Antibacterial and Antifungal Activities of Spices

62 Pages·2017·0.43 MB·English

International Journal o f
Molecular Sciences
Review
Antibacterial and Antifungal Activities of Spices
QingLiu1,XiaoMeng1,YaLi1,Cai-NingZhao1,Guo-YiTang1andHua-BinLi1,2,*
1 GuangdongProvincialKeyLaboratoryofFood,NutritionandHealth,DepartmentofNutrition,
SchoolofPublicHealth,SunYat-senUniversity,Guangzhou510080,China;
[email protected](Q.L.);[email protected](X.M.);[email protected](Y.L.);
[email protected](C.-N.Z.);[email protected](G.-Y.T.)
2 SouthChinaSeaBioresourceExploitationandUtilizationCollaborativeInnovationCenter,
SunYat-senUniversity,Guangzhou510006,China
* Correspondence:[email protected];Tel.:[phone]
Received:16May2017;Accepted:11June2017;Published:16June2017
Abstract: Infectious diseases caused by pathogens and food poisoning caused by spoilage
microorganismsarethreateninghumanhealthallovertheworld. Theefficaciesofsomeantimicrobial
agents, which are currently used to extend shelf-life and increase the safety of food products in
foodindustryandtoinhibitdisease-causingmicroorganismsinmedicine,havebeenweakenedby
microbialresistance. Therefore,newantimicrobialagentsthatcouldovercomethisresistanceneed
tobediscovered. Manyspices—suchasclove,oregano,thyme,cinnamon,andcumin—possessed
significantantibacterialandantifungalactivitiesagainstfoodspoilagebacterialikeBacillussubtilis
andPseudomonasfluorescens,pathogenslikeStaphylococcusaureusandVibrioparahaemolyticus,harmful
fungilikeAspergillusflavus,evenantibioticresistantmicroorganismssuchasmethicillinresistant
Staphylococcus aureus. Therefore, spices have a great potential to be developed as new and safe
antimicrobialagents. Thisreviewsummarizesscientificstudiesontheantibacterialandantifungal
activitiesofseveralspicesandtheirderivatives.
Keywords: spice;antibacterialactivity;antifungaleffect;antimicrobialproperty;essentialoil;clove;
oregano;thyme
1. Introduction
Microbial pathogens in food may cause spoilage and contribute to foodborne disease
incidence, and the emergence of multidrug resistant and disinfectant resistant bacteria—such as
Staphylococcusaureus(S.aureus),Escherichiacoli(E.coli),andPseudomonasaeruginosa(P.aeruginosa)—has
increasedrapidly,causingtheincreaseofmorbidityandmortality[1]. Weakacidssuchasbenzoic
and sorbic acids [2], which are commonly applied in food industry as chemical preservatives to
increasethesafetyandstabilityofmanufacturedfoodsonitswholeshelf-lifebycontrollingpathogenic
and spoilage food-related microorganisms [3], can result in the development of microbiological
resistance [4]. Moreover, chemical preservatives cannot completely eliminate several pathogenic
bacteria like Listeria monocytogenes (L. monocytogenes) in food products or delay the growth of
spoilagemicroorganisms[5]. Naturalproducts,assubstitutesofsyntheticchemicalpreservatives,are
increasinglyacceptedbecausetheyareinnatelybettertoleratedinhumanbodyandhaveinherent
superioritiesforfoodindustry[4]. Theantimicrobialactivitiesofnaturalproductsarenecessarytobe
studiedandappliedinfoodindustry.
Morbidityandmortalityaremainlycausedbyinfectiousdiseasesallovertheworld. TheWorld
HealthOrganizationreportedthat55millionpeoplediedworldwidein2011,withone-thirdofthe
deathsowingtoinfectiousdiseases[6]. Antibioticresistantmicroorganismscanincreasemortality
ratesbecausetheycansurviveandrecoverthroughtheirabilitytoacquireandtransmitresistance
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Int.J.Mol.Sci.2017,18,1283 2of62
afterexposuretoantibioticdrugs,whichareoneofthetherapiestoinfectiousdiseases[7]. Antibiotic
resistant bacteria threaten the antibiotic effectiveness and limit the therapeutic options even for
commoninfections[8]. Thedeclineinresearchanddevelopmentofnewantibacterialagents,which
areabletoinhibitantibioticresistantdisease-causingmicroorganismssuchasS.aureus,aggravates
theemergingantibioticresistance[9]. Therefore,muchattentionshouldbepaidtonaturalproducts,
whichcouldbeusedaseffectivedrugstotreathumandiseases,withhighefficacyagainstpathogens
andnegligiblesideeffects.
Spiceshavebeenusedasfoodandflavoringsinceancienttimes[10],andasmedicineandfood
preservativesinrecentdecades[11,12]. Manyspices—suchasclove,oregano,thyme,cinnamon,and
cumin—havebeenappliedtotreatinfectiousdiseasesorprotectfoodbecausetheywereexperimentally
provedtopossessantimicrobialactivitiesagainstpathogenicandspoilagefungiandbacteria[10,13,14].
Moreover,thesecondarymetabolitesofthesespicesareknownasantimicrobialagents,themajority
ofwhicharegenerallyrecognizedassafematerialsforfoodwithinsignificantadverseeffects[11].
