Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens

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Food

Microbiology

Antimicrobial

activities

of

six

essential

oils

commonly

used

as

condiments

in

Brazil

against

Clostridium

perfringens

Marcela

Radaelli

_,

_Bárbara

_Parraga

_da

_Silva

_,

_Luciana

_Weidlich

_,

_Lucélia

_Hoehne

_,

Adriana

Flach

_,

_Luiz

_Antonio

_Mendonc¸a

_Alves

_da

_Costa

_,

_Eduardo

_Miranda

_Ethur

a_{CentrodeCiênciasBiológicasedaSaúde,CentroUniversitárioUNIVATESLajeado,RS,Brazil} b_{CentrodeCiênciasExataseTecnológicas,CentroUniversitárioUNIVATES,Lajeado,RS,Brazil} c_{DepartamentodeQuímica,UniversidadeFederaldeRoraimaBoaVista,RR,Brazil}

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r

t

i

c

l

e

i

n

f

o

Received12August2014 Accepted2October2015 Availableonline2March2016 AssociateEditor:MarizaLandgraf

Keywords: Food-bornedisease Antimicrobialactivity Clostridiumperfringens Spices Essentialoils

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Despiterecentadvancesinfoodproductiontechnology,food-bornediseases(FBD)remain achallengingpublichealthconcern.Inseveralcountries,includingBrazil,Clostridium per-fringensisamongthefivemaincausativeagentsoffood-bornediseases.Thepresentstudy determinesantimicrobialactivitiesofessentialoilsofsixcondimentscommonlyusedin Brazil,viz.,OcimumbasilicumL.(basil),RosmarinusofficinalisL.(rosemary),Origanummajorana

L.(marjoram),Mentha×piperitaL.var.Piperita(peppermint),ThymusvulgarisL.(thyme)and

PimpinellaanisumL.(anise)againstC.perfringensstrainA.Chemicalcompositionsoftheoils weredeterminedbyGC–MS(gaschromatography–massspectrometry).Theidentitiesofthe isolatedcompoundswereestablishedfromtherespectiveKovátsindices,anda compari-sonofmassspectraldatawasmadewiththosereportedearlier.Theantibacterialactivity wasassessed fromminimum inhibitoryconcentration(MIC)andminimumbactericidal concentration(MBC)usingthemicrodilutionmethod.Minimuminhibitoryconcentration valueswere1.25mgmL−1_{forthyme,5.0mgmL}−1_{forbasilandmarjoram,and10mgmL}−1

forrosemary,peppermintandanise.Alloilsshowedbactericidalactivityattheirminimum inhibitoryconcentration,exceptaniseoil,whichwasonlybacteriostatic.Theuseof essen-tialoilsfromthesecommonspicesmightserveasanalternativetotheuseofchemical preservativesinthecontrolandinactivationofpathogensincommerciallyproducedfood systems.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:eduardome@univates.br(E.M.Ethur).

http://dx.doi.org/10.1016/j.bjm.2015.10.001

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Food-bornedisease(FBD)ischaracterizedbydiverse symp-tomsthatarisefromconsumptionofcontaminatedfoodsor beverages. Despitethe recent advances infood production technologyandprocessing,FBDremainsamajorcauseof mor-bidityandmortality, constitutingbothan importantpublic healthconcernandasignificanteconomicproblemataglobal level.1–4

One of the most common causes of FBD is the Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens(familyClostridiaceae),whichiswidelydistributed intheenvironmentandinfoodstuffs.Thesporesofthis bac-terium,comparedtothevegetativecells,areveryrobustand resistanttoheating,drying,pHandcertaintoxiccompounds. This allows the microorganism to persist until conditions become favorable for germination and growth. There are five strain-types of C. perfringens, designated as A–E. Each ofthem producesaunique spectrum ofexotoxins.Type A strainsofthebacteriumcausefoodpoisoninginthe classi-calform,whilethestrainsoftypeCcausenecroticenteritis –a disorder that can besevere and isoften fatalbut, for-tunately, its occurrenceisrare. Themicroorganism prefers substrates,suchasmeatproducts,poultry,andsauces,which contain high level of moisture and rich in protein. These are the main classes of food materials involved in occur-renceofthis disease.Specificfactors thatfavorthe spread ofthisagentareprolongedcoolingandnon-refrigerated stor-age,whereinsausage,cannedfish,pate,cheeseandfermented oysterprovide idealconditions forthe developmentofthe bacterium.1,2,5

