Inhibition of foodborne pathogen bacteria by essential oils extracted from citrus fruits cultivated in Sicily

Food Control 26 (2012) 326e330

Contents lists available at SciVerse ScienceDirect

Food Control journal homepage: www.elsevier.com/locate/foodcont

Short communication

Inhibition of foodborne pathogen bacteria by essential oils extracted from citrus fruits cultivated in Sicily Luca Settanni a, Eristanna Palazzolo b, Valeria Guarrasi a, c, Aurora Aleo d, Caterina Mammina d, Giancarlo Moschetti a, Maria Antonietta Germanà a, * a

DEMETRA Department, University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy SAGA Department, University of Palermo, Viale delle Scienze 13, 90128 Palermo, Italy Institute of Biophysics at Palermo, Italian National Research Council, Via U. La Malfa 153, 90146 Palermo, Italy d Department of Sciences for Health Promotion “G. D’Alessandro”, Section of Hygiene, University of Palermo, Palermo, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 December 2011 Received in revised form 17 January 2012 Accepted 27 January 2012

The antagonistic activity of the essential oils (EOs) extracted by hydrodistillation from the fruit peel of several citrus genotypes (pummelo, grapefruit, orange, kumquat, mandarin and lemon) was evaluated against foodborne pathogen bacteria (43 strains of Listeria monocytogenes, 35 strains of Staphylococcus aureus and 14 strains of Salmonella enterica). Five commercial EOs were used for comparison. Most of the EOs were more effective against the Gram-positive bacteria rather than Salmonella. EOs of lemon genotypes 14 and 15 showed the best results in terms of number of strains inhibited and width of the inhibition zone. The most susceptible strain of each species (L. monocytogenes 133, St. aureus 473 and Salmonella Newport 50404) was used as indicator for the evaluation of the minimum inhibition concentration (MIC) of the EOs 14 and 15. The last two EOs, EO 16 (showing the weakest inhibition power among lemon EOs), and the commercial EO L (lemon) were analysed for their chemical composition. A total of 30 major compounds were identified by gas chromatography/mass spectrometry (GC/MS) and the oxygenated monoterpenes were suggested to be implicated in the process of bacterial inhibition by citrus EOs. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Antibacterial activity Citrus fruits Essential oils Foodborne pathogens

1. Introduction Essential oils (EOs) and extracts of various plants, herbs, spices and fruits show antimicrobial potential (Nychas, Tassou, & Skandamis, 2003; Oussalah, Caillet, Saucier, & Lacroix, 2006). The composition of EOs include a complex mixture of several compounds. When the inhibitory activities are directed towards foodborne pathogens, EOs may be employed in the food preservation, since they are referred to as GRAS (Generally Recognised As Safe) (Viuda-Martos, Ruiz-Navajas, Fernández-López, & PérezÁlvarez, 2008). In the last years, consumers have become aware of the health concerns of the food ingredients determining an increase in the request of foods processed without the addition of synthetic chemical preservatives. Thus, the use of EOs may provide a “natural” alternative to the chemical preservation of foods. Citrus EOs have been industrially applied in many products, including foods and beverages, cosmetics and medicines (Uysal, Sozmen, Aktas, Oksal, & Odabas Kose, 2011) and the activities against some of the most important foodborne pathogens have

* Corresponding author. Tel.: þ39 091 23896094; fax: þ39 091 6515531. E-mail address: [email protected] (M.A. Germanà). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.01.050

been proved (Fisher & Phillips, 2006; Kim, Marshall, & Wei, 1995). Since citrus EOs are mainly located in the fruit peel, their extraction is economically sustainable, because the fruit peel constitutes a waste for the fruit juice industry (Tirado, Stashenko, Combariza, & Martinez, 1995). The objectives of this study were: to evaluate the inhibitory potential of EOs extracted from the fruit peel of several citrus varieties cultivated in Sicily against the most common foodborne pathogens; and to determine the chemical composition of the most effective EOs. 2. Materials and methods 2.1. Citrus samples and EO extraction Citrus fruit samples used in this study (Table 1) were collected in April 2011. Samples 1, 2, 3, 4, 5, 6, 9, 10 and 11 were provided by the Azienda “Sperimentale Palazzelli” C.R.A.eCentro di ricerca per l’agrumicoltura e le colture mediterranee Contrada Palazzelli Scordia (CT, Italy), while samples 7, 8, 12, 13, 14, 15, 16, 17 and 18 were collected in the orchard “Parco d’Orleans”eAgricultural Faculty of Palermo (Italy). Commercial mandarin, lemon,