Therefore, spices could be candidates to discover and develop new antimicrobial agents against
foodborneandhumanpathogens.
This review summarizes the scientific studies on the antibacterial and antifungal activities of
spicesandtheirderivatives,andsomesuggestionsandprospectsareofferedforfuturestudies.
2. Clove
Clove (Eugenia caryohyllata), belonging to family Myrtaceae, is widely used in medicine as
antisepticagainstinfectiousdiseaseslikeperiodontaldiseaseduetotheantimicrobialactivitiesagainst
oralbacteria[15]. Cloveisalsocommonlyappliedinfoodindustryasanaturaladditiveorantiseptic
toincreaseshelf-lifeduetotheeffectiveantimicrobialactivitiesagainstsomefoodbornepathogens[16].
Themainactivecomponentofcloveoilandextractwasfound,i.e.,eugenol[15,17].
2.1. AntimicrobialActivitiesofClove
Antimicrobialactivitiesofclovewaterextractwerestudiedinvitroandinvivoagainstpathogenic
microorganisms (S. aureus and E. coli, in a model of pyelonephritis) [18]. An invitro study
was conducted with the agar well diffusion method, and the results suggested that clove water
extract showed antibacterial activity against S. aureus (minimum inhibitory concentration (MIC):
2 mg/mL) and E. coli (MIC: 2.5 mg/mL). While invivo, the study was conducted in 40 adult
male albino rats, and the results confirmed the efficacy of clove extract as natural antimicrobials.
The direct antimicrobial activities of ultra-fine powders of ball-milled cinnamon and clove were
tested by Kuangetal.[19] against E. coli, S. aureus, Brochothrix thermosphacta (B. thermosphacta),
Lactobacillusrhamnosus(L.rhamnosus),andPseudomonasfluorescens(P.fluorescens)frommeat,usingbroth
dilutionmethod. Clovepowdershowedstronginhibitoryeffectsonfivemicroorganismstestedwith
theMICsrangingfrom1.0%w/v(L.rhamnosusandB.thermosphacta)to2.0%w/v(P.fluorescens),and
theinhibitoryeffectswerepositivelyassociatedwiththeconcentrationsofpowder,whichincreased
from0.5%to2.5%w/v.
Clovecoulddestroycellwallsandmembranesofmicroorganisms,andpermeatethecytoplasmic
membranesorenterthecells,theninhibitthenormalsynthesisofDNAandproteins[16]. Eugenol,
themajorcomponentofclove,couldinhibittheproductionofamylaseandproteasesinBacilluscereus
(B.cereus)andhastheabilityofcellwalldeteriorationandcelllysis[20].
2.2. ComparisonofAntimicrobialActivitiesofCloveandOtherSpices
Badeietal.[21]testedtheantimicrobialactivitiesofcardamom,cinnamonandcloveessential
oils(EOs)againstnineGram-positivebacterialstrains,fourGram-negativebacterialstrains,seven
molds,andtwoyeasts,comparedwithphenol,usingthediscdiffusionmethod. CloveEOshowed
the highest antimicrobial activity, and the antimicrobial spectra (diameter of inhibition zones) of
10% clove EO was 1.48 times as that of 10% phenol. Schmidt et al. [22] evaluated the antifungal
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effects of eugenol-containing EOs of 4 spices on 38 Candida albicans (C. albicans) isolates, of which
12 were isolated from oropharynges, 16 from vaginas, and 10 from damaged skin, using the
microdilutionmethod. CloveEOpossessedthestrongestantifungalactivitiesagainstallC.albicans
strainsamongthetestedspices. Pureeugenolaloneexhibitedweakerantifungalactivitiesthanclove
leaf EO. Angiendaetal.[23] investigated the antimicrobial activities of EOs of four spices against
Salmonella typhimurium (S. typhimurium), E. coli, B. cereus, and Listeria innocua (L. innocua) by agar
diffusiontest. CloveEOshowedthemosteffectiveinhibitionagainstbothGram-positivebacteria
andGram-negativebacteriacomparedwiththreeotherEOs,withtheMICsrangingfrom1.25%v/v
(B. cereus) to 2.50% v/v (S. typhimurium and E. coli). Lomarat et al. [17] reported the antimicrobial
activitiesofEOsfromninespicesagainsthistamine-producingbacteriaincludingMorganellamorganii
(M.morganii),bydeterminingMICsandminimumbactericidalconcentrations(MBCs)usingthebroth
dilutionassay,andalsofoundtheantibacterialcompoundsofEOsbybioautography-guidedisolation.
TheresultsindicatedthatthecloveEOwasthemosteffectiveagainstM.morganiiamongninetested
spiceswithMIC0.13%v/vandMBC0.25%v/v. Theeugenolwasidentifiedastheactivecomponent
ofcloveEObythinlayerchromatographybioautographyassay.
Shanetal.[24]testedtheantibacterialactivitiesofethanolextractsfromfivespicesandherbs
against L. monocytogenes, S. aureus, and Salmonella enterica (S. enterica) in raw pork by counting
bacterial enumeration. When treated with clove extract, raw pork samples were found with the
fewestcoloniesoftestedbacteria. Bayoubetal.[25]reportedtheantimicrobialactivitiesofethanol
extracts of 13 plants including clove against L. monocytogenes, the MICs were determined by agar
well diffusion test. The results showed that clove extract was the most effective inhibitor against
L.monocytogenescomparedwiththeother12selectedplantethanolextracts,withtheMIC0.24mg/mL.
