Antimicrobials Against Campylobacter Jejuni Cect Djenane2012

7/24/2019 Antimicrobials Against Campylobacter Jejuni Cect Djenane2012

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PERSPECTIVES ON THE USE OF ESSENTIAL OILS ASANTIMICROBIALS AGAINST CAMPYLOBACTER JEJUNICECT7572 IN RETAIL CHICKEN MEATS PACKAGED INMICROAEROBIC ATMOSPHEREjfs_342 37..47

DJAMEL DJENANE1, JAVIER YANGELA2, DIEGO GMEZ2 and PEDRO RONCALS2,3

1Facult des Sciences Biologiques et des Sciences Agronomiques, Department: Biochimie et Microbiologie, Universit Mouloud Mammeri,

Tizi-Ouzou, Algeria2Department: Produccin Animal y Ciencia de los Alimentos, Universidad de Zaragoza. C/ Miguel Servet, 177-50013 Zaragoza, Spain

3Corresponding author. TEL:

+34-976-76-15-82; FAX:+34-976-76-15-90;

EMAIL: [email protected]

Received for Publication February 23, 2011

Accepted for Publication July 28, 2011

doi:10.1111/j.1745-4565.2011.00342.x

ABSTRACT

The chemical composition of the essential oils (EOs) ofInula graveolens, Laurusnobilis, Pistacia lentiscus and Satureja montana was analyzed using a gaschromatography-mass spectrometry technique. The main components of EOsobtained were, respectively, bornyl acetate, 1,8-cineole,b-myrcene and carvacrol.EOs were screened for their ability to inhibit the growth ofCampylobacter jejuniCECT 7572 using the standard agar-disk diffusion assay. The results obtained, fol-lowed by measurements of minimal inhibitory concentrations, indicated that I. gra-veolenswas most active (F =53.3 mm), with the lowest MIC value againstC. jejuni(2mL/mL).EOs were testedin chicken stored in microaerobic conditions at 3 2C,experimentally inoculated with the pathogen at a level of 5 105 cfu/g. C. jejunicounts in treated samples were 0.74.7 log10 cfu/g lower (P< 0.05) than the controlsthroughout storage. The latter reached numbers of about 8 log10cfu/g after 1 week.Lipid oxidation (thiobarbituric acid reactive substances [TBARS]) and sensoryfreshness odor were also determined. Samples treated with any EO had the lowestTBARS values (P< 0.05). The presence of EOs significantly extended fresh meat

odor. The results of the bioassays, together with the chemical profile of the EOs,support the possibility of using all EOs as potent natural preservatives to contributein the reduction of experimentally inoculated C. jejuni in chicken meat.

PRACTICAL APPLICATIONS

Theresults revealed forthe first time in a chicken meat systemthe potential ofI. gra-veolens, L. nobilis, P. lentiscusandS. montanaEOs in inhibitingC. jejuni. This sug-gests the possibility that they, particularlyI. graveolens, could be used as naturalpreservatives in chicken meat for reducing food hazards caused by this pathogen,which is now recognized as the leading cause of bacterial foodborne gastroenteritis.

INTRODUCTION

Campylobacterspecies causecampylobacteriosis in humans,agastrointestinal tract infection (Suzuki and Yamamoto 2009;Wegener 2010). Campylobacter contamination of chickencarcasses is common, and poultry meat is generally recog-nizedtoplayasignificantroleinhuman Campylobacterinfec-tion (Zilbauer et al. 2008). The most important pathogenic

strains belong to thegroupof thermotolerant Campylobacter,particularlyCampylobacter jejuni(Sonet al. 2007).C. jejunihas recently overtakenSalmonellaspp. as the major reportedsource of foodborne bacterial diseases within the EuropeanUnion (European Food Safety Authority 2009). C. jejuniispart of normal enteric microbiota in animals (cattle, chickenand pigs) and can be transmitted to humans throughcontaminated foods (Aslim and Yucel 2008). A positive

Journal of Food Safety ISSN 1745-4565

37Journal of Food Safety32(2012) 3747 2011 Wiley Periodicals, Inc.


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correlation was observed between the number ofC. jejunipresent in the caeca and the number of bacteria present oncarcasses andcut products (Reich et al.2008).Contaminationof carcasses with C. jejuni occurs particularly duringscalding,defeathering, evisceration and chilling operations (Nautaet al. 2009).

Several recent studies described in detail the antimicrobialproperties of some EOs against a number of relevant, food-borne, pathogenic bacteria, which may be envisaged asnatural alternatives to chemical-based antibacterial for foodsafety and preservation (Bakkaliet al. 2008; Solomakoset al.2008; Djenane et al. 2011a,b,c). Despite the potential of manycommon plants and EOs is considerable, knowledge of thisarea and studies on their biological activities remain scarce.Most of the data published on the antimicrobial properties ofplant EOs are fragmented and employ only basic screeningtechniques. Moreover, most studies on the antimicrobialaction of plant extracts have been conductedin vitro, so thatlittle information exists regarding the antimicrobial activityof EOs in food systems (Koutsoudaki et al. 2005; Oussalahet al. 2007; Oke et al. 2009).

