Essential oils: their antibacterial properties and potential

31 Pages·2004·0.43 MB·English

InternationalJournalofFoodMicrobiology94(2004)223–253
www.elsevier.com/locate/ijfoodmicro
Review
Essential oils: their antibacterial properties and potential
applications in foods—a review
Sara Burt*
DepartmentofPublicHealthandFoodSafety,FacultyofVeterinaryMedicine,UniversityofUtrecht,P.O.Box80175,
3508TDUtrecht,TheNetherlands
Received14November2003;accepted3March2004
Abstract
Invitrostudieshavedemonstratedantibacterialactivityofessentialoils(EOs)againstListeriamonocytogenes,Salmonella
typhimurium,EscherichiacoliO157:H7,Shigelladysenteria,BacilluscereusandStaphylococcusaureusatlevelsbetween0.2
and 10 Al ml(cid:1)1. Gram-negative organisms are slightly less susceptible than gram-positive bacteria. A number of EO
componentshasbeenidentifiedaseffectiveantibacterials,e.g.carvacrol,thymol,eugenol,perillaldehyde,cinnamaldehydeand
cinnamicacid,havingminimuminhibitoryconcentrations(MICs)of0.05–5Alml(cid:1)1invitro.Ahigherconcentrationisneeded
to achieve the same effect in foods. Studies with fresh meat, meat products, fish, milk, dairy products, vegetables, fruit and
cookedricehaveshownthattheconcentrationneededtoachieveasignificantantibacterialeffectisaround0.5–20Alg(cid:1)1in
foodsandabout0.1–10Alml(cid:1)1insolutionsforwashingfruitandvegetables.EOscomprisealargenumberofcomponentsand
itislikelythattheirmodeofactioninvolvesseveraltargetsinthebacterialcell.ThehydrophobicityofEOsenablesthemto
partitioninthelipidsofthecellmembraneandmitochondria,renderingthempermeableandleadingtoleakageofcellcontents.
PhysicalconditionsthatimprovetheactionofEOsarelowpH,lowtemperatureandlowoxygenlevels.Synergismhasbeen
observed between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol. Synergy between EO
componentsandmildpreservationmethodshasalsobeenobserved.SomeEOcomponentsarelegallyregisteredflavouringsin
theEUandtheUSA.UndesirableorganolepticeffectscanbelimitedbycarefulselectionofEOsaccordingtothetypeoffood.
D2004Elsevier B.V. All rightsreserved.
Keywords:Essentialoils;Antibacterial;Preservatives;Foodbornepathogens
1. Introduction from a food borne disease each year and in 2000 at
least two million people died from diarrhoeal disease
In spite of modern improvements in slaughter worldwide (WHO, 2002a). There is therefore still a
hygiene and food production techniques, food safety needfornewmethodsofreducingoreliminatingfood
is an increasingly important public health issue borne pathogens, possibly in combination with exist-
(WHO, 2002a). It has been estimated that as many ingmethods(thehurdleprinciple; Leistner,1978).At
as 30% of people in industrialised countries suffer the same time, Western society appears to be experi-
encing a trend of ‘green’ consumerism (Tuley de
Silva, 1996; Smid and Gorris, 1999), desiring fewer
* Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](S.Burt). synthetic food additives and products with a smaller
0168-1605/$-seefrontmatterD2004ElsevierB.V.Allrightsreserved.
doi:10.1016/j.ijfoodmicro.2004.03.022
224 S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253
impact on the environment. Furthermore, the World Thepurposeofthispaperistoprovideanoverview
Health Organization has recently called for a world- of the published data on the antibacterial activity of
wide reduction in the consumption of salt in order to those EOs and their components that could be con-
reduce the incidence of cardio-vascular disease sidered suitable for application in or on foods, and to
(WHO, 2002b). If thelevel of salt inprocessed foods describe their possible modes of action. The current
is reduced, it is possible that other additives will be knowledge on potential antagonists and synergists is
needed to maintain the safety of foods. There is presented; legal and safety aspects are discussed and
therefore scope for new methodsofmaking foodsafe areasforfutureresearchareproposed.Althoughsome
which have a natural or ‘green’ image. One such data are presented on spoilage flora, this paper will
possibility is the use of essential oils as antibacterial focus chiefly on the antibacterial effect of EOs on
additives. food borne pathogens and, in particular, those for
Essentialoils(EOs)(alsocalledvolatileorethereal which food animals are the major reservoir.
oils; Guenther, 1948) are aromatic oily liquids ob-
tained from plant material (flowers, buds, seeds, 1.1. Historical use of essential oils
leaves, twigs, bark, herbs, wood, fruits and roots).