Cuietal.[26]testedtheantimicrobialactivitiesof90plantextracts(waterand99.5%ethanolextracts)
againstClostridiumspp. Clovewaterextractwasfoundwiththegreatestantimicrobialactivityagainst
Clostridium botulinum in trypticase peptone glucose yeast extract broth (pH = 7.0) among all the
water extracts, and the MICs of clove extract ranged from 0.1% to 0.2% against Clostridium spp.
Antimicrobialeffectsof3extracts(ethylacetate,acetone,andmethanolextracts)of12plantswere
testedon2fungi(Kluyveromycesmarxianus(K.marxianus)andRhodotorularubra(R.rubra))and8bacteria
(Klebsiellapneumoniae(K.pneumoniae),Bacillusmegaterium(B.megaterium),P.aeruginosa,S.aureus,E.coli,
Enterobactercloacae(E.cloacae),Corynebacteriumxerosis(C.xerosis),andStreptococcusfaecalis(S.faecalis))
bythediscdiffusionmethod[27]. Cloveexhibitedthemosteffectiveinhibitoryimpacts. Themethanol
extractfromcloveshowedinhibitionagainstmicroorganisms(diameterofinhibitionzones(DIZs):
8–24mm)testedexceptK.pneumoniae. Theacetoneextractshowedinhibitionagainstmicroorganisms
(DIZs: 8–18 mm) tested except R. rubra and K. pneumoniae. The ethyl acetate extract only showed
antibacterialactivityagainstB.megaterium(DIZ:7mm). Liangetal.[28]observedtheantimicrobial
activitiesofsevenspices,anddifferentconcentrationsofextractsandEOsineachspicewereusedto
testtheeffectsonthegrowthofspoilagemicroorganismsinappleciderbytotalplatecounts. Clove
productsshowedthestrongestantimicrobialactivitiescomparedwithotherspicestested.Nearlyseven
logreductionofmicroorganismswasobservedat0.8%v/vintheciderforcloveoiland2%w/wfor
clovepowderatroomtemperature. Badheetal.[29]testedtheantimicrobialactivitiesofmanyspice
andherbpowdersagainstS.aureus,S.typhimurium,E.coli,andB.cereusatrefrigeratedtemperature
(8±2◦C)forintervalsof0,3,6,12,24,and48h. Theresultsindicatedthatattheconcentrationof2%,
clovepowdershowedhighesteffectonS.aureusfollowedbyE.coliandS.typhimurium,andat24h
underrefrigeration,clovepowderledtoasignificantreductionofbacteriacounting.
2.3. TheApplicationofCloveasAntimicrobialAgentsinFoodPackaging
CloveEOanditsfunctionalextractshavebeenincorporatedintofilms,theantimicrobialactivities
ofwhichhavebeenevaluatedinsomestudies.Inastudy,chitosanathigh,moderateandlowmolecular
masswereelaboratedwithantimicrobialfilmswhichwereincorporatedwithEOsandextractsfrom
twospices[30]. ThentheantimicrobialeffectsofthefilmswereinvestigatedonE.coli,S.typhimurium,
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S.aureus,B.cereus,andL.monocytogenes. Thefilmspreparedbylowmolecularmasschitosanwith
2%EOandethylheptanoateextractfromcloveshowedantimicrobialactivitiesagainstamajority
ofthetestedstrains. Inanotherstudy,theresearcherstestedtheantimicrobialactivitiesofEOsand
functionalextractsofcumin,clove,andelecampaneagainstE.coli,S.typhimurium,B.cereus,S.aureus,
andL.monocytogenesbydeterminingtheMICsandMBCs[31]. Theyalsoevaluatedtheantibacterial
activitiesofediblefilmspreparedbyEOsandfunctionalextractsofspicesbasedonchitosanpolymeric
structureagainstthesamebacteriabydeterminingtheDIZs. CloveEOshowedthebestinhibitory
effectswiththeMICof500mg/Lonallthebacteriatested,cloveextractsshowedverysimilarMICs
to those of EO, except ethyl caproate extract of clove against L. monocytogenes (MIC of 750 mg/L)
andethylheptanoateextractofcloveagainstB.cereus(MICof250mg/L).Amongthechitosanfilms
addedwithEOs,onlycloveshowedinhibitionzonesofalltestedbacteriaexceptL.monocytogenes. The
ethylheptanoateextractofclovefilmalsopossessedantibacterialactivitiesagainstalltestedbacteria,
weakerthanthoseofcloveEOthough. Liuetal.[32]evaluatedtheantimicrobialactivitiesofspice
EOsagainstmicrobialpopulationsinchilledporkstoredinPEfilmantimicrobialpackageusingthe
diskdiffusionmethodtodeterminetheDIZsandserialdilutionassaytodeterminetheMICs. Clove
EOwasthemosteffectiveagainstmicroorganismstestedamongallthespiceEOstested. TheMICsof
cloveEOwere0.10%,0.10%,and0.30%v/vagainstEnterobacteriaceae,S.aureus,andPseudomonassp.,
respectively. SpiceEOspossessedtheabilitytodecreasethenumberofspoilagepopulations,butnot
thespeciesdiversityofspoilagemicrobiota.