The main objectives of this study were (1) to determinechemical composition of the steam-distilled EOs of Inula

graveolens, Laurus nobilis, Pistacia lentiscus and Saturejamontanaby gas chromatography-mass spectrometry (GC-MS); (2) to investigate the antimicrobial activity of these EOsagainst the food pathogenC. jejunibyin vitrodisk diffusionmethods, as well as to determine their minimum inhibitoryconcentration (MIC); (3) to study their antimicrobial effectin a chicken meat system; and (4) to investigate the effect ofEO addition on the sensory properties of meat throughout

storage.

MATERIALS AND METHODS

Plant Material and Essential Oils Extraction

Theaerial parts ofI. graveolens and L. nobilis were collectedatTizi-Ouzou province (Algeria), from March to July 2009 andauthenticated by the Department of Biology, UniversityMouloud Mammeri of Tizi-Ouzou (Algeria). The wholefresh plants were then extensively washed with distilledwater (20C) to remove epiphytic hosts normally found on the

surface andwere dried in the darkness at 25C. Only the leaveswere recuperated for subsequent extraction to obtain theirEOs. EOs from P. lentiscusand S. montana were purchasedfromFlorameAromathrapie (St Rmy de Provence,France).Both EOs were certified by Ecocert SAS F32600 (France) andwere considered 100% pure and natural, obtained fromMediterranean biological culture.

The I. graveolens and L. nobilis EOs were obtained fromdried leaves and plant parts by steam hydrodistillation in aClevenger-type apparatus for 3 h (Groupe Pharmaceutique

SAIDAL, Filiale Biotic, Algiers, Algeria). The EOs obtainedwere separated from water and dried over anhydrous sodiumsulphate (Na2SO4). The isolated EOs were preserved in dark-nessinasealedvialat1Cuntilitsanalysisoritsuseinbioassays.

Analysis of Essential Oils

GC Analysis. GC analyses of EOs obtained from driedmaterial were performed using a Hewlett Packard 6890gas chromatograph (Hewlett-Packard Company, Palo Alto,CA) equipped with a flame ionization detector (FID) anda Stabilwax (polyethylene glycol) column (30 m0.32 mminternal diameter, 1-mm film thickness; Centre de Rechercheen Analyses Physico-Chimiques [CRAPC]; USTHB, Algiers,Algeria).

The operating conditions were as follows: injector anddetector temperatures, 250 and 280C, respectively; carriergas, N2 at a flow rate of 1 mL/min; oven temperatureprogram, 3 min isothermal at 50C, raised at 2C/min to 220C,and finally held isothermal for 15 min. The identities of theseparatedcomponents on the polar column were determinedby comparing their retention indices relative to aliphatichydrocarbons injected under the above temperature programwith literature values measured on columns with identicalpolarities.

GC-MS Analysis. The Gas chromatography-mass spec-trometry (GC-MS) analysis was performed using a Hewlett-Packard 6890 series GC systems coupled to a quadrupolemass spectrometer (model HP 5973) equipped with anHP5 MS capillary column (5% phenyl methylsiloxane,

30 m0.25 mm, 0.25-mm film thickness; CRAPC, USTHB).For GC-MS detection, an electron ionization system withionization energy of 70 eV was used over a scan range of30550 atomic mass units. Helium was the carrier gas, at aflow rate of 0.5 mL/min. Injector and detector MS transferline temperatures were set at 250 and 280C, respectively; thetemperature of the ion source was 230C. Column tempera-ture was initially kept at 60C for 8 min, then graduallyincreased to 280C at 2C/min, and finally held isothermal for30 min. The volume of injections was 0.2mL of a hexaneoilsolution, injected in the splitless mode. The identity of thecomponents was assigned by matching their spectral data

with those detailed in the Wiley 7N, National Institute ofStandards and Technology (NIST) 02 and NIST 98 libraries.The results were also confirmed by the comparison of theirretention indices, relative to C7-C29 n-alkanes assayed underGC-MS in the same conditions as the oils. Some structureswere further confirmed by available authentic standardsanalyzed under the same conditions described earlier. Thepercentage composition of the oils was computed by thenormalization method from the GC peak areas, calculatedas the mean value of two injections from each EO.

ANTIMICROBIALSFORC. JEJUNIIN CHICKEN D. DJENANEET AL.

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Antibacterial Activity Assays

Bacterial Strain and Culture Conditions. The bacterialstrain of gram-negativeC. jejuni studied was provided by theSpanish Type Culture Collection. Strain used was C. jejuniCECT 7572. Bacterial strain was grown in Bolton broth

(CM0983) supplemented with 5% laked horse blood(SR0048; BioMrieux, Marcy lEtoile, France) and selectivesupplement(SR0183)at 42C in microaerobic conditions (5%O2,10%CO2 and85%N2). One milliliterof stock culture wasstandardized through two successive 24-h growth cycles at42C in 9 mL of brainheart infusion broth (BHIB; Oxoid,Basingstoke, U.K.). After 48 h, 100mL of the suspension wasthen inoculated in fresh BHIB and incubated at 42C for 12 hto obtain a working fresh culture containing about5105 cfu/mL, determined by measuring transmittance at600 nm (Spectrophotometer: Spectronic 20 Bausch & LombU.K. Ltd., Kingston-Upon-Thames, Surrey, U.K.). The strainwas maintained frozen(-80C)in cryovials (Cryobanks,Mast,Merseyside-Liverpool,U.K.) containing an antifreezing agentto preserve the viability of the cells during storage and weresubcultured every antibacterial test.