Many of the spices commonly used in food not only improvepalatabilityandflavorbutalsoassistinthe preser-vationofthe fooditself.Theantimicrobialactivity ofsuch spices is bestowed mainly by the essential oils that con-tainterpenoidshydrocarbons,alcohols,aldehydes,ketones, phenolics and their derivatives. The quantity, quality, and chemicalprofilesoftheessentialoilsderivedfrom asingle plant species can vary considerably according to the geo-graphicorigin,climaticconditions,soilcomposition,thepart oftheplantused,andthe ageandseasonwhenthe mate-rial is collected. Besides, drying process and storage time canalter, quantitatively, and qualitatively, the essential oil composition.6_{Whiletheantimicrobialactivitiesofessential}

oilsarewellreported,themechanismsoftheiractionarenot yetfullyunderstood,althoughtheremightbeseveraldifferent microbialtargetsites.7,8

Inthepresentstudy,theessentialoilsofsomeofthespices mostcommonlyusedinBrazil,namely,OcimumbasilicumL. (basil),RosmarinusofficinalisL.(rosemary),Origanummajorana

L. (marjoram), Mentha × piperita L. var. Piperita (pepper-mint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise),wereassessedwithregardtotheirantimicrobial activ-itiesagainstC.perfringensstrainA.Thecriteriaforselection of plants were their popular use as spices, availability to commonpeople,andensuringthatorganicproduction meth-ods have been used in accordance with the Law: 10,831.9

Thechemicalcompositionsoftheessentialoilswere deter-minedbyGC–MS(gaschromatography–massspectrometry),

andminimuminhibitoryandbactericidalconcentrationswere determinedusingthemicrodilutionmethod.

Materials

and

methods

Plantmaterials

Dried,fragmentedleavesofbasil,rosemary,marjoram, pep-permint,andthymeanddriedfruitsofanisewerepurchased commerciallyinSãoPaulo,Brazil,inOctober2013.All sam-ples wereacquired inaccordance withthe termsofexpiry mentionedonthelabels.

Extractionofessentialoils

Dried leavesofbasil,marjoram,peppermint, rosemary and thyme, andthe driedfruitsofanisewere powdered (10–20 mesh) and samples were extracted by hydro-distillation (plant:waterratio1:10,w/v)for3.5hinamodifiedClevenger apparatus.Theoilyphasewasremoved,driedoveranhydrous sodiumsulfate,andstoredinafreezerat–20◦C. Microbiolog-icalanalyseswereperformedsevendaysaftertheextraction.

Chemicalcompositionsofessentialoils

Essential oil samples were submitted for GC–MS analysis totheLaboratoryofChromatography,EnergyResearch Cen-ter, Federal University of Roraima, Boa Vista, RR, Brazil. Theanalyseswere performedusingShimadzu GC2010 sys-tem withan autoinjector AOC-20i and Plus massdetector QP2110, and equipped with an HP5-MS fused silica capil-larycolumn(30m×0.25mm×0.25␮m).Thechromatographic conditionswereasfollows:carriergas,heliumataflowrate of1.02mLmin–1_{;oventemperatureprogrammedinitiallyat}

60◦Candincreasedto310◦Catarampof3◦Cmin–1_;injector

temperature,220◦C;injectormodeinsplitratioof1:20with 2mLmin–1_{purge;MSinterfacetemperature,280}◦_C;ionsource

temperature,260◦C;andionizationenergy,70eV.Theoil sam-ples(15mg)weredissolvedin1.5mLofpurifiedethylacetate and1␮Lvolumeofthatwasinjectedforanalysis.Theisolated compoundswereidentifiedbytheirrespectiveKováts reten-tionindicesdeterminedinreferencetoaseriesofn-alkanes,

andverifiedbyacomparisonofmassspectraldatawiththose obtainedusingpurestandardsandwiththosereportedinthe literature,10 _{and eventually by comparingtheir mass}

spec-tra withthe GC–MSspectrallibrary(Wiley8andFFNSC1.2 libraries).