They can be obtained by expression, fermentation, Although spices have been used for their perfume,
enfleurage or extraction but the method of steam flavour and preservative properties since antiquity
distillation is most commonly used for commercial (Bauer et al., 2001), of the known EOs, only oil of
production of EOs (Van de Braak and Leijten, 1999). turpentine was mentioned by Greek and Roman
The term ‘essential oil’ is thought to derive from the historians (Guenther, 1948). Distillation as a method
namecoinedinthe16thcenturybytheSwissreform- of producing EOs was first used in the East (Egypt,
erofmedicine,ParacelsusvonHohenheim;henamed India and Persia) (Guenther, 1948) more than 2000
the effective component of a drug Quinta essentia yearsagoandwasimprovedinthe9thcenturybythe
(Guenther,1948).Anestimated3000EOsareknown, Arabs (Bauer et al., 2001). The first authentic written
of which about 300 are commercially important— account of distillation of essential oil is ascribed to
destinedchieflyfortheflavoursandfragrancesmarket Villanova (ca. 1235–1311), a Catalan physician
(Van de Braak and Leijten, 1999). It has long been (Guenther, 1948). By the 13th century EOs were
recognised that some EOs have antimicrobial proper- being made by pharmacies and their pharmacological
ties (Guenther, 1948; Boyle, 1955) and these have effects were described in pharmacopoeias (Bauer et
been reviewed in the past (Shelef, 1983; Nychas, al., 2001) but their use does not appear to have been
1995) as have the antimicrobial properties of spices widespread in Europe until the 16th century, from
(Shelef, 1983) but the relatively recent enhancement which time they were traded in the City of London
of interest in ‘green’ consumerism has lead to a (Crosthwaite, 1998). Publishing separately in that
renewal of scientific interest in these substances century on the distillation and use of EOs, two
(Nychas, 1995; Tuley de Silva, 1996). Besides anti- Strassburgphysicians,BrunschwigandReiff,mention
bacterial properties (Deans andRitchie, 1987; Carson only a relatively small number of oils between them;
et al., 1995a; Mourey and Canillac, 2002), EOs or turpentine, juniper wood, rosemary, spike (lavender),
theircomponentshavebeenshowntoexhibitantiviral clove, mace, nutmeg, anise and cinnamon (Guenther,
(Bishop, 1995), antimycotic (Azzouz and Bullerman, 1948).AccordingtotheFrenchphysician,DuChesne
1982; Akgu¨l and Kivan(cid:1)c, 1988; Jayashree and Sub- (Quercetanus), in the 17th century the preparation of
ramanyam, 1999; Mari et al., 2003), antitoxigenic EOs was well known and pharmacies generally
(Akgu¨l et al., 1991; Ultee and Smid, 2001; Juglal et stocked 15–20 different oils (Guenther, 1948). The
al.,2002),antiparasitic(Pandeyetal.,2000;Pessoaet use of tea tree oil for medical purposes has been
al., 2002), and insecticidal (Konstantopoulou et al., documented since the colonisation of Australia at the
1992; Karpouhtsis et al., 1998) properties. These end of the 18th century, although it is likely to have
characteristics are possibly related to the function of been used by the native Australians before that
these compounds in plants (Guenther, 1948; Mah- (Carson and Riley, 1993). The first experimental
moud and Croteau, 2002). measurement of the bactericidal properties of the
S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253 225
vapours of EO is said to have been carried out by De Extraction by means of liquid carbon dioxide under
la Croix in 1881 (Boyle, 1955). However, in the low temperature and high pressure produces a more
course of the 19th and 20th centuries the use of EOs natural organoleptic profile but is much more expen-
in medicine gradually became secondary to their use sive (Moyler, 1998). The difference in organoleptic
for flavour and aroma (Guenther, 1948). profileindicatesadifferenceinthecompositionofoils
obtained by solvent extraction as opposed to distilla-
1.2. Current use of EOs tion and this may also influence antimicrobial prop-
erties. This would appear to be confirmed by the fact
ThegreatestuseofEOsintheEuropeanUnion(EU) that herb EOs extracted by hexane have been shown
is in food (as flavourings), perfumes (fragrances and to exhibit greater antimicrobial activity than the
aftershaves) and pharmaceuticals (for their functional corresponding steam distilled EOs (Packiyasothy
properties)(BauerandGarbe,1985;VanWelie,1997; and Kyle, 2002). EOs are volatile and therefore need
VandeBraakandLeijten,1999).Thewell-knownuse to be stored in airtight containers in the dark in order
ofEOinaromatherapyconstituteslittlemorethan2% to prevent compositional changes.