Collectively, clove EO and extracts could prevent against some food spoilage and foodborne
pathogens(Table1),especiallyGram-positivebacteria. TheMICsofclovewerelessthan2.5%against
testedmicroorganismslikeP.fluorescens,S.typhimurium,E.coli,B.cereus,andL.innocua. Generally
speaking,thequalitiesofthepaperscitedaregoodandtheresultsarereliable.
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Table1.Antibacterialandantifungalactivitiesofclove.
TypeofSamples BacteriaandFungi MainResults Reference
Bothinvivoandinvitroresultsconfirmedtheefficacyofclove
Cloveandcinnamonwaterextracts StaphylococcusaureusandEscherichiacoli [18]
extractasnaturalantimicrobials.
Clovepowdershowedastronginhibitoryeffectwiththe
E.coli,S.aureus,Brochothrixthermosphacta,
Ultra-finepowdersofball-milledclove minimuminhibitoryconcentrations(MICs)rangingfrom1.0%to [19]
Lactobacillusrhamnosus,Pseudomonasfluorescens
2.0%w/v.
Cardamom,cinnamon,cloveessential
13bacterialstrains,7moldsand2yeasts CloveEOpossessedthehighestantimicrobialactivities. [21]
oils(EOs)andphenol
CloveEOpossessedthestrongestactivitiesagainstallC.albicans
4spiceEOs Candidaalbicans [22]
strains.
Salmonellatyphimurium,E.coli,Bacilluscereus, CloveEOshowedthemosteffectiveinhibitionwiththeMICs
4spiceEOs [23]
Listeriainnocua rangingfrom1.25%to2.50%v/v.
9spiceEOs Morganellamorganii CloveEOwasthemosteffectivewithMICof0.13%v/v. [17]
Ethanolextractsfrom5spicesandherbs Listeriamonocytogenes,S.aureus,Salmonellaenterica Cloveextractwasthemosteffectiveagainstbacteriatested. [24]
CloveextractwasthemosteffectivewiththeMICof0.24
13plantethanolextracts L.monocytogenes [25]
mg/mL.
Clovewaterextractwasthemosteffectiveamongallthewater
90plantextracts Clostridiumspp. [26]
extractswiththeMICrangingfrom0.1%to0.2%.
Kluyveromycesmarxianus,Rhodotorularubra,Klebsiella
Ethylacetate,acetoneandmethanol pneumoniae,Bacillusmegaterium,Pseudomonasaeruginosa,
Clovepossessedthemosteffectiveinhibitoryeffects. [27]
extractsof12plantspecies S.aureus,E.coli,Enterobactercloacae,
Corynebacteriumxerosis,Streptococcusfaecalis
Cloveproductshadthestrongestantimicrobialactivities
7spices,theirextractsandEOs Microorganismsinapplecider [28]
comparedwithotherspicestested.
2%levelofclovepowderwasmoreeffectiveagainstS.aureus
Manyspiceandherbpowders S.aureus,S.typhimurium,E.coli,B.cereus [29]
followedbyE.coliandS.typhimuriumunderrefrigeration.
Thefilmsoflowmolecularweightchitosanwithaconcentration
EOsandfunctionalextractsofcumin E.coli,S.typhimurium,S.aureus,B.cereus,
of2%ofEOofcloveandcloveethylheptanoateextracthad [30]
andclove. L.monocytogenes
antimicrobialactivitiesagainstmoststrainstested.
EOsandfunctionalextractsofcumin, E.coli,S.typhimurium,B.cereus,S.aureus, ChitosanfilmsaddedwithcloveEOshowedthebestinhibitory
[31]
clove,andelecampane L.monocytogenes effectswiththeMICsof500mg/L.
Enterobacteriaceae,S.aureus,Pseudomonassp.,Lacticacid
3spiceEOs CloveEOwasthemosteffectiveagainstmicroorganismstested. [32]
bacteria,Brocithrixthermosphacta
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3. Oregano
Oregano(Origanumvulgare),belongingtofamilyLamiaceae,hasbeenusedasfoodseasoningand
flavoringforalongtime. Themajorcomponentsassociatedwithantimicrobialactivitiesinoregano
EOwereprovedtobecarvacrolandthymol[33].
3.1. AntimicrobialActivitiesofOregano
Babacan et al. [34] evaluated the antimicrobial activities of oregano extract against various
Salmonella serotypes by evaluating the bacterial growth with disc diffusion method. The results
showed that DIZs of oregano were 15, 19, and 16 mm for Salmonella gallinarum (S. gallinarum),
Salmonellaenteritidis(S.enteritidis),andS.typhimurium,respectively. Santoyoetal.[35]observedthe
antimicrobialactivitiesofEO-richfractionsoforeganowhichwereselectivelyprecipitatedinthesecond
separatorindifferentconditionsagainstsixmicroorganismstrains(S.aureus,Bacillussubtilis(B.subtilis),
E.coli,P.aeruginosa,C.albicans,andAspergillusniger(A.niger)),usingthediskdiffusionandbroth
dilutionmethods. Theresultsshowedthatallofthesupercriticalfluidextractionfractionsexhibited
antimicrobialeffectsontestedmicroorganisms,andthemostefficientfractionwasobtainedwith7%
ethanolat150barand40◦C.DeSouzaetal.[36]evaluatedtheeffectsofheating(atthetemperatures
of60,80,100,and120◦C,atadurationof1hforeach)ontheantimicrobialactivitiesoforeganoEO
against9microorganismstrains(C.albicans,Candidakrusei(C.krusei),Candidatropicalis(C.tropicalis),
B. cereus, E. coli, S. aureus, Yersinia enterocolitica (Y. enterocolitica), S. enterica, and Serratia marcescens
(S. marcescens)), using the solid medium diffusion procedure. The results indicated that heating
treatmentshowednosignificanteffectsontheantimicrobialactivitiesofEO,withtheDIZsandMICs
ofheatedEOclosetothoseofEOkeptatroomtemperature(MICsrangingfrom10to40µL/mL).