ForGC-FID,HP-5MScolumn(30m×0.25×0.25␮m)atsame temperatureasthatofGC–MS,usinghydrogenandnitrogen carriergas,wasused.TheFIDtemperaturewas260◦C.The relativecompositionsoftheoilswerecalculatedfromthepeak areas(uncorrectedforspecificresponsefactors)oftheisolated compounds.

Antimicrobialactivitiesofessentialoils

Theminimum inhibitoryconcentration(MIC) andthe MBC of the oil samples were assessedagainst type A strain of

C.perfringensATCC13124(agasgangreneisolate)usingthe microdilutionmethod.

Standardizationofinoculum

C.perfringens(ATCC13124)wasrevivedaccordingtostandard proceduresintryptosesulfite-cycloserineagar(Oxoid)with d-cycloserine(Sigma)underanaerobicconditionsat36±1◦Cfor 24h.11_{Thebacterialconcentrationintheinoculumwas}

stan-dardizedat0.5ontheMcFarlandturbidityscale,equivalentto 108_CFUmL–1_{.Analiquot(1mL)ofthissuspensionwas}

trans-ferredtoasteriletubeandthevolumewasadjustedto10mL withsodiumchloridesolution(0.8%,w/v)toobtaina concen-trationof107_CFUmL–1_{.Workinginoculumswerepreparedby}

transferring200␮Laliquots ofthissuspensiontothreetest tubes and adjusting the volumes to 10mL withreinforced clostridialmedium(RCM;Oxoid)togivefinalconcentrations of2.0×105_CFUmL–1_.12

Minimalinhibitoryconcentrations

Allmicrobiological assayswere performedunderanaerobic conditions. MIC determinationswere performedin 96-well microplates accordingtoprocedures describedbythe Clin-icaland LaboratoryStandardsInstitute.13 _{Eachessentialoil}

(200mg)wasdissolvedindimethylsulfoxide(40␮L)andthe volumewas madeto 5mLwith sterileRCMcontaining 1% Tween80toprovideastocksolutioncontaining40mgmL–1

of oil. Serial twofold dilutions of each essential oil stock were made with RCM to yield final concentrations ran-gingfrom20to0.625mgmL–1_{.Thedilutedsamples(100␮L)}

were transferred to microplate wells and mixed well with themicropipette.Chloramphenicolwasemployedasa pos-itivecontrolintheconcentrationrange0.1–0.003125mgmL–1_,

while the negative controls comprised sterile RCM either alone or with dimethyl sulfoxide (at concentrations used in the dilutions). In order to ascertain aseptic condi-tions,the controlwells contained sterileRCM but without inoculum. The inoculated microplates were incubated at 36±1◦C for 48hunder anaerobic conditions; and the bac-terial growth was confirmed by adding 10␮L of a sterile 0.5%aqueoussolutionoftriphenyltetrazoliumchloride(TTC, Sigma–Aldrich) and incubating at 36◦C for 30min.14 _The

viable bacterial cells reduced the yellow TTC to pink/red 1,3,5-triphenylformazan(TPF).Allassayswereperformedin triplicate.

Minimumbactericidalconcentrations

MBCs were determined by inoculating the assay mixtures from the wells showingnomicrobial growth onto the sur-faceofsterileShahidi-FergusonPerfringensagarmediumas recommended bythe Ministério da Agricultura,Pecuária e Abastecimento.15 _{Theplates wereincubated under}

anaero-bic conditions for24h in an oven maintained at 36±1◦C and subjectedtovisual inspection.Thepresenceof micro-bial growth on the medium indicated that the essential oil possessed bacteriostatic activity, while the absence of the growth implied bactericidal activity of the oil sam-ple.

Results

and

discussion

Yieldsandchemicalprofilesoftheessentialoils

Theyieldsoftheessentialoilswere0.24%forbasil,1.57%for rosemary,0.47%formarjoramoil,0.49%forpeppermint,0.97% forthymeand1.29%foranise.