L. Settanni et al. / Food Control 26 (2012) 326e330

grapefruit, blood orange and blond orange EOs were kindly provided by Eurofood s.r.l. of Capo d’Orlando (ME, Italy). Citrus fruit peels were subjected to hydrodistillation using a Clevenger-type apparatus (Comandè, Palermo, Italy) and the EOs were collected for 3 h in hexane.

Table 1 List of citrus peel EOs used in the antimicrobial assays. EO

Species

Variety

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 P A AR L M

Pummelo (Citrus maxima (Burm.) Merrill) Grapefruit (Citrus paradisi Macf.) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Kumquat (Fortunella margarita) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Mandarin (Citrus reticulata Blanco) Lemon (Citrus limon L. Burm.) Lemon (Citrus limon L. Burm.) Lemon (Citrus limon L. Burm.) Lemon (Citrus limon L. Burm.) Lemon (Citrus limon L. Burm.) Orange (Citrus sinensis L. Osbeck) Grapefruit (Citrus paradisi Macf.) Orange (Citrus sinensis L. Osbeck) Orange (Citrus sinensis L. Osbeck) Lemon (Citrus limon L. Burm.) Mandarin (Citrus reticulata Blanco)

Reinking pummelo Pink Tarocco Meli Lane Late Nucellar C2611 Ovale Valencia Frost Valencia Lane Late VCR Tarocco S. Alfio Frost Washington Navel Washington Navel 3033 Mandarino Tardivo di Ciaculli Femminello apireno Femminello Santa Teresa Monachello Femminello Continella Femminello vecchio clone Valencia Grapefruits (commercial) Blond oranges (commercial) Blood oranges (commercial) Lemons (commercial) Mandarins (commercial)

327

2.2. Bacterial strains The strains used in this study (Tables 2e4), associated with foodborne diseases, belonged to the culture collection of the Section of Hygiene, Department of Sciences for Health Promotion “G. D’Alessandro” (Palermo, Italy). All strains were subcultured in Brain Heart Infusion (BHI) agar (Oxoid, Milan, Italy) and incubated overnight at 37  C. 2.3. Screening of the antibacterial activity EOs were tested against a concentration of approximately 107 CFU mL1 of each bacterial strain applying the paper disc diffusion method reported by Militello et al. (2011). Sterile water and streptomycin (10%, w/v) were used as negative and positive control, respectively. Petri dishes were incubated at 37  C for 24 h

Table 2 Inhibitory activitya of citrus EOs against Listeria monocytogenes. Strain

129b 130b 131b 132b 133b 134b 135b 136b 137b 138b 139b 140b 179c 180d 182d 184e 185f 186g 187h 188i 1BOl 2BOm 3BOm 4BOm 5BOm 6BOm 7BOm 8BOm 11BOn 12BOm 13BOo 14BOo 15BOo 16BOo 17BOo 18BOo 19BOo 20BOo 21BOo 22BOp 23BOp 24BOp