OreganocouldbindtosterolsinthefungalmembranesofC.albicansstrains[37],buttheexact
mechanismsofactiononothermicroorganismsaretobefurtherstudied. Carvacrol,oneofthemajor
componentsoforegano,couldinteractwithcellmembranesthroughchangingthepermeabilityfor
smallcations[38]. AsthechemicalcompoundsinEOandextractsoforeganoarecomplex,theycould
inhibitmicroorganismsthroughdifferentcelltargets.
3.2. ComparisonofAntimicrobialActivitiesofOreganoandOtherSpices
Ozcanetal.[39]investigatedtheantifungalactivitiesoffourspicedecoctionsagainstsixmolds
(Fusarium oxysporum f. sp. phaseoli, Macrophomina phaseoli (M. phaseoli), Botrytis cinerea (B. cinerea),
Rhizoctoniasolani(R.solani),Alternariasolani(A.solani),andAlternariaparasiticus(A.parasiticus)). The
resultsshowedthatthemycelialgrowthwere100%inhibitedby10%oreganodecoctioninculture
medium.Ai-Turkietal.[40]testedtheantimicrobialactivitiesofaqueousextractsoffourplantsagainst
E.coliandB.subtilisusingthediscdiffusionmethod. Oreganoextractshowedthebestantibacterial
effectsontwobacteriacomparedwiththreeotherspiceextracts,andB.subtilisshowedmoresensitivity
thanE.coli.Marquesetal.[41]assessedtheantimicrobialactivitiesoftheEOsoforeganoandmarjoram
againstS.aureusisolatedfrompoultrymeatusingthediskdiffusionmethod,andtheMICsandMBCs
weretestedusingthemicrodilutiontechnique. AlltheS.aureusstrainsweresusceptibletooreganoEO
withtheMICsrangingfrom6.25to25µL/mL,butfouroftheisolateswereresistanttoampicillinand
onewasresistanttotetracycline. Bozinetal.[42]investigatedtheantimicrobialactivitiesof3spiceEOs
against13bacterialstrainsusingthehole-plateagardiffusionmethodand6fungibythemicrodilution
technique. The results indicated that the most effective antibacterial activities were expressed by
oreganoEO,evenonmultiresistantstrainsofP.aeruginosaandE.coli. Viuda-Martosetal.[43]studied
theantimicrobialactivitiesofEOsfromsixspicesagainstsixbacteria(Lactobacilluscurvatus(L.curvatus),
Lactobacillus sakei (L. sakei), Staphylococcus carnosus (S. carnosus), Staphylococcus xylosus (S. xylosus),
Enterobactergergoviae(E.gergoviae)andEnterobacteramnigenus(E.amnigenus)),usingthediscdiffusion
method. OreganoEOwasthemosteffectiveagainstbacteriatested,withDIZsrangingfrom35.29mm
(S. xylosus) to 57.90 mm (E. amnigenus). Santurio et al. [44] reported the antimicrobial activities of
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EOsofeightspicesagainstE.colistrainsisolatedfrompoultryandcattlefaecesbydeterminingthe
MICsusingthebrothmicrodilutiontechnique. Theresultsshowedthatthemosteffectiveagainstall
E.colistrainsinthestudywasoreganoEO.Khosravietal.[45]investigatedtheantifungalactivities
ofArtemisiasieberiandoreganoEOsagainstCandidaglabrata(C.glabrata)isolatedfrompatientswith
vulvovaginalcandidiasisbydeterminingtheMICsandminimalfungicidalconcentrations(MFCs),
usingthebrothmacrodilutionmethod.TheresultsindicatedthattheEOsinhibitedalltestedC.glabrata
isolates concentration-dependently, with the MICs ranging from 0.5 to 1100 µg/mL (mean: 340.2
µg/mL) for oregano. Dal Pozzo et al. [46] studied the antimicrobial activities of 7 spice EOs, and
somemajorityconstituentsofthesespicessuchascarvacrol,thymol,cinnamaldehyde,andcineole
against33Staphylococcusspp. isolatesfromherdsofdairygoats,bydeterminingtheMICsandMBCs
usingthebrothmicrodilutionmethod. Oreganoandthymepossessedequallystrongantimicrobial
activitiesamongEOs. Santosetal.[47]evaluatedtheantimicrobialactivitiesoffourspicesagainst
severalbacterialikeS.aureusandE.coliisolatedfromvongoleandbacteriastandardATCC(American
TypeCultureCollection): E.coli,S.aureus,andSalmonellacholeraesuis(S.choleraesuis),bydetermining
theMICsusingdiffusiontest. OreganoandcloveEOspresentedantimicrobialactivitiesagainstall
tested bacteria, but oregano presented larger DIZs of 26.7 mm (E. coli) and 29.3 mm (S. aureus).