AsshowninTable1,themostabundantcompoundsinall oftheessentialoilswereoxygenatedcompounds,especially oxygenatedmonoterpenesandphenylpropanoids.The com-bination ofoxygenated monoterpenes and sesquiterpenes, phenylpropanoidsandalcoholsaccounted82.17%ofthe oxy-genatedcompoundsforO.basilicum,96.56%forR.officinalis,

69.39%forO.majorana,96.05%forMenta×piperita,92.96%for

T.vulgarisand97.87%forP.anisum.

Intheanalyzedoilsamples,mostofthecompoundswere identified unambiguouslyfrom the Kováts indexand mass spectral data(Table1). For basil oil, themajor compounds were linalool, methyl chavicol and 1,8-cineole, with trace amountsof␣-trans-bergamoteneandepi-␣-cadinol.Hussain, Anawar, Sherazi,and Przybylskianalyzedthe essentialoils fromtheaerialpartsofO.basilicumharvestedindifferent sea-sonsandfoundsignificantvariationsinthemajorcompounds includinglinalool(56.70–60.60%),␣-bergamotene(7.60–9.20%), ␥-cadinene (3.20–5.40%) and epi-␣-cadinol (8.60–12.40%).16

Linalool was reported asthe major component(66.40%) of theoilextractedfromoneofthethreebotanicalvarietiesand cultivarsofO.basilicum,whiletheothertwocontained ␣-trans-bergamotene(6.84–7.96%).17

Rosemaryoilcontainstwomajorcompounds,1,8-cineole andcamphor,togetherwithborneoland␣-terpineolinmuch smaller amounts. In a recent evaluation of the antimicro-bial activityofR.officinalis,Jiangetal.identified1,8-cineole (26.54%), ␣-pinene (20.14%), camphor (12.88%), camphene (11.38%) and ␤-pinene (6.95%) asthe mainconstituents of the essential oil. In marjoram oil, the major components were terpinen-4-ol,trans-sabinenehydrate,␥-terpinene and ␣-terpineol.18_{Busattaetal.evaluatedtheantimicrobial}

activ-ityoftheessentialoilfromdriedleavesofO.majoranausedin aprocessedfood,andfoundmainconstituentsas terpinen-4-ol(30.41%),␥-terpinene(13.94%),cis-sabinenehydrate(9.64%), and␣-terpineol(4.47%).19

In peppermint oil, the major constituents are menthol and carvonewithminuteamountsofmenthoneand men-thylacetate.TyagiandMalikreportedthemainconstituents ofM.piperitaoilasmenthol(19.10%),iso-menthone(14.80%), menthone(14.80%),limonene(10.60%),iso-menthol(8.80%), menthyl acetate (6.60%), ␤-pinene (5.60%) and ␣-pinene (4.80%).20 _{The essential oil ofthyme analyzed herein}

con-tained borneol and ␣-terpineol as the major compounds, together withtraceamountsofthymolandcarvacrol.Rota, Herrera, Martinez, Sotomayor, and Jordan determined the chemicalcompositionandantimicrobialactivityofthe essen-tial oilofT.vulgaris(thymolchemotype) andidentified the mainconstituentsasthymol(57.70%),p-cymene(18.70%)and carvacrol(2.80%).21

Theoilderivedfrom the driedfruitsofanisecontained almost entirely (95.59%)E-anethole. Tepeet al.studied the antioxidative andantimicrobialactivitiesofP. anisetumand

Table1–Chemicalcompositionoftheessentialoilsobtainedfromthespices. Compoundsa _{Kovátsindex Ocimum}