Citrus samples 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

P

A

AR

M

L

     e   e    e e e e e e  e þ      e e  e e e         þ 

 e       e e  þ e  e  e   e       e e  e e e         þ 

 e e e  e   e   e e e e  e e    þ  þ    e  e e     e     þ 

      þ  e þ  þ e    e e   þ þ  þ  þ  e  e      e     þ 

e e  e   þþ þ  þ e e   e þ e   e  e e e e  e e  e e e   e e e  e e  

e     þ þ   e    e e þ e  e   e     e e e e e      e   e e 

e e e e    e e e e e  e e þ e e e e  e e  e  e e e e e e e   e e e e e e e

  þ   þ              e þ  e  e  e e            e  

e e       e e e   e e  e  e e þ    e   e e e e e e  e e    e e e

e  e   e e   e   þ  e   e e  e   e e  e e e   e e  e e  e e   

e  e   e e      þ  e  e   þ e       e              

  þ þ þ  þ   þ  þ þ þ  þ e þ þ   þ    þ   þ   þ   þ  þ  þ  þ þ

   þ        þ    þ þþ þþ  þ þ þ  þ    þ þ  þ þ         þ 

þþ þ þ þ þþ þþ þþ þ þ þ þþ þþ þþ þþ þþ þþ þþ þþ þ þþ þþ þ þþ þþ þ þ þþ þþ þ þþ þþ þþ þ þ þ þ þþ þþ þ þþ þ þþ

þþ þ þ þþ þþ þþ þþ þ þ þ þ þþ þ þþ þþ þþ þþ þþ  þþ þþ þ þþ þþ þ þ  þ þ þ þþ þþ þþ þ  þ þ þþ þ þ þ þþ

e þ  þ             þ þ   þ þ          þ þ        þ 

 þ þ þ þ þ  þ   þ þ  þ þ  þ þþ þ þ þ þ þ þ þ   þ þ   þ        þ þ þ

e  e   e        e e e e e e       e e e e e e   e e       

e e e e e e e e  þ   e e e e e e e e  þ e e e e þ e  e þ þ e þ e e e e e  e e

e e e e e e e e  e  e e e e e e e e e e  e e e e e e e e e e e e e e e e e e e e

e e e   e  e  þ   e e e e e e e e e  e e e e e e e e e e e e e e e e e   þ

    þ     þ     þ e e e e þ  þ e  e        e         

    þ     þ   þ þ   þ   þ þ þþþ     þ  þ  þ þ þ    þ     

Source of isolation: b, human stools; c, salmon; d, Ricotta cheese; e, rice salad; f, beef; g, mozzarella salad; h, roasted chicken; i, green salad; l, chopped meat; m, salami; n, meat factory; o, Gorgonzola cheese; p, Taleggio cheese. a e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter); þþþ, huge inhibition (>18 mm diameter).

328

L. Settanni et al. / Food Control 26 (2012) 326e330

Table 3 Inhibitory activitya of citrus EOs against Staphylococcus aureus. Strain

C1/5634-MSSAb C4/6561.1-MSSAc C6/5145-MSSAb C38/249.1-MSSAb C45/12425-MSSAb 195-MRSAd TUM-MRSd 9-MRSd 14-MRSAd 189-MRSAd 1313-MRSAd 1369-MRSAd 581-MRSAd 340-MRSAd 4ADI MRSAd 7ADI MSSAd 14LU MRSAd 16 MSSAd 19 MRSAd 20ADI MRSAd 21ADI MRSAd 62 MRSAd 68 MRSAd 106 MRSAd 109 MRSAd 156 MRSAd 168 MRSAd 206 MSSAd 473 MRSAd 493 MRSAd 637 MRSAd 734 MSSAd 735 MSSAd 750 MSSAd E36GI MRSAd

Citrus samples 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

P

A

AR

M

L

          þ  þ    þ  þ þ þ þ    e þ   þ þ  þ  

e e   e    e  þ     e         e e       e  

e e   e e  e e e e e    e       e e e e         

 e              e  e      e e e þ        e

e e e e e e e e e e e e e e e e e e e     e   e e e e e e e  e

   e  e   e e e     e  e  e      e   e e e e   e

e e e e e e e e e e e e e e  e e e  e       e   e  e e  e

   e e   e e e e e e e  e e e        þ    e  e e e 

   e e e e e e e e e e e e e e e e e    e e  e e e e e e e e e

e e  e  e e e e e e e e e e e  e e      e    e  e e   e

e e  e  e e e e    e e e   e e e e    e    e  e  e  e

                    þ         þ   þ þ 

þ  þ  þ  þ       þ þ  þ   þ þ   þ þ þ þ  þ þ þ þ  þ 

þþ þþ þþ þþ þþ þ þþ þ þ þ þþ þþ þþþ þþ þþ e þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þ þþþ þþ þþ þþþ þ þþ þþ

