial efficacy exhibited by the rosemary essential oil from spontaneous populations located in the southeast of Spain. The Lamiaceae family is one of the most important as regards the production of essential oils with antimicrobial and antioxidant properties (Tsao & Zhou, 2007, chap. 18, pp. 364e387). Many articles can be found in the scientific literature regarding the antibacterial activity of rosemary essential oil against food-borne pathogens (Alves de Azeredo et al., 2011; Ivanovic, Misic, Zizovic, & Ristic, 2012; Jiang et al., 2011; Roldán et al., 2010). The antibacterial activity of one differs from that of another as a consequence of the different chemotype composition. For this reason it is necessary to define the chemotype e or the order of abundance between the components that defines the same e that best acts against the most common food-borne pathogens. This was the main goal of the present study, and, based on the diameter of inhibitions obtained, the effectiveness of rosemary essential oil against S. typhimurium is favoured by high eucalyptol content, while high concentrations of

Table 5 Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) (mL mL1) of different rosemary essential oil chemotypes. Chemotype

E-aP-C

E-C-aP

C-E-aP

C-aP-E

aP-E-C

S. typimurium

E. coli

L. monocytogenes

S. aureus

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 1 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 5 1 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 1 <0.5 <0.5 <0.5 <0.5 <0.5

5 2 5 2 5 2 5 2 2 5 e e e e e e e e e e e e e e e

5 2 5 2 5 2 5 2 2 5 e e e e e e e e e e e e e e e

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

<0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

E ¼ eucalyptol; C ¼ camphor; aP ¼ a-pinene.

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