basilicum (area%)d Rosmarinus officinalis (area%) Origanum majorana (area%) Mentha× piperita(area%) Thymusvulgaris (area%) Pimpinella anisum(area%) KIexpb KIlitc Sabinene 981 975 3.14 ␤-Pinene 984 979 0.56 0.44 0.23 0.43 0.19 Myrcene 993 991 0.42 0.55 3-Octanol 997 991 0.18 ␣-Phellandrene 1007 1003 0.19 ␣-Terpinene 1017 1017 0.11 4.84 p-Cymene 1024 1025 1.39 1.56 0.93 ␤-Phellandrene 1029 1030 1.51 Limonene 1029 1029 0.32 0.25 1,8-Cineole 1031 1031 9.55 57.39 0.19 2.13 ␥-Terpinene 1059 1060 8.27 0.41 cis-Sabinene hydrate 1067 1070 2.62 Terpinolene 1089 1089 1.56 trans-Sabinene hydrate 1100 1098 10.14 Tetrahydrolinalool 1101 1099 0.83 Linalool 1104 1099 42.10 2.33 trans-Pinene hydrate 1122 1123 2.52 cis-Pinenehydrate 1140 1144 1.64 Camphor 1144 1146 0.59 27.94 0.23 Camphenehydrate 1149 1150 0.12 Menthone 1154 1153 6.22 Iso-menthone 1164 1163 1.90 Borneol 1166 1169 0.55 5.74 47.59 Neo-menthol 1166 1166 3.16 Menthol 1178 1172 35.33 Terpinen-4-ol 1178 1177 0.65 0.82 40.85 0.85 1.48 Iso-menthol 1185 1182 0.30 ␣-Terpineol 1192 1189 1.19 3.99 7.25 0.36 25.04 cis-Piperitol 1197 1196 0.66 Dihydrocarveol 1198 1194 2.19 cis-Dihydrocarvone 1199 1193 3.17 Safranal 1199 1197 1.50 Methylchavicol 1201 1196 11.85 trans-Dihydrocarvone 1205 1201 0.66 0.24 trans-Piperitol 1209 1208 0.80 trans-Carveol 1221 1217 0.18 cis-Carveol 1234 1229 0.40 Pulegone 1240 1237 2.60 Carvone 1244 1243 0.26 28.39 Carvacrolmethyl ether 1245 1245 1.10 trans-Sabinene hydrateacetate 1253 1256 0.32 Z-Anethole 1254 1253 0.07 Piperitone 1255 1253 0.61 Linalylacetate 1256 1257 1.56 2-Phenylethyl acetate 1256 1258 0.71 Neo-menthyl acetate 1276 1274 0.25 Isobornylacetate 1286 1286 0.81 0.56 2.19 E-Anethole 1287 1285 1.04 95.59 Menthylacetate 1295 1295 4.46 Thymol 1296 1290 5.31 Carvacrol 1306 1298 5.52 Z-Methyl cinnamate 1306 1300 0.50

Table1–(Continued)

Compoundsa _{Kovátsindex Ocimum}

basilicum (area%)d Rosmarinus officinalis (area%) Origanum majorana (area%) Mentha× piperita(area%) Thymusvulgaris (area%) Pimpinella anisum(area%) KIexpb KIlitc Eugenol 1359 1359 0.60 E-Methyl cinnamate 1384 1379 2.00 ␤-Boubornene 1385 1388 0.37 ␤-Elemene 1393 1391 0.59 Methyleugenol 1407 1404 3.19 E-Caryophyllene 1420 1419 0.97 0.25 4.70 1.21 3.66 ␣-trans-Bergamotene 1437 1435 6.89 Aromadendrene 1440 1441 0.39 ␣-Humulene 1454 1455 0.62 ␤-Chamigrene 1478 1478 0.20 Bicyclogermacrene 1497 1494 2.18 ␣-Bulnesene 1506 1510 0.44 GermacreneD 1515 1485 0.30 ␣-Cadinene 1515 1514 2.89 ␤-Cadinene 1525 1523 0.25 Spathulenol 1578 1578 0.61 0.84 0.62 Caryophyllene oxide 1584 1583 1.12 Viridiflorol 1592 1590 0.88 ␣-Cadinol 1656 1654 0.17 epi-␣-Cadinol 1663 1640 6.89 0.81 Groupedcomponents(%) Monoterpene hydrocarbons 0.56 2.36 21.85 0.75 1.78 Oxygenated monoterpenes 56.53 96.56 68.55 93.16 91.03 1.50 Sesquiterpene hydrocarbons 12.79 0.25 6.88 1.58 4.21 0.20 Oxygenated sesquiterpenes 7.50 0.84 1.67 1.93 Alcohols 0.18 Phenyl-propanoids 18.14 1.04 96.37 Total hydrocarbons compounds 13.35 2.61 28.73 2.33 5.99 0.20 Totaloxygenated compounds 82.17 96.56 69.39 96.05 92.96 97.87 Total 95.52 99.17 98.12 98.38 98.95 98.07