þþ þþ þ þþ þþ þ e þ þ þ þþ  þþ þþ þþ e þþ þþ þ þþ þþ þþ þþ þþ þþ þþ þþ þ þþþ þþ þþ þþþ þ þþ þþ

þ      e   e e e        þ     þ þ þ    þ    

þ  þ þ þ  þ þ þ þ þ   þ þ  þ þ þ þ þ þ þ þ þþ þþ þ  þ þ þ þþ   þ

e e e e  e e e e  e e e e e e e e e e e e e e e e   e e e e e e e

e e e e e e e e  e e e e e e e e e e  e e e e e e  e   e    

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

e e e e e e e e e e e e e e e e e e  e e e e e e e e e e e e e e e e

e e  e  e e   e e e e     e  e         þ  þ    

   e þ    þ    þþ  þ   e  e     þ þ   þþ þ þ    

Source of isolation: b, cheese; c, raw milk; d, human stools. a e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter); þþþ, huge inhibition (>18 mm diameter).

and the inhibitory activity was evaluated as positive if a definite clear area was detected around the paper disc. Each test was performed in duplicate and the experiments were repeated twice.

of EO antibacterial performances (Burt, 2004). MIC is defined as the lowest concentration of an active compound inhibiting visible growth of test organisms (Karapinar & Aktug, 1987). The EOs were serially diluted (dilution factor ¼ 2) in acetone (SigmaeAldrich, Milan, Italy). Each test tube, containing 990 mL of broth medium and 10 mL of EO dilution, was inoculated with approximately 106 CFU mL1 of a given sensitive strain. Acetone alone was used as negative control.

2.4. Determination of the minimum inhibitory concentration The antibacterial activity was measured as minimum inhibitory concentration (MIC), which represents the most common expression Table 4 Inhibitory activitya of citrus EOs against Salmonella enterica. Strain

S. S. S. S. S. S. S. S. S. S. S. S. S. S.

Abony 50398b Agona 50360b Blockley 50314b Bredeney 50374b Derby 50399b Enteritidis 50339c Hadar 50272d Infantis 50270b Muenchen 50393b Napoli 50376b Newport50404b Panama 50347b Saintpaul 50415b Thompson 50280e

Citrus samples 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

P

A

AR

M

L

e e e e e e e e e e  e e e

e e e e e e e e e e e e e e

e e e e e e e e e e  e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e  e e e

 e þ þ e þ þ  e þ þ e þ e

þþ þ þþ þþ þ þþ þþ þ þþ þþ þþ þþ þþ e

þþ þ þþ þþ þþ þþ þþ þ þþ þþ þþ þþ þþ 

e e e e e þ e e e þ þ e e e

þ  þþ þ þþ þþ þ þ þþ þþ þþ þþ þ e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

e e e e e e e e e e e e e e

Source of isolation: b, human stools; c, food preparation; d, egg; e, chicken-pork meat. a e, no inhibition; , inhibition (<9 mm diameter); þ clear inhibition (9e12 mm diameter); þþ, strong inhibition (13e15 mm diameter).