of the total market (Van de Braak and Leijten, 1999). Numerous publications have presented data on the
Individual components of EOs are also used as food composition of the various EOs. The major compo-
flavourings, either extracted from plant material or nentsoftheeconomicallyinterestingEOsaresummar-
syntheticallymanufactured(Oosterhavenetal.,1995). ised by Bauer et al. (2001). Detailed compositional
The antibacterial properties of essential oils and analysisisachievedbygaschromatographyandmass
their components are exploited in such diverse com- spectrometryoftheEOoritsheadspace(Salzer,1977;
mercialproductsasdental root canal sealers(Manabe Scheffer and Baerheim Svendsen, 1981; Wilkins and
etal.,1987),antiseptics(BauerandGarbe,1985;Cox Madsen, 1991; Daferera et al., 2000; Juliano et al.,
et al., 2000) and feed supplements for lactating sows 2000;Jerkovicetal.,2001;Delaquisetal.,2002).EOs
and weaned piglets (Van Krimpen and Binnendijk, can comprise more than sixty individual components
2001; Ilsley et al., 2002). A few preservatives con- (Senatore, 1996; Russo et al., 1998). Major compo-
taining EOs are already commercially available. nentscanconstituteupto85%oftheEO,whereasother
‘DMC Base Natural’ is a food preservative produced componentsarepresentonlyasatrace(Senatore,1996;
by DOMCA S.A., Alhend´ın, Granada, Spain and Bauer et al., 2001). The phenolic components are
comprises 50% essential oils from rosemary, sage chiefly responsible for the antibacterial properties of
and citrus and 50% glycerol (Mendoza-Yepes et al., EOs(Cosentinoetal.,1999).Themajorcomponentsof
1997).‘ProtectaOne’and‘ProtectaTwo’areblended a number of EOs with antibacterial properties are
herbextractsproducedbyBavariaCorp.Apopka,FL, presented in Table 1 and the structural formulae of a
USA and are classed as generally recognized as safe number of antibacterial components are presented in
(GRAS) food additives in the US. Although the Fig. 1. These components have either been shown to
precise contents are not made known by the manu- haveantibacterialactivityorthedataontheirmodeof
facturer, the extracts probably contain one or more action is discussed in this paper. There is some evi-
EOs and are dispersed in solutions of sodium citrate dencethatminorcomponentshaveacriticalparttoplay
and sodium chloride, respectively (Cutter, 2000). in antibacterial activity, possibly by producing a syn-
Further physiological effects of EOs are made use of ergistic effect between other components. This has
in widely differing products such as commercial been found to be the case for sage (Marino et al.,
potato sprout suppressants (Hartmans et al., 1995) 2001), certain species of Thymus (Lattaoui and Tan-
and insect repellents (Carson and Riley, 1993). taoui-Elaraki, 1994; Paster et al., 1995; Marino et al.,
1999)andoregano(Pasteretal.,1995).
The composition of EOs from a particular species
2. Composition of EOs of plant can differ between harvesting seasons and
betweengeographicalsources(ArrasandGrella,1992;
Steam distillation is the most commonly used Marotti et al., 1994; McGimpsey et al., 1994; Cosen-
method for producing EOs on a commercial basis. tino et al., 1999; Marino et al., 1999; Juliano et al.,
226 S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253
Table1
MajorcomponentsofselectedaEOsthatexhibitantibacterialproperties
Common Latinnameofplantsource Majorcomponents Approximate References
nameofEO %compositionb
Cilantro Coriandrumsativum Linalool 26% (Delaquisetal.,2002)
(immatureleaves) E-2-decanal 20%
Coriander Coriandrumsativum(seeds) Linalool 70% (Delaquisetal.,2002)
E-2-decanal –
Cinnamon Cinnamomumzeylandicum Trans-cinnamaldehyde 65% (Lens-Lisbonneetal.,1987)
Oregano Origanumvulgare Carvacrol Trace-80% (Lawrence,1984;Prudentetal.,1995;
Thymol Trace-64% Charaietal.,1996;Sivropoulouetal.,1996;
g-Terpinene 2–52% Kokkinietal.,1997;Russoetal.,1998;
p-Cymene Trace-52% Dafereraetal.,2000;Demetzosand
Perdetzoglou,2001;Marinoetal.,2001)
Rosemary Rosmarinusofficinalis a-pinene 2–25% (Dafereraetal.,2000,2003;Pintoreetal.,2002)
Bornylacetate 0–17%
Camphor 2–14%
1,8-cineole 3–89%
Sage SalviaofficinalisL. Camphor 6–15% (Marinoetal.,2001)
a-Pinene 4–5%
h-pinene 2–10%
1,8-cineole 6–14%
a-tujone 20–42%
Clove(bud) Syzygiumaromaticum Eugenol 75–85% (Baueretal.,2001)
Eugenylacetate 8–15%
Thyme Thymusvulgaris Thymol 10–64% (Lens-Lisbonneetal.,1987;
Carvacrol 2–11% McGimpseyetal.,1994;
g-Terpinene 2–31% Cosentinoetal.,1999;Marinoetal.,1999;
p-Cymene 10–56% Dafereraetal.,2000;Julianoetal.,2000)
aEOswhichhavebeenshowntoexertantibacterialpropertiesinvitroorinfoodmodelsandforwhichthecompositioncouldbefoundin
theliterature.
bPercentagesoftotalvolatilesroundeduptothenearestwholenumber.