a _{AllcompoundswereidentifiedbyKovátsindexandmassspectraldatabycomparingvaluesreporterintheliterature.}

b _{Kovátsindexexperimental.}

c _{Kovátsindexliterature.}10

d_{Relativeproportionsaspercentageofthetotalpeakarea.}

found E-anethole as the main constituent, accounting for 82.80%ofthetotaloil.22

Thedifferencesbetweentheessentialoilprofilesreported hereinandthoseinotherstudiescanbeattributedtotheuse ofcommerciallyavailabledriedplantmaterialsratherthan thefreshplantparts.

Antimicrobialactivitiesofessentialoils

Incubationoftheassayplates for48hwassufficientforall negative controls to show significant microbial growth (in the form of well-dispersed colonies),while the wells with positive control (chloramphenicol) and the system control (RCMwithoutinoculum)remainedclearwithnoobservable

colonyformation.Thepresenceofviablemicroorganismsin microplatewellsviaindicationofcolonyformationwas veri-fiedbyreductionofyellowTTCtopinkTPF;thiscolorchange wasnotobservedinthewellswithoutanymicrobialgrowth.In thisway,theexactconcentrationofeachoftheessentialoils abletoinhibitthegrowthofC.perfringenswasdetermined.The valuesobtainedforMICandMBCareshowninTable2.

TheessentialoilfromleavesofT.vulgarisshowedthe low-estMICandMBCvalues(1.25mgmL–1_{)againstC.perfringens,}

sincebothvaluesaresimilar,theoilmustbeconsideredto possessstrongbactericidalactivity.Al-BayatireportedanMIC valueof0.5mgmL–1_{forthymeoilagainstKlebsiellapneumoniae,}

0.25mgmL–1againstSalmonellatyphi,0.125mgmL–1againstS. typhimurium,0.625mgmL–1_{againstEscherichiacoliandProteus}

Table2–Minimalinhibitoryconcentration(MIC)and minimalbactericideconcentration(MBC)ofessentialoils againstClostridiumperfringens.a

Essentialoils MIC(mgmL–1_{) MBC(mgmL}–1₎

Rosmarinusofficinalis 10.0 10.0 Mentha×piperita 10.0 10.0 Origanummajorana 5.0 5.0 Ocimumbasilicum 5.0 5.0 Thymusvulgaris 1.25 1.25 Pimpinellaanisum 10.0 20.0

a _{Chloramphenicolwasemployedaspositivecontrol.TheMBCto}

chloramphenicolwas0.003125mgmL–1_{inallexperiments.}

mirabilis,and0.312mgmL–1_{againstStaphylococcusaureusand}

P.vulgaris.23

TheessentialoilsfromleavesofO.basilicumandO.majorana

showedMICandMBCvaluesof5.0mgmL−1againstC. perfrin-gens,indicatingthattheseoilspossessbactericidalproperties. Lv,Liang,Yuan,andLideterminedMICvaluesof1.25mgmL–1

forbasil oilagainstE.coliand S.aureus,and 0.625mgmL–1

againstBacillussubtilis.24_{Inastudyonmarjoramoil,Busatta}

etal.determinedMICvaluesof2.3mgmL–1_against Enterococ-cusfaecalis,Serratiasp.andStreptococcusmutans,0.92mgmL–1

againstAeromonas sp.,E. coli, K. pneumoniaeand Salmonella choleraesuis,0.782mgmL–1_{againstShigellaflexneriand} Staphy-lococcusaureus,and0.069mgmL–1_{againstB.subtilis.}19

TheMICandMBCvaluesforR.officinalisandM.piperitaoils againstC.perfringens wereconsiderably higher(10mgmL–1₎

than the other samples tested suggesting that these oils possess much weaker bactericidal activities. In the case ofrosemary oilextracted from fresh plantmaterial, Okoh, Sadimenko, and Afolayan reported anMIC of 3.75mgmL–1 and an MBC of 7.5mgmL–1 _{against S. aureus, an MIC of}