L. Settanni et al. / Food Control 26 (2012) 326e330

2.5. Determination of EO composition Samples were analysed by gas chromatography/mass spectrometry (GC/MS) (EI) on a GCMS-QP2010 (Shimadzu, Milan, Italy) using a fused silica capillary column, SLB-5MS (5% diphenyl:95% methylsiloxane) : 30 m  0.25 i.d.  0.25 mm film thickness (Supelco, Milan, Italy); temperature program 50e280  C at 3  C min1; carrier gas He at a constant linear rate: 30 cm s1 (30.6 kPa); 1.0 mL of solution (1:10 v/v, essential oil/hexane) injected on a splitesplitless injector; injector temperature 250  C; injection mode split (split ratio 100:1). MS scan conditions: source temperature 200  C, interface temperature 250  C, EI energy 70 eV; mass scan range 40e400 amu. Data were handled through the use of GCMS-Solution software and the peak identification was carried out with NIST21,107,147 Library according to a similarity larger than 90%. 3. Results and discussion 3.1. Inhibition of bacterial growth The antibacterial activity of the EOs analysed is shown in Tables 2e4. The active extracts were mainly effective against the Gram-positive bacteria (Tables 2 and 3), while only three lemon EOs (13, 14 and 17) clearly inhibited the growth of Salmonella (Table 4). This finding is not surprising, since also other studies showed that Gram-positive bacteria were more susceptible to EOs of different origin, including citrus fruits, than Gram-negative bacteria (Al-Reza, Rahman, Lee, & Kang, 2010; Burt, 2004; Calsamiglia, Busquet, Cardozo, Castillejos, & Ferret, 2007; Davidson & Naidu, 2000). The presence of the outer membrane provides a strong impermeable barrier for the Gram-negative bacteria (Nikaido, 1994). The commercial EOs were characterised by a lower inhibitory power than Sicilian EOs, although commercial lemon and mandarin EOs showed some antagonistic properties vs Listeria and staphylococci. Salmonella growth was not inhibited by any commercial EO. In general, the strains belonging to the Gram-positive species showed a different level of susceptibility to the treatments. Undoubtedly, the most interesting results were displayed by EOs 14 and 15 which inhibited all 42 Listeria monocytogenes and almost all 35 St. aureus strains tested (34 strains by EO 14 and 33 by EO 15). For the majority of the susceptible strains, the diameter of the halo was in the range 13e15 mm (scored as strong inhibition). Except for S. Thompson 50280, which was quite resistant to the EOs, all other Salmonella strains were strongly inhibited by EOs 14, 15 and 17. L. monocytogenes 133, St. aureus 473 and S. Newport 50404 were found highly sensitive to the EOs 14 and 15 and, for this reason, they were chosen for the calculation of the MIC (Table 5). In particular, S. Newport 50404 showed the most interesting result, because it was characterised by the highest susceptibility, despite it is a Gramnegative bacterium. Previous studies showed that the inhibition of some Gram-negative (Escherichia coli and Campylobacter jejuni) strains was comparable with that of Gram-positive bacteria (Fisher & Phillips, 2006). Fisher and Phillips (2008) suggested that the antibacterial effects of citrus EOs are not uniform among bacteria and could depend on the compounds and the species/isolate under study. MIC data of EOs 14 and 15 were of paramount importance, since these two EOs were effective at concentrations extremely low. The first aspect to be considered for the application of EOs in foods is represented by their impact on the organoleptic properties of the final product, because the main constituents of EOs are responsible for their fragrance. Hence, naturally derived preservatives can alter the taste of foods or exceed acceptable flavour thresholds (Hsieh,

329

Table 5 MIC of Sicilian lemon EOs 14 and 15 against the most sensitive pathogenic strains. MIC (mL mL1)

Strains

L. monocytogenes 133 St. aureus 473 S. Newport 50404

EO 14

EO 15

0.156 0.039 0.019

0.156 0.078 0.019

Mau, & Huang, 2001; Nazer, Kobilinsky, Tholozana, & DuboisBrissonneta, 2005). Moreover, once the antibacterial activity is evaluated in vitro, a major problem to the in situ efficacy of a given EO derives from its interaction with the food components and/or other additives. For this reason, Burt (2004) suggested that a higher concentration of EOs could be needed to achieve the same effect in food as in vitro. Thus, the high inhibitory activity of EOs at low concentrations determines a lower amount of EOs to be added to the food matrices, limiting the unwanted changes of the sensory features.