2000;Faleiroetal.,2002).This can be explained, at (Kokkinietal., 1997).Generally,EOs produced from
least in part, by the formation of antibacterial herbsharvestedduringorimmediatelyafterflowering
substances from their precursors. p-Cymene (1-meth- possessthestrongestantimicrobialactivity(McGimp-
yl-4-(1-methylethyl)-benzene) and g-terpinene (1- sey et al., 1994; Marino et al., 1999). Enantiomers of
methyl-4-(1-methylethyl)-1,4-cyclohexadiene) are the EO components have been shown to exhibit antimi-
precursors of carvacrol (2-methyl-5-(1-methylethyl)- crobialactivitytodifferentextents(Lis-Balchinetal.,
phenol) and thymol (5-methyl-2-(1-methylethyl)phe- 1999).ThecompositionofEOsfromdifferentpartsof
nol)inspeciesofOriganumandThymus(Cosentinoet thesameplantcanalsodifferwidely.Forexample,EO
al.,1999;Jerkovicetal.,2001;Ulteeetal.,2002).The obtained from the seeds of coriander (Coriandrum
sum of the amounts of these four compounds present sativum L.) has a quite different composition to EO
in Greek oregano plants has been found to be almost ofcilantro,whichisobtainedfromtheimmatureleaves
equal in specimens derived from different geographi- of thesame plant (Delaquiset al.,2002).
calregions(Kokkinietal.,1997)andtoremainstable
in plants harvested during different seasons (Jerkovic
etal.,2001).ThesameistrueofThymusvulgarisfrom 3. In vitro tests of antibacterial activity
Italy(Marinoetal.,1999).Thisindicatesthatthefour
compounds are biologically and functionally closely Tests of antimicrobial activity can be classified as
associated and supports the theory that thymol is diffusion,dilutionorbioautographicmethods(Rioset
formed via p-cymene from g-terpinene in T. vulgaris al., 1988). The principles and practice of these test
S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253 227
Fig.1.StructuralformulaeofselectedcomponentsofEOs.
methods are explained in the literature (Barry, 1976; and incubation time and temperature (Rios et al.,
Davidson and Parish, 1989; Hodges and Hanlon, 1988), comparison of published data is complicated
1991)butitappearsthatnostandardisedtesthasbeen (Janssenetal.,1987;Friedmanetal.,2002).Anumber
developed for evaluating the antibacterial activity of of reviewers have surveyed the methods used for
possible preservatives against food-related microor- antibacterial activity studies carried out with EOs
ganisms,althoughtheneedforsuchhasbeenindicated (Koedam, 1977a,b; Shelef, 1983; Janssen et al.,
(DavidsonandParish,1989).TheNCCLSmethodfor 1987; Rios et al., 1988; Nychas, 1995). In papers
antibacterialsusceptibilitytesting,whichisprincipally published since, the number of variations on culture
aimed at the testing of antibiotics has been modified medium, size of inoculum, choice of emulsifier and
fortestingEOs(Hammeretal.,1999;NCCLS,2000). basic test method has further increased.