ScreeningofEO. ScreeningofEOsforantibacterialactivitywas done by the disk diffusion method. Petri dishes were pre-pared by pouring 20 mL of Mueller Hinton (MH) agarmediumandallowedtosolidify.Platesweredriedfor30 minina biological safety cabinet with vertical laminar flow and0.1 mLof standardizedinoculumssuspensionwas pouredanduniformly extended. The inoculums were allowed to dry for

5 min. To prepare the stock solution of the samples, the pureEOs were dissolved in 5% (v/v) dimethyl sulfoxide (DMSO;Sigma-Aldrich Qumica S.A., Madrid, Spain). Then sterilefilter paper disk (6-mm diameter, Filter LAB ANOIA, testingpaper, Barcelona, Spain) was impregnated with 05mL EO,using a capillary micropipette (Finnpipette, Thermo FischerScientific Inc., Helsinki, Finland). The dishes were left for15 min at room temperature to allow the diffusion of the EO,andthentheywereincubatedat42Cfor2448 hinmicroaero-bic conditions. At the end of the period, the diameter of theclear zone around thedisk wasmeasured witha caliper (WihadialMax, Schonach, Germany, ESD-Uhrmessschieber, CH)

and expressed in millimeters (disk diameter included) as itsantimicrobial activity. The sensitivity to the different EOswasclassifiedbythediameteroftheinhibitionhalosasfollows:not sensitive (-) for diameter less than 8 mm; sensitive (+)for diameter 914 mm; very sensitive (++) for diameter1519 mm and extremely sensitive (+++) for diameter largerthan 20 mm (Ponceet al. 2003). Negative controls were pre-paredusingthesamesolventemployedtodissolvethesamples.Standard reference antibiotic, gentamicin (10mg/disk; SigmaAldrich Qumica S.A.), was used as positive control to

determine the sensitivity of the tested microorganism. Eachassay in thisexperiment was replicated three times.

Microdilution Assays. The MIC values were also studiedforthe targetbacterium, which wasdetermined as sensitivetothe EOs in disk diffusion assay, as described in the earlier

section. The inoculum ofC. jejuniwas prepared from 12-hbroth cultures andsuspension was adjusted to 0.5 McFarlandstandard turbidity to give a final density of 3105 cfu/mL.I. graveolens,L. nobilis,P. lentiscus and S. montana EOs dis-solved in 0.5% DMSO were first diluted to the highest con-centration (32mL/mL) to be tested, and then serial twofolddilutions were made in a concentration range from 32 to0.3125mL/mL in 10-mL sterile test tubes containing MHbroth. MIC values of all EOs against C. jejuni were deter-mined based on a microwell dilution method. The 96-wellplates (Iwaki brand, Asahi Techno Glass, Funabashi, CHB,Japan) were prepared by dispensing into each well 95mL ofMH broth and 5mL of the inoculum. A 100-mL aliquot fromall EOs extracts initially prepared at the concentration of32mL/mL was added into the first wells. Then, 100mL fromtheir serial dilutions was transferred into consecutive wells.The last well containing 195mL of nutrient broth withoutcompound and 5mL of the inoculums on each strip was usedas negative control.The final volume in each well was 200mL.After incubation at 42Cfor 1824 h under microaerobic con-ditions (5% O2, 10% CO2 and 85% N2; Genbox microaer,BioMrieux) in 2.5-L anaerobic jars (Oxoid, Basingstoke,U.K.) with agitation; the wells were then examined for evi-dence of growth and MICs (mL/mL) values were determinedas the lowest EO concentration that inhibited visible growth

of the tested microorganism, which was indicated by absenceof turbidity. The negative control was set up with DMSO inamount corresponding to the highest quantity present in thetest solution (0.5%). The tests were performed in duplicateand repeated twice.

Inhibitory Effect of the Essential Oils against

C. jejuniInoculated in Chicken Meat

Preparation of Chicken Meat. Chicken muscles wereaseptically cut by means of a sterile meat knife from chickencarcasses at about 6 h postslaughter (local supermarket,

Alcampo,Zaragoza, Spain) and transported to the laboratoryunderrefrigeratedconditions within30 min.After the asepticremovalof theouter surface,meatwasasepticallyprepared bymeans of a sterile steel meat preparation.