Hyun et al. [48] tested the antibacterial effects of various spice EOs including oregano on total
mesophilic microorganisms in products (fresh leaf lettuce and radish sprouts) using the dipping
method. Onespeciesoforegano(intheUSA)EOshowedthebesteffectsonmaintainingreduced
levels of total mesophilic microorganisms in fresh leaf lettuce and radish sprouts compared with
thecontrol.
3.3. TheApplicationofOreganoasAntimicrobialAgentsinFoodPackaging
TheantimicrobialeffectsofpureEOsoffourspicesandchitosan-EOsfilmsonL.monocytogenes
andE.coliwereevaluatedinvitrobyagardiffusiontest[49]. TheantimicrobialactivitiesofEOsalone
andincorporatedinthefilmsweresimilarfollowingtheorder: oregano>>coriander>basil>anise.
Whenusedininoculatedbolognasamplesat10◦Candstoredforfivedays,purechitosanfilmsled
to2logreductionofL.monocytogenes,3.6–4logreductionofL.monocytogenes,and3logreductionof
E.coliwereobservedinfilmsincorporatedwith1%and2%oreganoEO.
Alltheabovestudiesareofgoodquality, andoreganoshowedstrongantimicrobialactivities
againstmicroorganismstrainssuchasStaphylococcusspp. andS.aureusisolateswithlargerDIZsand
lowerMICs,MBCs,andMFCscomparedwithseveralotherspices(Table2). Futurestudiescould
focusontheapplicationoforeganoanditsEOinfoodindustry,andalsothepossiblemodeofaction.
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Table2.Antibacterialandantifungalactivitiesoforegano.
TypeofStudy BacteriaandFungi MainResults Reference
Salmonellagallinarum,Salmonellaenteritidis,
Oreganoextract OreganoextracthadantibacterialeffectsonSalmonellaserotypes. [34]
S.typhimurium
S.aureus,B.subtilis,E.coli,P.aeruginosa,C.albicans, Allofthesupercriticalfluidextractionfractionsshowed
EO-richfractionsoforegano [35]
Aspergillusniger antimicrobialactivitiesagainstalltestedmicroorganisms.
C.albicans,Candidakrusei,Candidatropicalis,B.cereus,
Heatingtreatmentshowednosignificanteffectsonthe
OreganoEO E.coli,S.aureus,Yersiniaenterocolitica,S.enterica, [36]
antimicrobialactivitiesofEO.
Serratiamarcescens
F.oxysporumf.sp.phaseoli,Macrophominaphaseoli,
The10%leveloforeganodecoctionwas100%inhibitiveto
4spicedecoctions Botrytiscinerea,Rhizoctoniasolani,Alternariasolani, [39]
mycelialgrowthintheculturemedium.
Alternariaparasiticus
Oreganoextracthadthehighestantibacterialactivitiesagainstall
4plantaqueousextracts E.coliandB.subtilis [40]
testedbacteria.
OreganoandmarjoramEOs S.aureusisolatedfrompoultrymeat. AlltheisolatestestedweresensitivetoEOoforegano. [41]
3spiceEOs 13bacterialstrainsand6fungi OreganoEOshowedthemosteffectiveantibacterialactivities. [42]
Staphylococcusxylosus,Staphylococcuscarnosus,
6spiceEOs Lactobacillussakei,Lactobacilluscurvatus,Enterobacter OreganoEOwasthemosteffective. [43]
gergoviae,Enterobacteramnigenus
8spiceEOs E.colistrainsisolatedfrompoultryandcattlefaeces. OreganoEOwasthemosteffectiveagainstE.coli. [44]
Candidaglabrataisolatedfrompatientswith TheMICsoforeganoEOrangedfrom0.5to1100µg/mLforall
OreganoandA.sieberiEOs [45]
vulvovaginalcandidiasis. testedC.glabrataisolates.
OreganoandthymeEOspossessedtheequalandstrongest
7spiceEOsandthemajorityconstituents 33Staphylococcusspp.isolates [46]
antimicrobialactivitiesamongEOs.
S.aureusandE.coliisolatedfromvongoleandbacteria Oreganopresentedantimicrobialactivitiesagainstalltested
4spiceEOs [47]
standardATCC:E.coli,S.aureus,Salmonellacholeraesuis bacteria.
Oregano-2(intheUSA)oilwasthemosteffectiveatmaintaining
VariousspiceEOs Microorganismsinfreshleaflettuceandradishsprouts. [48]
thereducedlevelsoftotalmesophilicmicroorganisms.
PureEOsof4spicesandchitosan-EOs BothoreganoEOaloneandincorporatedinthefilmspossessed
L.monocytogenesandE.coli [49]
films thebestantimicrobialactivities.
Int.J.Mol.Sci.2017,18,1283 9of62
4. Thyme
Thyme(Thymusvulgaris), belongingtofamilyLamiaceae, isasubshrubnativetothewestern
Mediterraneanregion. Thymeiswidelyusedasaspicetoaddspecialflavortofoods. Inrecentstudies,
thymewasfoundtopossessefficientantimicrobialactivitiesandwasusedinsomefoodstoextendthe
shelf-life[50].