3.2. Chemical characterization of EOs The EOs 14 and 15, which showed the highest inhibitory activities, EO 16, that within the lemon EO group displayed the lowest inhibitory efficacy, and the commercial EO L were chemically analysed by GC and GCeMS. The qualitative and quantitative

Table 6 Chemical composition of commercial and Sicilian lemon EOs. Chemical compounds

Monoterpene hydrocarbons a-Thujene a-Pinene Camphene Sabinene b-Pinene b-Myrcene a-Terpinene (þ)-4-Carene o-Cymene D-Limonene p-Cymene a-Terpinolene Oxygenated monoterpenes 1-Octanol Linalol Nonanal (þ)-Citronellal Nonanol 4-Terpineol a-Terpineol cis-Geraniol b-Citronellale b-Citral Nerol a-Citral Neryl acetate Geranyl acetate Sesquiterpene hydrocarbons Caryophyllene a-Bergamotene b-Bisabolene Others Tetratetracontane

LRIa

%b EO L

EO 14

EO 15

EO 16

1419 1493 1502

77.75 0.58 2.34 0.07 1.95 14.12 2.10 t 0.29 0.07 55.32 0.25 0.56 6.18 t 0.14 0.21 0.17 t 0.06 0.20 0.03 t 1.21 0.03 2.18 1.02 0.96 1.96 0.34 0.64 0.98

68.74 0.32 1.49 0.05 1.71 11.77 1.57 0.20 t 1.28 49.65 0.18 0.52 19.21 0.22 0.76 0.24 0.13 0.16 1.13 1.61 2.12 0.45 3.18 2.90 4.29 1.24 0.78 1.27 0.16 0.43 0.68

67.61 0.36 1.64 0.05 1.89 13.19 1.51 t 0.32 1.62 46.31 0.14 0.58 19.35 0.31 0.66 0.26 0.11 0.27 1.43 2.17 2.86 0.35 2.46 3.94 3.32 0.71 0.50 1.47 0.18 0.50 0.79

78.23 0.34 1.50 0.03 1.53 10.29 1.97 0.06 0.18 2.59 60.90 0.21 0.47 7.69 0.19 0.55 0.17 0.05 0.07 0.73 0.98 0.90 0.18 0.67 1.09 0.89 0.68 0.54 1.54 0.13 0.55 0.86

1689

0.02

t

0.36

t

930 939 954 976 981 990 1014 1020 1024 1029 1026 1088 1090 1098 1015 1145 1155 1177 1188 1255 1314 1337 1347 1358 1365 1383

t, trace (<0.02%). a Linear retention index on SLB-5MS column. b Percentage of each compound on the total oil (computed from the total GC peak area).

330

L. Settanni et al. / Food Control 26 (2012) 326e330

composition of the EOs is reported in Table 6. Chemical compounds were classified by phytochemical groups and listed in order of their elution. A total of 82 chemicals were identified, most of which (52) were detected in trace (percentage lower than 0.10). The total recovery was: 85.91% for EO L, 89.22% for EO 14, 88.79% for EO 15 and 87.46% for EO 16. All EOs were dominated by the monoterpene hydrocarbon fraction that reached almost the 78% for the EOs L and 16 and almost 68% for EOs 14 and 15. On the contrary, the oxygenated monoterpene fraction of EOs 14 and 15 (more than 19%) was about three times that of EOs L and 16. Sesquiterpene hydrocarbons did not exceed 2%, while among the other compounds, tetratetracontane was detected at a percentage higher than 0.10 only in EO 15. The efficacy of EOs 14 and 15 may be imputable to the oxygenated monoterpenes, in particular to 4-terpineol, a-terpineol, cis-geraniol, b-citral, nerol and a-citral. Our results confirmed previous observations that this class of chemicals, especially phenolic substances, exhibits strong antimicrobial activity than hydrocarbon monoterpenes (Knobloch, Weigand, Weis, Schwarm, & Vigenschow, 1986; Sokovi c & van Griensven, 2006; Sokovic, Tzakou, Pitarokili, & Couladis, 2002). Hydrocarbon monoterpenes are characterised by a low water solubility which limits their diffusion through the medium and their inactivity is closely related to their limited hydrogen bound capacity (Griffin, Markham, & Leach, 2000). Acknowledgements Authors wish to thank Eurofood s.r.l. of Capo d’Orlando (ME, Italy) for providing the commercial EO samples and Dr. G. Russo, Dr. Reforgiato Recupero and S. Recupero (Centro di ricerca per l’agrumicoltura e le colture mediterranee) for providing some citrus samples. References Al-Reza, S. M., Rahman, A., Lee, J., & Kang, S. C. (2010). Potential roles of essential oil and organic extracts of Zizyphus jujuba in inhibiting food-borne pathogens. Food Chemistry, 119, 981e986. Burt, S. (2004). Essential oils: their antibacterial properties and potential application in foods e a review. International Journal of Food Microbiology, 94, 223e253. Calsamiglia, S., Busquet, M., Cardozo, P. W., Castillejos, L., & Ferret, A. (2007). Invited review: essential oils as modifiers of rumen microbial fermentation. Journal of Dairy Science, 90, 2580e2595. Davidson, P. M., & Naidu, A. S. (2000). Phyto-phenols. In A. S. Naidu (Ed.), Natural food antimicrobial systems (pp. 265e293). Boca Raton, FL: CRC Press.