Treatment of Chicken Meat. Prior to meat inoculationwithC. jejuniand the addition of EOs, chicken muscles werealso examined forany contamination by bacteria or thetestedpathogen (results not shown). To evaluate the antimicrobialactivity of all EOs in a chicken meat system, a sufficient

D. DJENANEET AL. ANTIMICROBIALS FORC. JEJUNI IN CHICKEN



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amount of fresh chicken meat was prepared following goodpractices and was tested using the twofold MICs value foundfor all EOs. After the aseptic removal of the outer surface. Atotal of 80 meat samples wereobtained. Twoindividual dupli-cates of each sample were performed in all cases. For micro-biological study, meat samples (40100 2 g) were placed

individually in stomacher bags and inoculated with strain ofC. jejuniat a level of 3105 cfu/g. The inoculated sampleswere homogenized to ensure proper distribution of thepathogen. Following homogenization, the individual EO wasadded to the inoculated samples. Addition of EO was doneat twofold MIC values (0.20.6%, respectively). To attainuniform distribution of the added compounds, treated meatsamples were further homogenized, as previously described.All stomacher bags with samples from all treatments werewrapped in a pouch made of a polyethylene and polyamidelaminate (Sidlaw Packaging-Soplaril, Barcelona, Spain) ofwater vapor permeability 57 g/m2/24 h at 23C and oxygenpermeability 4050 mL/m2/24 h atm at 23C. The pouch wasfilled with 1.5 L of a gas mixture of 5% O2+10% CO2+85%N2, supplied by Abell-Linde S.A. (Barcelona, Spain), ther-mosealed and stored in the dark at 3 2Cfor 8 days.

Microbial analyses of samples for populations ofC. jejuniwere carried out at 2-day intervals up to the eighth day ofrefrigerated storage. However, for the oxidation study, theremainder (40100 2 g) meat samples were placed intopolystyrene trays. The 40 portions were divided into fivegroups of eight. The first group (control) was sprayed withsterile distilled water, using 1.5 mL of solution to 100 g ofmeat. The final four groups were similarly sprayed respec-tively with each EO solution, respectively. Samples from all

treatments were wrapped and stored under aerobic condi-tions at 3 2Cfor 8 days.

On days 2, 4, 6 and 8 of storage, two packs containing eachsample from each group were opened. One pouch from eachof the set was used for microbiological sampling, while theother two were used for sensory analysis and for chemicalanalyses.

Bacterial Enumeration. Microbiological analyses ofsamples for populations of C. jejuni were carried out at2-day intervals up to the eighth day of refrigerated storage(3 2C). At each sampling time, samples (25 g) of chicken

muscle in the stomacher bags were aseptically added with225 mL of sterile peptone water. The contents were macer-ated in the stomacher (Stomacher 400-Circulator. Seward.Worthing, U.K.) for 1 min at room temperature. Resultingslurries were serially diluted (1:10) in sterile peptone water.Sample dilutions (0.1 mL) were spread plated on appro-priate media in duplicate. The selective media used fornumeration ofC. jejuniwas modified charcoal cefoperazonedeoxycholate agar (mCCDA, Oxoid; CM0739+SR0155).The mCCDA plates were incubated for 48 h at 42C in 2.5-L

gas jars with CampyGen microaerophilic generating gaspacks (BioMrieux). Triplicate sets of plates were preparedon each occasion and each experiment repeated three times.Counts were expressed as the log10of colony forming units(cfu) per gram.

Thiobarbituric Acid Reactive Substances. Lipid oxida-tion was measured in triplicate by the 2-thiobarbituric acid(TBA) method of Pfalzgrafet al. (1995). Meat samples of 10 gwere taken andmixed with 20 mL trichloroacetic acid (10%),using an Ultra-Turrax T25 macevator (Janke & Kunkel,Staufen, Germany).Samples were centrifuged at 2,300g for30 min at 5C; supernatants were filtered through quantitativepaper (MN 640 W, Machinery-Nagel GmbH & Co. KG,Dren, Germany). 2 mL of the filtrate was taken and mixedwith 2 mL of thiobarbituric acid (20 mM); tube contentswere homogenised and incubated at 97C for 20 min inboiling water. Absorbance wasmeasured at 532 nm.The con-

centration of the samples was calculated using a calibrationcurve. TBARS values were expressed as milligrams of mal-onaldehyde per kilogram of sample.

SensoryAnalysis. Samples of chickenmeat were evaluatedfor freshness odor by a sixth-member trained panel.Panelists were selectedamongstudentsandstaff of thedepart-ment and trained according to the American Meat ScienceAssociation guidelines (AMSA 1995). The attribute freshnessodor wasevaluated using a 5-point scale.Odorscores referredto the intensity of freshness odor, inversely associated to meatspoilage: 5=very desirable odor, 4=desirable odor,

3=slightly desirable odor, 2=moderately undesirable odorand1=very undesirableodor (Djenaneet al.2001).

Statistical Analysis. Variance analyses were used to testthe significant difference among the results from the antibac-terial assays, sensory and chemical analysis (SPSS 10.0 soft-ware package, SPSS Inc., Chicago, IL, USA; SPSS 1995).Means and standard errors (SE) of the samples were calcu-lated. Three replicates were performed for each treatment.Differences between means were tested through least squaredifference and values of P< 0.05 were considered signifi-cantly different.