7.5mgmL–1_{andanMBC>7.5mgmL}–1 _{againstE.coli,andan}

MIC of1.88mgmL–1 _{and an MBCof 7.5mgmL}–1 _againstB. subtilis.25_{Forpeppermintoil,TyagiandMalikfoundanMICof}

1.13mgmL–1_{andanMBCof4.5mgmL}−1_{againstE.coli,anMIC}

valueof2.25mgmL–1_{andanMBCvalueof9.0mgmL}–1_against Pseudomonasaeruginosaand P.fluorescens,and anMIC value of1.13mgmL–1_{andanMBCvalueof2.25mgmL}–1_againstB. subtilisandS.aureus.20

Forthe essentialoilfromfruitsofPimpinellaanisum,the MBC value (20mgmL–1_{) was twofold higher than the MIC}

value indicating only bacteriostaticactivity of the oil at a concentrationof10mgmL–1_{. Al-Bayatireported MIC values}

of 0.125mgmL–1 _{for anise oil against S. aureus and} Pro-teus mirabilis, 0.25mgmL–1 _{against Salmonella typhimurium,}

0.5mgmL–1 _{againstS.typhiandvalues>0.5mgmL}–1 _against K.pneumoniae,PseudomonasaeruginosaandE.coli.23

Although the mechanisms associated with the antimi-crobialactivities ofessential oils are not fully understood; numerous modes of action have been proposed involv-ing, for example, degradation of the bacterial cell wall, modificationofproteinsofthecytoplasmicmembrane, alter-ationofmembranepermeability,inactivationofextracellular enzymes,reductionofintracellular ATP,leakage ofcellular contents,coagulationofcytoplasm,andinterruptionof elec-tronflow and active transport.7,8,26 _{However, somestudies}

have reported the specific mechanism of action for some

oil constituents. Thymol and carvacrol are believed to act byincreasingthepermeability ofcellmembranes.27 _Inthis

context,Ultee,Bennik,andMoezelaarshowedthatcarvacrol accumulatesinthelipidphaseofthemembranebychanging theconformationofthephospholipidbilayer,whichcauses expansion of the membrane and leakage of ions, thereby increasingmembranefluidityandpermeability.28_p-Cymene

alsoaccumulatesinlargeamountsandactsbycausing expan-sion of the membrane phospholipids by increasing spaces throughwhichionleakagemightoccur.28_Inthecaseof

car-vone,thecompoundisbelievedtobepartitionedinthelipid membrane,therebydisturbingselectivebarrierfunctionand theconservationofmetabolicenergy.29_{Terpinen-4-ol,onthe}

other hand,has been shown toinhibit cellular respiration and todamagethe structureofthe cellmembrane attenu-atingitsroleasapermeablebarrier.30_{However,ingeneral,it}

isbelievedthattheantimicrobialefficacyofanessentialoil isnotassociatedexclusivelywithaspecificconstituentbut ratherasynergisticeffectofalloftheconstituentscontained.

Conclusions

Theresultsobtainedinthisstudydemonstratethatthe essen-tialoilsofbasil,rosemary,marjoram,peppermint,thymeand aniseexhibitinvitroantimicrobialactivitiesagainstC. perfrin-gens.TheessentialoilfromT.vulgarisshowedthelowestMIC valueamongalloftheoilstestedandwasthemosteffective bactericideagainstoneofthemaincausesoffoodpoisoning inBrazil.Theuseofessentialoilsfromcommonlyemployed spicesclearlyoffersanalternativetothechemical preserva-tivesinthecontrolandinactivationofpathogensinfood,but furtherstudiesareneededinordertoverifydirectapplication in commercially produced food systems. The results sug-gestthat theoxygenatedcompound, especiallyoxygenated monoterpenes andphenylpropanoids,mightberesponsible forthe antimicrobialactivity against C.perfringens, but the synergisticeffectsofthesechemicalswithotherminor con-stituentsoftheessentialoilshouldalsobeconsidered.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

TheauthorswouldliketothanktheMicrobiologyLaboratory atUnivatesfortheC.perfringensstrainand technical assis-tancewithmicrobiologicalassays.

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