4.1. AntimicrobialActivitiesofThyme
A study evaluated the antimicrobial activities of thyme EO against bacteria (B. subtilis,
S.aureus,Staphylococcusepidermidis(S.epidermidis),P.aeruginosa,E.coli,andMycobacteriumsmegmatis
(M.smegmatis))andfungalstrains(C.albicansandCandidavaginalis)[51]. ThymeEOshowedeffective
bactericidalandantifungalactivitiesagainsttestedmicroorganismstrainswithMICsrangingfrom75
to1100µg/mLforbacteria,andfrom80to97µg/mLforfungi. Inanotherstudy,EOsobtainedfrom
thymeharvestedatfourontogeneticstagesweretestedfortheirantibacterialactivitiesagainstnine
strainsofGram-negativebacteriaandsixstrainsofGram-positivebacteriausingthebioimpedance
methodtotestthebacteriostaticactivitiesandplatecountingtechniquetostudytheinhibitoryeffects
by direct contact [52]. The results indicated that all the thyme EOs had significant bacteriostatic
activitiesagainstthemicroorganismstested. Furthermore,theantimicrobialactivitiesofEOsoffour
Thymusspecies(T.vulgaris, T.serpyllum, T.pulegioides,andT.glabrescens)weredeterminedbyagar
diffusionmethod[53].T.vulgarisandT.serpyllumEOswerethemostefficientastheyinhibitedallthe
testedbacteria(P.aeruginosa,Cronobactersakazakii(C.sakazakii),L.innocua,andStreptococcuspyogenes
(S.pyogenes))andyeasts(C.albicansandSaccharomycescerevisiae(S.cerevisiae))bothatoriginaland
half-dilutedconcentrations.P.aeruginosa,L.innocua,andS.pyogeneswerehighlyandequallysensitive
totheThymusoils,whileC.sakazakiiexhibitedlimitedsensitivity,andthesensitivityofthetwoyeast
strainsweresimilartothatofC.sakazakii,butS.cerevisiaewasalittlemoresensitivethanC.albicans.
Themajoractivecompoundofthymeisthymol,whichexerteditsantimicrobialactionthrough
bindingtomembraneproteinsbyhydrophobicbondingandhydrogenbonding,andthenchanging
thepermeabilityofthemembranes[20]. Thymolalsodecreasedintracellularadenosinetriphosphate
(ATP)contentofE.coliandincreasedextracellularATP,whichcoulddisruptthefunctionofplasma
membranes[54]. AsthymolwasprovedtoactdifferentlyagainstGram-positiveandGram-negative
bacteria[20],theexactmechanismsofantimicrobialactionshouldbefurtherstudied.
4.2. ComparisonofAntimicrobialActivitiesofThymeandOtherSpices
Al-Turkietal.[55]reportedtheantibacterialactivitiesofthyme,peppermint,sage,blackpepper
andgarlichydrosolsagainstB.subtilisandS.enteritidis,usingtheagardiskdiffusionmethod. Thyme
hydrosoldemonstratedmoresignificantinhibitoryeffectsonB.subtilisandS.enteritidisthansage,
peppermint, and black pepper hydrosols, with the mean DIZs 20 mm for B. subtilis and 15 mm
forS.enteritidis. Accordingtoanotherstudy,theantimicrobialeffectsofthesixplanthydrosolson
S.aureus,E.coli,S.typhimurium,P.aerugenosa,andC.albicansweretestedbydeterminingthemicrobial
growthzonesonhydrosolagarplatesandcontrolagarplates[56]. Theresultsshowedthatat15%
thymehydrosolcompletelyinhibitedE.coliandS.typhimurium,butC.albicanswasinactivetothe
tested hydrosols. Girova et al. [57] assessed the antimicrobial activities of five plant EOs against
psychrotrophicmicroorganisms(P.fluorescens,Pseudomonasputida(P.putida),P.fragi,B.thermosphacta,
andC.albicans)isolatedfromspoiledchilledmeatproductsandsomereferencestrains(P.fluorescens
ATCC17397,P.putidaNBIMCC(NationalBankforIndustrialMicroorganismsandCellCultures)561,
P.aeruginosaATCC9027,andC.albicansATCC10231)usingthemethodofdiscdiffusionandserial
brothdilution. TheresultsindicatedthattheantimicrobialeffectsoftheEOswereequalat37◦Cand
4◦C.ThymeEOexhibitedthehighestantimicrobialactivitieswiththeMICsrangingfrom0.05%to
0.8%w/v. Hajlaouietal.[58]observedtheanti-Vibrioalginolyticus(V.alginolyticus)activitiesoffive
aromaticplantEOsusingagarwelldiffusiontest,andtheMICsandMBCswereexaminedusingthe
brothmicrodilutionsusceptibilitymethod. ThymeEOwasprovedtobethemostefficientagainst13
Int.J.Mol.Sci.2017,18,1283 10of62
V.alginolyticusstrainscomparedwith4otherEOs,withtheMICsrangesof0.078–0.31mg/mLand
MBCsrangesof0.31–1.25mg/mL.Also,Viuda-Martosetal.[59]assessedthegrowthinhibitionofsome
indicatorsofspoilagebacteriastrains(L.innocua,S.marcescens,andP.fluorescens)andtheconcentration
effectsoffivespiceEOsusingtheagardiscdiffusionmethod. OnlytheEOofthymeshowedinhibitive
effectsonalltestedbacteriaatalladdeddoses(100%,50%,25%,12.5%,and5%). Aliakbarluetal.[60]
evaluatedtheantibacterialactivitiesofEOsfromthyme,Thymuskotschyanus,Ziziphoratenuior,and
Ziziphoraclinopodioides,againsttwoGram-positivebacteria(B.cereusandL.monocytogenes)andtwo
Gram-negative bacteria (S. typhimurium and E. coli), using the agar disc diffusion and micro-well
dilutionassay. TheEOofthymeshowedthehighestantibacterialactivities,withthewidestinhibition
zonesandthelowestMICs(0.312–1.25µL/mL),andB.cereuswasthemostsensitivebacteriumtested.