Fisher, K., & Phillips, C. A. (2006). The effect of lemon, orange, and bergamot essential oils and their components on the survival of Campylobacter jejuni, Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in food systems. Journal of Applied Microbiology, 101, 1232e1240. Fisher, K., & Phillips, C. A. (2008). Potential antimicrobial uses of essential oils in foods: is citrus the answer? Trends in Food Science and Technology, 19, 156e164. Griffin, G. S., Markham, L. J., & Leach, N. D. (2000). An agar dilution method for the determination of the minimum inhibitory concentration of essential oils. Journal of Essential Oil Research, 12, 249e255. Hsieh, P.-C., Mau, J.-L., & Huang, S.-H. (2001). Antimicrobial effect of various combinations of plant extracts. Food Microbiology, 18, 35e43. Karapinar, M., & Aktug, S. E. (1987). Inhibition of foodborne pathogens by thymol, eugenol, menthol and anethole. International Journal of Food Microbiology, 4, 161e166. Kim, J., Marshall, M. R., & Wei, C.-I. (1995). Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry, 46, 1739e1745. Knobloch, K., Weigand, H., Weis, N., Schwarm, H. M., & Vigenschow, H. (1986). Action of terpenoids on energy metabolism. In E. J. Brunke (Ed.), Progress in essential oil research (pp. 429e445). Berlin: Walter de Gruyter. Militello, M., Settanni, L., Aleo, A., Mammina, C., Moschetti, G., Giammanco, G. M., et al. (2011). Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil. Current Microbiology, 62, 1274e1281. Nazer, A. I., Kobilinsky, A., Tholozana, J.-L., & Dubois-Brissonneta, F. (2005). Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium: a synergistic effect? Food Microbiology, 22, 391e398. Nikaido, H. (1994). Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science, 264, 382e388. Nychas, G. J. E., Tassou, C. C., & Skandamis, P. (2003). Antimicrobials from herbs and spices. In S. M. Roller (Ed.), Natural antimicrobials for the minimal processing of foods (pp. 176e200). New York: Woodhead Publishers, CRC Press. Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2006). Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control, 18, 414e420. Sokovi c, M., Tzakou, O., Pitarokili, D., & Couladis, M. (2002). Antifungal activities of selected aromatic plants growing wild in Greece. Nahrung, 46, 317e320. Sokovi c, M., & van Griensven, L. J. L. D. (2006). Antimicrobial activity of essential oils and their components against the three major pathogens of the cultivated button mushroom, Agaricus bisporus. European Journal of Plant Pathology, 116, 211e224. Tirado, C. B., Stashenko, E. E., Combariza, M. Y., & Martinez, J. R. (1995). Comparative study of Colombian citrus oils by high-resolution gas chromatography and gas chromatography-mass spectrometry. Journal of Chromatography A, 697, 501e513. Uysal, B., Sozmen, F., Aktas, O., Oksal, B. S., & Odabas Kose, E. (2011). Essential oil composition and antibacterial activity of the grapefruit (Citrus Paradisi L.) peel essential oils obtained by solvent-free microwave extraction: comparison with hydrodistillation. International Journal of Food Science and Technology, 46, 1455e1461. Viuda-Martos, M., Ruiz-Navajas, Y., Fernández-López, J., & Pérez- Álvarez, J. (2008). Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Control, 19, 1130e1138.