RESULTS AND DISCUSSION

Chemical Composition of the EOs

Steam distillation is the most commonly used method forproducing EOs on a commercial basis. The average values ofhydrodistillation extraction yields of plant EOs from I. gra-veolens and L. nobilis werefoundtobe0.15and0.084%(v/w),respectively. The results obtained in the qualitative and quan-




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titative analysesaccording to their elution orderon a HP5 MScapillary column are shown in Table 1.

From the oil ofI. graveolens, 30 constituents were identi-fied, representing 87.36% (area percent) of the total oil,among which bornyl acetate (40.85%), borneol (21.04%),camphene (8.17%) and 1,8-cineole (2.41%) were the major

compounds. The most abundant chemical category were theoxygenated monoterpenes (65.82%), followed by monoter-pene hydrocarbons (13.22%), oxygenated sesquiterpenes(5.05%) and sesquiterpene hydrocarbons (3.27%).

Forty-seven constituents have been identified from the oilofL. nobilis, representing 87.08% of the total oil, among

TABLE 1. MAIN CONSTITUENTS (%) OF THE ESSENTIAL OILS OF INULA, LAUREL, PISTACIA AND SATUREJA SPECIES, AS IDENTIFIED BY GAS

CHROMATOGRAPHY-MASS SPECTOMETRY ANALYSIS

Retention time (min) Compound Inula graveolens Laurus nobilis Pistacia lentiscus Satureja montana

1 5.177 Tricyclene 0.64

2 5.320 a-Thujene 0.70 0.73

3 5.527 a-Pinene 7.15 5.54 0.79

4 5.932 Camphene 8.17 0.81 3.15 0.515 6.640 Sabinene 8.12

6 6.844 b-Pinene 1.05 3.17 5.10 0.96

7 7.287 Myrcene 0.65 1.04

8 7.335 b-Myrcene 0.80 15.18

9 7.763 a-Phellandrene 3.83

10 8.232 a-Terpinene 0.58 2.78 1.33

11 8.577 p-Cymene 1.03 1.64 11.77

12 8.742 Limonene 0.64

13 8.773 1,8-cineole 2.41 40.25 15.02

14 9.130 trans-b-ocimene 0.92

15 9.544 cis-b-ocimene 1.68

16 9.887 Isoamyle butyrate 0.51

17 10.034 g-terpinene 0.85 4.10 6.72

18 10.392 Trans-4-thujanol 1.0519 11.349 Terpinolene 2.21

20 12.020 Linalool 4.95 1.97

21 14.725 Menthone 0.59

22 15.377 Borneol 21.04 1.75

23 16.063 Terpinen-4-ol 1.87 6.41 1.04

24 16.854 a-Terpineol 1.52 2.16 2.97

25 19.982 Thymol methyl ether 0.95

26 20.877 Farnesol 4.10

27 22.330 Bornyl acetate 40.85 1.88

28 23.330 Carvacrol 29.19

29 23.854 Thymol 15.41

30 24.740 Myrtenyl acetate 0.73

31 26.310 Terpenyl acetate 10.15

32 26.944 Eugenol 2.10

33 28.563 Geranyl acetate 0.53

34 30.215 Caryophyllene 0.71 4.03 5.38

35 32.135 a-caryophyllene 0.84

36 33.802 Germacrene-D 0.87

37 34.759 g-elemene 0.75

38 35.040 a-cadinene 1.27 0.56

39 35.716 b-bisabolene 0.87

40 36.428 d-cadinene 1.80 0.23

41 39.611 Caryophyllene oxide 2.05 0.98

Monoterpenes hydrocarbons 13.22% 25.86% 53.74% 43.76%

Oxygenated monoterpenes 65.82% 49.23% 31.34% 12.54%

Sesquiterpene hydrocarbons 3.27% 11.98% 13.62% 31.63%

Oxygenated sesquiterpenes 5.05% 6.87%

Total identified 84.62% 87.08% 98.7% 94.8%

Only components percentage at >0.5 were represented.




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which 1,8-cineole (40.25%), terpenyl acetate (10.15%), sab-inene (8.12%), a-pinene (7.15%) and linalool (5%) werefound as the most abundant components. Oxygenatedmonoterpenes (49.23%) were the most abundant chemicalcategory, followed by monoterpene hydrocarbons (25.86%)and sesquiterpene hydrocarbons (11.98%).

Fifty-seven constituents were detected from the oil ofP. lentiscus, representing 98.7% of the total oil, among which

b-myrcene (15.18%), 1,8-cineole (15.02%), terpinen-4-ol(6.41%) and a-b-pinene (5.54% and 5.10% respectively)were the major ones. The most abundant chemical categorywere monoterpene hydrocarbons (53.74%), followed by oxy-genated monoterpenes (31.34%) and sesquiterpene hydro-carbons (13.62%).