Researchers adapt experimental methods to better The minimum inhibitory concentration (MIC) is
representpossiblefutureapplicationsintheirparticular cited by most researchers as a measure of the anti-
field. However, since the outcome of a test can be bacterial performance of EOs. The definition of the
affectedbyfactorssuchasthemethodusedtoextract MIC differs between publications and this is another
the EO from plant material, the volume of inoculum, obstacle to comparison between studies. In some
growth phase, culture medium used, pH of the media cases, the minimum bactericidal concentration
228 S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253
(MBC) or the bacteriostatic concentration is stated, The strength of the antibacterial activity can be
bothtermsagreeingcloselywiththeMIC.Alistofthe determined by dilution of EO in agar or broth. The
most frequently used terms in antibacterial activity published studies using dilution in agar have used
testingofEOsispresentedinTable2.Inaddition,the differentsolventstoincorporatetheEOinthemedium
term‘minimumcidalconcentration’hasbeenusedbut (Prudent et al., 1995; Pintore et al., 2002), different
is not defined (Hammer et al., 1999). The terms volumes of inoculum (1–100 Al) (Juven et al., 1994;
‘minimumlethaldilution(orconcentration)’(Janssen, Prudent et al., 1995), which is sometimes dotted
1989; Janssen et al., 1987) and ‘minimum inhibitory (Pintore et al., 2002) and sometimes streaked (Farag
dilution’ (Janssen, 1989) appear to have fallen out of et al., 1989) onto the agar surface. Despite these
use, at least in food microbiology literature. variations, the MICs of EOs determined by agar
A summary of the techniques used to test the dilution generally appear to be in approximately the
antibacterial activity of EOs is presented in Table 3. sameorderofmagnitude(Faragetal.,1989;Prudentet
ScreeningofEOsforantibacterialactivityisoftendone al.,1995;Pintoreetal.,2002).Inbrothdilutionstudies
by the disk diffusion method, in which a paper disk anumberofdifferenttechniquesexistfordetermining
soaked with EO is laid on top of an inoculated agar the end-point—the most used methods are that of
plate.Thisisgenerallyusedasapreliminarycheckfor optical density (OD) (turbidity) measurement and the
antibacterial activity prior to more detailed studies. enumeration of colonies by viable count. The former
FactorssuchasthevolumeofEOplacedonthepaper method has the advantage of being automated; the
disks, the thickness of the agar layer and whether a latter is labour intensive. The measurements of con-
solventisusedvaryconsiderablybetweenstudies.This ductance/conductivityandend-pointdeterminationby
meansthatthismethodisusefulforselectionbetween visual monitoring have been less often used. A new
EOs butcomparison ofpublisheddata isnotfeasible. microdilutionmethodfordeterminingtheMICofoil-
The agar well test in which the EO is deposited into basedcompoundsusestheredoxindicatorresazurinas
wellscutintotheagarcanbeusedasascreeningmethod a visual indicator of the MIC. The results compare
when large numbers of EOs and/or large numbers of favourably with those obtained by viable count and
bacterialisolatesaretobescreened(Deansetal.,1993; OD measurement and the method is more sensitive
Dorman andDeans,2000).Inorder tomakebacterial than the agar dilution method (Mann and Markham,
growth easier to visualise, triphenyl tetrazolium chlo- 1998).Apatentedcolourindicatorbasedonresazurin
ridemaybeaddedtothegrowthmedium(Elgayyaret has been used to determine the MICs for methanolic
al.,2001;MoureyandCanillac,2002). extractsofplantmaterials(Salvatetal.,2001)andEOs
Table2
Termsusedinantibacterialactivitytesting
Term Definition,withreferencetoconcentrationofEO Reference
Minimuminhibitory Lowestconcentrationresultinginmaintenanceorreduction (Carsonetal.,1995a)
concentration(MIC) ofinoculumviability
Lowest concentration required for complete inhibition of (Wanetal.,1998;CanillacandMourey,2001)
testorganismupto48hincubation
Lowestconcentrationinhibitingvisiblegrowthoftest (KarapinarandAktug,1987;Onawunmi,1989;
organism Hammeretal.,1999;Delaquisetal.,2002)