Hyunetal.[48]investigatedtheantibacterialeffectsofseveralEOson18pathogenicbacteriaand15
spoilagebacteriabyagardiscdiffusiontest. Theresultsshowedthatthyme-1(T.vulgaris)EOand
thyme-2(T.vulgarisctlinalool)EOexertedthehighestantibacterialactivitiesagainst18pathogenic
bacteriastrainscomparedwithotherspices,exceptforP.aeruginosa. Thyme-1EOalsodemonstrated
thebestantibacterialeffectsonspoilagebacteria. Inaddition,theantimicrobialeffectsof17spices
andherbsagainstShigellastrainsweretestedinanotherstudy[61]. TheMICsweredeterminedby
theagardilutionmethodwithdriedgroundspicesandherbsaddedtothebrothandagar,andthe
results showed that MICs of thyme were 0.5–1% w/v for the Shigella strains. The study also used
various combinations of temperatures (12, 22, and 37 ◦C), pH values (5.0, 5.5, and 6.0), and NaCl
concentrations(1%,2%,3%,and4%w/v),andtheinclusionorexclusionofthymeorbasilat1%w/vin
aMueller–Hintonagarmodelsystemtotesttheinhibitoryeffectsofthymeandbasil. Inthepresenceof
thyme,Shigellaflexneri(S.flexneri)didnotdevelopColony-FormingUnits(CFU)duringtheseven-day
incubationperiodfor16ofthe18testedcombinations.
Somestudiescomparedtheantimicrobialactivitiesofdifferentextractsofthyme.Martinsetal.[62]
evaluatedandcomparedtheantimicrobialactivitiesoftheinfusion,decoction,andhydroalcoholic
extracts prepared from thyme against S. aureus, S. epidermidis, E. coli, Klebsiella spp., P. aeruginosa,
Enterobacteraerogenes(E.aerogenes),Proteusvulgaris(P.vulgaris),andEnterobactersakazakii(E.sakazakii)
usingthediscdiffusionhalotest. ForGram-positivespecies,thymeextractsonlypresentedactivity
againstS.epidermidis,andhydroalcoholicextractshowedalowerantibacterialactivitythandecoction
andinfusionextracts,whichshowedthesimilaractivities. ForGram-negativespecies,thymeextracts
showed antimicrobial activities in the order of E. coli > P. vulgaris, P. aeruginosa > E. aerogenes =
E. sakazakii; decoction and hydroalcoholic extracts had similar effects against the bacteria except
P.aeruginosa,whilethelowestactivitywasobservedininfusionextracts. Moreover,theantifungal
effectsofthymeEO,hydrosolandpropolisextractsonnaturalmycobiotaonthesurfaceofsucukwere
evaluated[63]. Theresultsshowedthatpotassiumsorbate(15%w/v,inwater),thymeEO(10mg/mL,
indimethylsulfoxide),andpropolisextract(50mg/mL,indimethylsulfoxide)reducedby4.88,2.45,
and2.05logCFU/ginyeast-moldcountingcomparedwithsterilewater,respectively.
Aman et al. [64] analyzed the polyphenolic fractions and oil fractions of oilseeds from
4 spices, including thyme, for their antimicrobial activities against 35 bacterial strains. The
results showed that oil fractions of all spice oilseeds were more active than their polyphenolic
fractions,andthymeoilfractionhadthehighestantibacterialactivitiescomparedwithotherspice
oilseeds. Aznar et al. [65] studied the growth of Candida lusitaniae (C. lusitaniae) on different
concentrationsofnisin(0.1–3mmol/L),thymol(0.02–1.5mmol/L),carvacrol(0.02–1mmol/L),or
cymene(0.02–3mmol/L)inbroths(pH=5,25◦C),andalsoevaluatedtheinhibitoryactivityofthymol
againstC.lusitaniaeintomatojuice. Thymol,carvacrol,andcymenetotallyinhibitedtheyeastgrowth
formorethan21daysat25◦Cwhentheconcentrationswerehigherthan1mmol/L.Comparedwith
thecontrolwithoutthymol,theactivityofthymolagainstC.lusitaniaeintomatojuicewassignificant.
Inconclusion,theresultsobtainedfromanumberofinvestigationswithgoodqualityindicated
thatthymepossessedeffectiveantimicrobialactivitiesagainstseveralpathogenicandspoilagebacteria
andfungi,likeS.aureusandE.coli,withlowMICs(≤1100µL/mL)(Table3).