FromtheoilofS. montana, 44 compounds were identified,representing 94.8%, among which carvacrol (29.19%),thymol (15.41%), p-cymene (11.77%) and g-terpinene(6.72%) were the main constituents. The oil was rich inmonoterpene hydrocarbons (43.76%), followed by the ses-quiterpene hydrocarbons (31.63%), the oxygenated mono-terpenes (12.54%) and the oxygenated sesquiterpenes(6.87%). Our study supports the view that bornyl acetate and1,8-cineole are major components of the EOs ofI. graveolensand L. nobilis, respectively, of Algerian origin.

In agreement with our results, Bokadia et al. (1986) andBarla et al. (2007)foundthat thecharacteristic compounds ofthe genusInulaandLauruswere monoterpenes and sesquit-erpenes. The profile of the volatile oils obtained was also inagreement with values reported from other countries.L. no-bilis oil from Buenos Aires (Argentina) contains sabinene(8%), 1,8-cineole (42.75%),linalool (13.23%) and a-terpinyl(7.6%) (Lira et al. 2009). However, it has been found thatP. lentiscus EO from Greece was characterized by a high

monoterpene hydrocarbon fraction (4568.3%), followed by

oxygenated monoterpenes (13.323.1%) and sesquiterpenehydrocarbons (9.228.1%) (Chryssavgi et al. 2008). Similarfindings have been reported by other authors (Zrira et al.2003). In agreement with our results, previous investigationson the chemical composition of the EO ofSaturejaspeciesindicatedthat it containscarvacrol andthymol as majorcom-ponents (Baser 2002).

The different qualitative and quantitative chemical com-

positions of these EOs with respect to previous investigationscould be related first and foremost to the different environ-mental conditions, genetics (degree of hybridization), geo-graphical origin and harvest period (Slavkovska et al. 2001;Mastelic and Jerkovic 2003).

Antimicrobial Activity (Disk Assay)

According to the results given in Table 2, the oil ofI. graveo-lens showed the largest inhibitory effects against target bacte-ria (F =53 mm), which was followed by that ofL. nobilis(37.3 5.5 mm; P< 0.05), while the lowest were those ofP. lentiscusandS. montana(25.3 1.52 and 25.8 0.2 mm,respectively;P< 0.05). The average zone of inhibition of theantibiotic gentamicin used as a positive control against thesame target bacteria was of only 21 2.6 mm. Those resultswere consistent with those of the microdilution broth assay(Table 3), since the EO ofI. graveolensexhibited a MIC valueof 0.2% and all other three EOs reached only a MIC of 0.6%.

Smith-Palmeret al. (2001) reported similar results regard-ing the effect of oils of bay and thyme onC. jejuni. The sameauthors recorded MICs closeto this valuewhenthey testedtheantimicrobial properties of the EOs of 21 plants and twoessencesagainst C. jejuni.Recently,Nannapaneni et al.(2009)foundthatsevenorangeoilfractionsshowedalargeinhibitory

effect on both C. jejuni and C. coli, exhibiting zones of

TABLE 2. ANTIBACTERIAL ACTIVITY OF THE

EOs FROMINULA GRAVEOLENS, LAURUS

NOBILIS, PISTACIA LENTISCUSANDSATUREJA

MONTANA, USING PAPER DISK DIFFUSION

METHOD, EXPRESSED BY DIAMETER (mm) OF

INHIBITION ZONE (INCLUDING THE DISK

DIAMETER, 6 mm)

f(mm)a*

I. graveolens L. nobilis P. lentiscus S. montana Gentamicine

Campylobacter

jejuniCECT 7572

53.3 9.0a* 37.3 5.5b 25.3 1.52c 25.8 0.2c 21.3 2.6c

All tests were performed in triplicate.

Values followed by the same letter, are not significantly different (P>

0.05).* af: Inhibition zone in diameter around the disks impregnated with essential oils. The diameter

(6 mm) of the disk is included.

TABLE 3. MINIMAL INHIBITORY

CONCENTRATIONS VALUES FROMINULA

GRAVEOLENS, LAURUS NOBILIS, PISTACIA

LENTISCUSANDSATUREJA MONTANA

ESSENTIAL OILSS USING BROTH

MICRODILUTION METHOD

Minimal inhibitory concentration (%)*

I. graveolens L. nobilis P. lentiscus S. montana

Campylobacter

jejuniCECT 7572

0.2 0.02 0.6 0.05 0.6 0.02 0.6 0.04

All tests were performed in triplicate.

* Minimal inhibitory concentrations values expressed by % (v/v).




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inhibition of up to 80 mm. On the other hand, C. jejuniwas inhibited by bergamot (F =23 mm, MIC > 4%),lemon (F =18 mm, MIC > 4%), linalool (F > 90 mm,MIC=0.06%) and linalool vapor (Fisher and Phillips 2006).Manyreports confirmthat the EOsof plant species studied inourworkareamongthemostpotentantimicrobialagents(De

Corato et al.2010;Zhao et al.2010;Djenane et al.2011b,c).Theantibacterial effectof all four EOs may be attributedto

their high content of compounds with known antimicrobialactivity. The phenolic components are chiefly responsible forthe antibacterial properties of EOs, but, aldehydes and alco-hols also exhibit antimicrobial effects (Fitzgeraldet al. 2003).These components are able to disintegrate the outer mem-brane of gram-negative bacteria, releasing lipopolysaccha-rides and increasing the permeability of the cytoplasmicmembrane to adenosine triphosphate (Marino et al. 2001).The strength and spectrum of activity is known to varyaccording to the Gram type of the target bacteria; gram-positivebacteria were generallymore sensitiveto the effects ofthe EOs than gram-negative bacteria (Delaquis et al. 2002).