Lowestconcentrationresultinginasignificantdecreasein (Cosentinoetal.,1999)
inoculumviability(>90%)
Minimumbactericidal Concentrationwhere99.9%ormoreoftheinitialinoculum (Carson et al., 1995b; Cosentino et al., 1999;
concentration(MBC) iskilled CanillacandMourey,2001)
Lowestconcentrationatwhichnogrowthisobservedafter (Onawunmi,1989)
subculturingintofreshbroth
Bacteriostaticconcentration Lowest concentration at which bacteria fail to grow in (Smith-Palmeretal.,1998)
broth,butareculturedwhenbrothisplatedontoagar
Bactericidalconcentration Lowest concentration at which bacteria fail to grow in (Smith-Palmeretal.,1998)
broth,andarenotculturedwhenbrothisplatedontoagar
S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253 229
Table3
TestmethodsusedtomeasuretheantibacterialactivityofEOsandtheirconstituents
Purpose Testmethod References
Screeningfor Diskdiffusion (Faragetal.,1989;Aurelietal.,1992;Kimetal.,1995a,b;
antibacterial Sivropoulouetal.,1996;Firouzietal.,1998;Renzinietal.,
activity 1999;Senatoreetal.,2000;Elgayyaretal.,2001;
Skandamisetal.,2001;Cimangaetal.,2002;Faleiroetal.,
2002;PackiyasothyandKyle,2002;BurtandReinders,
2003;Wilkinsonetal.,2003)
Agarwells (DeansandRitchie,1987;Smith-Palmeretal.,1998;Wan
etal.,1998;DormanandDeans,2000;Rubertoetal.,2000)
Determinationof Agardilutionmethod (KarapinarandAktug,1987;Faragetal.,1989;Paster
strengthof etal.,1990;TingandDeibel,1991;Stecchinietal.,1993;
antibacterial Juvenetal.,1994;PanditandShelef,1994;Prudentetal.,
properties 1995;Ouattaraetal.,1997;Hammeretal.,1999;Pintore
etal.,2002;Wilkinsonetal.,2003;Moleyarand
Narasimham,1992;Negietal.,1999)
Brothdilution Visiblegrowth (Onawunmi,1989;Cosentinoetal.,1999;Elgayyaretal.,
2001;Delaquisetal.,2002;MoureyandCanillac,2002;
Bassoleetal.,2003)
Opticaldensity/turbidity (Shelefetal.,1984;IsmaielandPierson,1990;Kimetal.,
1995a;Sivropoulouetal.,1995;Sivropoulouetal.,1996;
Chaibietal.,1997;Ulteeetal.,1998;PolandSmid,1999;
Lambertetal.,2001;Skandamisetal.,2001;Ulteeand
Smid,2001)
Absorbance (Smith-Palmeretal.,1998;MejlholmandDalgaard,2002)
Colorimetric (Gilletal.,2002;BurtandReinders,2003)
Conductance/conductivity/impedance (Tassouetal.,1995;Wanetal.,1998;Marinoetal.,1999;
Tassouetal.,2000;Marinoetal.,2001)
Viablecount (Beuchat, 1976; Shelef et al., 1984; Tassou et al., 1995;
Sivropoulou et al., 1996; Hammer et al., 1999; Pol and
Smid, 1999; Koidis et al., 2000; Skandamis et al., 2000;
CanillacandMourey,2001;PeriagoandMoezelaar,2001;
Periagoetal.,2001;Friedmanetal.,2002)
Determinationof Time-killanalysis/Survivalcurves (Beuchat,1976;Shelefetal.,1984;TingandDeibel,1991;
rapidityand Aureli et al., 1992; Stecchini et al., 1993; Tassou et al.,
durationof 1995;Sivropoulouetal.,1996;Ulteeetal.,1998;Wan
antibacterial etal.,1998;PolandSmid,1999;PeriagoandMoezelaar,
activity 2001; Skandamis et al., 2001; Ultee and Smid, 2001;
Mejlholm and Dalgaard, 2002; Pintore et al., 2002; Burt
andReinders,2003;Cressyetal.,2003)
Observationof Scanningelectronmicroscopy (Lambert et al., 2001; Skandamis et al., 2001; Burt and
physicaleffects Reinders,2003)
ofantibacterial
activity
(Burt and Reinders, 2003) and the method can be Therapidityofabactericidaleffectortheduration
automatedbymeasuringtheendpointbyfluorescence of a bacteriostatic effect can be determined by time-
instead of visual means (Lancaster and Fields, 1996). killanalysis(survivalcurveplot)wherebythenumber
Triphenyltetrazoliumchloridehasbeenusedforvisual ofviablecellsremaininginbrothaftertheadditionof
end point determination in the study of tea tree oil in EO is plotted against time. The most frequently used
broth but, although it is an indicator of bacterial methods for this are measurement of OD and viable
growth, thecolourchange didnotfullycorrelatewith count after plating out onto agar. Damage to the
theMIC (Carson et al.,1995b). bacterial cell wall and loss of cell contents can be
230 S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253
studied by scanning electron microscopy (SEM) differencebetweencontrolandtreatedcellsaredueto
(Lambert et al., 2001; Skandamis et al., 2001; Burt theeffectoftheEOandnottothepreparationmethod.
and Reinders, 2003). Careful preparation of the sam- Several studies have used the measurement of OD
plesfor SEMisnecessarytoensure thattheobserved or conductance to perform further calculations rather
Table4
SelectedaMICsofessentialoilstestedinvitroagainstfoodbornepathogens
Plantfromwhich Speciesofbacteria MIC,approximate Reference(s)
EOisderived range(Alml(cid:1)1)b
Rosemary Escherichiacoli 4.5–>10 (Farag et al., 1989; Smith-Palmer et al., 1998;
Hammeretal.,1999;Pintoreetal.,2002)
Salmonellatyphimurium >20 (Hammeretal.,1999)
Bacilluscereus 0.2 (Chaibietal.,1997)
Staphylococcusaureus 0.4–10 (Farag et al., 1989; Smith-Palmer et al., 1998;
Hammeretal.,1999;Pintoreetal.,2002)
Listeriamonocytogenes 0.2 (Smith-Palmeretal.,1998)
Oregano E.coli 0.5–1.2 (Prudentetal.,1995;Hammeretal.,1999;
BurtandReinders,2003)
S.typhimurium 1.2 (Hammeretal.,1999)
Staph.aureus 0.5–1.2 (Prudentetal.,1995;Hammeretal.,1999)
Lemongrass E.coli 0.6 (Hammeretal.,1999)
S.typhimurium 2.5 (Hammeretal.,1999)
Staph.aureus 0.6 (Hammeretal.,1999)
Sage E.coli 3.5–5 (Farag et al., 1989; Smith-Palmer et al., 1998;
Hammeretal.,1999)
S.typhimurium 10–20 (Shelefetal.,1984;Hammeretal.,1999)
Staph.aureus 0.75–10 (Shelefetal.,1984;Faragetal.,1989;
Smith-Palmeretal.,1998;Hammeretal.,1999)
L.monocytogenes 0.2 (Smith-Palmeretal.,1998)
Clove E.coli 0.4–2.5 (Farag et al., 1989; Smith-Palmer et al., 1998;
Hammeretal.,1999)
S.typhimurium >20 (Hammeretal.,1999)
Staph.aureus 0.4–2.5 (Farag et al., 1989; Smith-Palmer et al., 1998;
Hammeretal.,1999)
L.monocytogenes 0.3 (Smith-Palmeretal.,1998)
Thyme E.coli 0.45–1.25 (Farag et al., 1989; Smith-Palmer et al., 1998;
Cosentinoetal.,1999;Hammeretal.,1999;
BurtandReinders,2003)
S.typhimurium 0.450–>20 (Cosentinoetal.,1999;Hammeretal.,1999)
Staph.aureus 0.2–2.5 (Farag et al., 1989; Smith-Palmer et al., 1998;
Cosentinoetal.,1999;Hammeretal.,1999)
L.monocytogenes 0.156–0.45 (Firouzi et al., 1998; Smith-Palmer et al., 1998;
Cosentinoetal.,1999)
Turmeric E.coli >0.2 (Negietal.,1999)
B.cereus 0.2 (Negietal.,1999)
Teabush(Lippiaspp.) E.coli 2.5–>80 (Bassoleetal.,2003)
Shigelladysenteria 5–>80 (Bassoleetal.,2003)
Staph.aureus 0.6–40 (Bassoleetal.,2003)
B.cereus 5–10 (Bassoleetal.,2003)
aEOsderivedfromplantsusedasherbs,spicesorinfusionsincookingwereselectedandMICsforaselectionofimportantfoodborne
pathogenscited.
bInthereferencesMICshavebeenreportedintheunitsppm,mgml(cid:1)1,%(v/v),All(cid:1)1andAgml(cid:1)1.Foreaseofcomparisonthesehave
beenconvertedtoAlml(cid:1)1,wherebyitwasassumedthatEOshavethesamedensityaswater.InsomereferencestheMICwastermedminimum
bactericidalorbacteriostaticconcentration(Smith-Palmeretal.,1998;Cosentinoetal.,1999;BurtandReinders,2003).
S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253 231
than directly stating the MIC. The OD of the test 2000;Lambert etal.,2001).Inonestudythepercent-
suspension and control may be used to calculate an age of EO resulting in a 50% decrease in the viable
inhibition index (Chaibi et al., 1997). Measurements count was determined from plots of percentage kill
of conductance can be used to calculate the period against concentration (Friedman et al., 2002). The
elapsing before growth can be detected, the detection diversityofwaysofreportingtheantibacterialactivity
time(DT),aftertreatmentofcellswithEO(Marinoet of EOs limits comparison between studies and could
al., 1999; Tassou et al., 2000; Marino et al., 2001). lead toduplication of work.
Comparison of the maximum specific growth rate Onefeatureoftestmethodsthatvariesconsiderably
(l ) of bacteria based on data from viable counts is whether or not an emulsifier or solvent is used to
max
or absorbance measurements has also been done in a dissolvetheEOortostabiliseitinwater-basedculture
number of studies (Ultee et al., 1998; Skandamis et media. Several substances have been used for this
al., 2000; Mejlholm and Dalgaard, 2002). A new purpose: ethanol (Beuchat, 1976; Deans and Ritchie,
method of calculating the MIC from OD measure- 1987;KarapinarandAktug,1987;Aurelietal.,1992;