The antimicrobial activities of the EOs are difficult to cor-relate to a specific compound due to their complexity andvariability. Synergism has been observed between variouscomponents of the same EO (Burt 2004). Some studies haveconcluded that whole EOs have a greater antibacterial activitythan the major components individually (Gill et al. 2002;Mourey and Canillac 2002). Nevertheless, some researchersreported that there is a relationship between the chemicalcomposition of the most abundant components in the EOand the antimicrobial activity. For example, 1,8-cineole(abundant in Algerian L. nobilis EO tested in this study) is

well known for its antimicrobial potential (Pattnaik et al.1997). Lis-Balchin and Deans (1997) showed that EOs con-taining large amounts of 1,8-cineole are better antilisterialagents than EOs that do not contain 1,8-cineole. The antimi-crobial effects of borneol (abundant in tested AlgerianI. gra-veolens EO)werealso reportedby Dorman and Deans (2000).

As a result of these findings,the higher antimicrobial activ-ity ofI. graveolensEO could be attributed to this particularchemotype, characterized by its complexity, with bornylacetate, borneol and camphene being the most abundant,which have a well-documented antibacterial and antifungalpotential (Marino et al. 2001; Holley and Patel 2005).

It has been indicated that the results of the disk diffusionmethod show a highly significant correlation with MICvalues (Okeet al. 2009). However, another report found thatthe MIC values of EOs on foodborne pathogens were lowlycorrelated with disk diffusion results (Djenaneet al. 2011a).This can be explained by the fact that the sensitivity dependson the type of bacteria, the type, composition and concen-tration of the EO, the solubility in media, and seasonal andintraspecific compositional variations. Generally, EOs pro-duced from herbs harvested during or immediately after

flowering possess the strongest antimicrobial activity(Marinoet al. 1999).

Antimicrobial Activity in Chicken Meat

Figure 1 depictsC. jejunigrowth inhibition in chicken meat

by the four tested EOs, applied at twofold MIC values, com-pared with the control.It appeared to be evident that, despitesome significant differences among them in a first phase ofbacterial inactivation, all EOs reached similar inhibitionactivities after 4 days of storage. The initial population of 5.6log10cfu/g ofC. jejuniincreased to 8.14 log10cfu/g by the endof storage in untreated samples. A reduction of 3.08, 3.50,3.08 and 2.08 log10cfu/g was recorded in 4 days of storage,respectivelybyI. graveolens, L. nobilis, P. lentiscus and S. mon-tana. Four days later (at day 8), reductions of 6.94, 6.14, 5.94and 5.94 log10cfu/g, were reached respectively byI. graveo-lens,L. nobilis,P. lentiscus and S. montana. As far as we are

aware,the antibacterial effect of these EOs against C. jejuni inchicken meat had not yet been reported.These results for the inhibition of C. jejuni in a meat

system were in good agreement with prior in vitro results,as well as with the MIC calculated values. However, theexpected higher effect ofI. graveolens was not confirmed.Several studies have reported the effect of a food matrix onmicrobial resistance to EOs, but no one of them quantifiedit nor explained the mechanism, although some suggestionshave been made.

Little research has been carried out on the effect ofInula,Laurus,Pistaciaand SaturejaEOs againstC. jejuni. Further

FIG. 1. SURVIVAL CURVES OFCAMPYLOBACTER JEJUNIBYINULA

GRAVEOLENS,LAURUS NOBILIS,PISTACIA LENTISCUSANDSATUREJA

MONTANAESSENTIAL OILS AT TWOFOLD MINIMUM INHIBITORY

CONCENTRATION VALUES IN CHICKEN MEAT STORED AT 3 2C

UNDER MICROAEROBIC CONDITIONS

() Control; ()S. montana; ()P. lentiscus; ( )I. graveolens; ()

L. nobilis. The error bars represent standard deviation.




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reviews of existing literature revealed that other herbs werealso effective in inhibitingC. jejuni. Lee et al. (2004) foundthat an aqueous leek extract wasvery effective against Campy-lobacterspecies. Previous studies using other EOs have shownantibacterial activity (Lis-Balchin andDeans 1997;Zhao et al.2010).

The effect of combining these EOs remains to be eluci-dated. Nevertheless, Djenaneet al. (2011c) reported that thecombination ofP. lentiscus and S. montana at low concentra-

tions (0.03%) exhibited a higher activity against Listeriamonocytogenes than individual EOs applied at higherconcen-trations (0.06%). Synergism between EOs and other param-eters in antimicrobial action must be therefore considered.Thus, further research is needed to evaluate the effectivenessof combined EOs in this and other food systems, as well as byusing active packaging (Camo et al. 2008), in order to assesstheir performance as natural antimicrobial agents in foodpreservation and safety.

TBA Reactive Substances andSensory Analysis

Figure 2 shows the results for TBARS indices during storageof chicken meat under microaerobic conditions. Lipid oxida-tion increased slightly with increasing time in all samples

until the fourth day of storage. Significant differences(P< 0.05) were evident among samples thereafter, dependingon the presence or not of the EOs. Untreated samples ofchicken meat showed thehighest valuesof TBARS(P< 0.05);they were well above 4 at day 8 of storage. On the other hand,samples treated with any of the EOs had significantly lowervalues(P< 0.05); they did not reach 1 mg malonaldehyde/kg,even after 8 days of storage.Martnez et al.(2006)foundthataTBARS index of 1.5 mg/kgis closely relatedto perceptible andunacceptable off-odor of meat by trained panel. Snchez-Escalante et al. (2003)and Djenane et al. (2002) reportedthatbeef patties and beef steaks, respectively, treated with rose-mary exhibited lower TBARS values and metmyoglobin

percentage than untreated samples during storage.The protective effect of meat against lipid oxidation by

antioxidant treatment had been already reported by Djenaneet al. (2003) and Camoet al. (2011). The antioxidant activityof EOs may be ascribed to the same chemical componentsthan those found in antioxidants. The polyphenols found inthe EOs ofI. graveolens, L. nobilis, P. lentiscus and S. montanamay act as radical scavenging agents (Sharififar et al. 2007;Tepe et al. 2007).

With a view to assess the possible effect of added EOs onthe sensory properties of meat, a sensory evaluation of meatodor was carried out throughout a storage period of 8 days.

Sensory scores for freshness odor are summarized in Table 4.Results showed that fresh meat odor intensity decreasedthroughout storage in all samples, though not at the samerate. Untreatedsamples were given scores below 3 by the pan-elists after 6 days of storage,whereas samples treated with anyof the EOs were given scores above 3, even at the end of thestorage period. Thus, any of the EOs significantly (P< 0.05)extended fresh chicken odor, reaching a maximum of 3.5,which may be considered as acceptable,after8 days of storage.

FIG. 2. TBARS (MILLIGRAMS OF MALONALDEHYDE PER KILOGRAM OF

MEAT) IN CHICKEN TREATED WITH ESSENTIAL OILS AND STORED AT

3 2C UNDER AEROBIC CONDITIONS

() Control; ()S. montana; ()P. lentiscus; ( )I. graveolens; ()

L. nobilis

The error bars represent standard deviation.

TABLE 4. EFFECT OF EOs ON FRESHNESS ODOR SENSORY SCORES (MEAN STANDARD DEVIATION) OF CHICKEN MEAT STORED AT 3 2C

Treatment

Days of storage

0 2 4 6 8

Freshness odors*

Control 5.00 00a 4.83 0.4a 3.66 0.5a 2.83 0.4a 1.50 0.5a

Inula graveolens(0.40%) 5.00 00a 5.00 00a 4.66 0.5b 4.00 00b 3.00 00b

Laurus nobilis(1.2%) 5.00 00a 5.00 00a 4.50 0.5b 3.66 0.8bc 3.00 0.6b

Pistacia lentiscus(1.2%) 5.00 00a 4.83 0.4a 4.83 0.4b 4.00 0.5b 3.50 0.5b

Satureja montana(1.2%) 5.00 00a 5.00 0.3a 4.66 0.5b 3.33 0.5bc 3.50 0.5b

Mean values in the same column and relating to freshness odor are significantly different when accompanied by different letters (P< 0.05).

* 5= Very desirable odor; 4= Desirable odor; 3 = Slightly desirable odor; 2 = Moderately undesirable odor; and 1= Very undesirable odor.




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It may, therefore, be concluded that from a practical point ofview, the use of EOs for chicken preservation is clearly advan-tageous since besides contributing to shelf-life extension; itcontributes to maintaining a pleasant fresh meat odor.

CONCLUSION

Our data support the possible use ofI. graveolens,L. nobilis,P. lentiscus and S. montana EOs, particularly that fromI. gra-veolens, for the preservation of chicken meat. By using thismethod, chicken meat can be stored in a modified atmo-sphere assuring a low risk associated toCampylobacter, at thesame time that lipid oxidation is inhibited, giving rise to ahigher sensory quality.

ACKNOWLEDGMENTS

The authors are grateful to Ministerio de Asuntos Exterioresy Cooperacin of Spain (AECID) and Ministre delEnseignement Suprieur et de la Recherche Scientifique ofAlgeria for financial assistances to this work within the Pro-grama de Cooperacin Interuniversitaria e InvestigacinCientfica PCI/MED Algeria-Spain (grant ALI A/011170/07;A/019342/08; A/023365/09; A/033506/10) and CNEPRU(F00520090025),respectively.

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