mentshasbeenfoundsuitablefortestingcombinations Moleyar and Narasimham, 1992; Juven et al., 1994;
of antibacterial substances (Lambert and Pearson, Lattaoui and Tantaoui-Elaraki, 1994; Pandit and She-
Table5
SelectedaMICsofessentialoilcomponentstestedinvitroagainstfoodbornepathogens
Essentialoil Speciesofbacteria MIC,approximaterange References
component (Alml(cid:1)1)b
a-Terpineol Escherichiacoli 0.450–>0.9 (Cosentinoetal.,1999)
Salmonellatyphimurium 0.225 (Cosentinoetal.,1999)
Staphylococcusaureus 0.9 (Cosentinoetal.,1999)
Listeriamonocytogenes >0.9 (Cosentinoetal.,1999)
Bacilluscereus 0.9 (Cosentinoetal.,1999)
Carvacrol E.coli 0.225–5 (Kimetal.,1995a;Cosentinoetal.,1999)
S.typhimurium 0.225–0.25 (Kimetal.,1995a;Cosentinoetal.,1999)
Staph.aureus 0.175–0.450 (Cosentinoetal.,1999;Lambertetal.,2001)
L.monocytogenes 0.375–5 (Kimetal.,1995a;Cosentinoetal.,1999;
PolandSmid,1999)
B.cereus 0.1875–0.9 (Cosentinoetal.,1999;PolandSmid,1999)
Citral E.coli 0.5 (Onawunmi,1989;Kimetal.,1995a)
S.typhimurium 0.5 (Kimetal.,1995a)
Staph.aureus 0.5 (Onawunmi,1989)
L.monocytogenes 0.5 (Kimetal.,1995a)
Eugenol E.coli 1.0 (Kimetal.,1995a)
S.typhimurium 0.5 (Kimetal.,1995a)
L.monocytogenes >1.0 (Kimetal.,1995a)
Geraniol E.coli 0.5 (Kimetal.,1995a)
S.typhimurium 0.5 (Kimetal.,1995a)
L.monocytogenes 1.0 (Kimetal.,1995a)
Perillaldehyde E.coli 0.5 (Kimetal.,1995a)
S.typhimurium 0.5 (Kimetal.,1995a)
L.monocytogenes 1.0 (Kimetal.,1995a)
Thymol E.coli 0.225–0.45 (Cosentinoetal.,1999)
S.typhimurium 0.056 (Cosentinoetal.,1999)
Staph.aureus 0.140–0.225 (Cosentinoetal.,1999;Lambertetal.,2001)
L.monocytogenes 0.450 (Cosentinoetal.,1999)
B.cereus 0.450 (Cosentinoetal.,1999)
aEOscomponentspresentinplantsusedincookingwereselectedandMICsforaselectionofimportantfoodbornepathogenscited.
bInthereferencesMICshavebeenreportedintheunitsmgml(cid:1)1,%(v/v),All(cid:1)1,Agml(cid:1)1andmmoll(cid:1)1.Foreaseofcomparisonthese
havebeenconvertedtoAlml(cid:1)1,wherebyitwasassumedthatEOshavethesamedensityaswater.Inonereference,theMICwastermed
minimumbactericidalconcentration(Cosentinoetal.,1999).
232 S.Burt/InternationalJournalofFoodMicrobiology94(2004)223–253
lef, 1994; Sivropoulou et al., 1995, 1996; Ouattara et sentedinTables4and5.Considering thediversityof
al., 1997; Marino et al., 1999; Pol and Smid, 1999; test methods, bacterial isolates (clinical or reference)
Marino et al., 2001; Packiyasothy and Kyle, 2002), and origins of the EOs used, the range of MICs
methanol (Onawunmi, 1989), Tween-20 (Kim et al., appears considerably narrow in most cases.
1995b; Mann and Markham, 1998; Hammer et al.,
1999), Tween-80 (Paster et al., 1990; Juven et al.,
1994;CarsonandRiley,1995;Cosentinoetal.,1999; 4. Tests of antibacterial activity of EOs in food
Mourey and Canillac, 2002; Bassole et al., 2003; systems
Wilkinson et al., 2003), acetone in combination with
Tween-80 (Prudent et al., 1995), polyethylene glycol Although, as mentioned previously, a small num-
(Pintore et al., 2002), propylene glycol (Negi et al., ber of food preservatives containing EOs is commer-
1999), n-hexane (Senatore et al., 2000), dimethyl ciallyavailable,untiltheearly1990sveryfewstudies
sulfoxide (Firouzi et al., 1998) and agar (Mann and of the activity of EOs in foods had been published
Markham,1998;Delaquisetal.,2002;Gilletal.,2002; (BoardandGould,1991).Sincethenafairnumberof
Burt and Reinders, 2003). However, a number of trials have been carried out with EOs in foods. An
researchers found it unnecessary to use an additive overview of the literature reporting studies on the
(Smith-Palmeretal.,1998;Wanetal.,1998;Cosentino antibacterial effect of EOs or their components in
etal.,1999; Renzini etal.,1999; Dorman and Deans, foodsispresentedinTable6.Reportsofstudiesusing
2000;Tassouetal.,2000;CanillacandMourey,2001; dilutedfoodsorfoodslurries(Poletal.,2001;Smith-
Elgayyaretal.,2001;Lambertetal.,2001;Cimangaet Palmer et al., 2001) and studies using dried herbs or
al., 2002; Mejlholm and Dalgaard, 2002). One study spicesortheirextracts(Tassouetal.,1996;Haoetal.,
employedvigorousshakinginphosphatesalinebuffer 1998a,b) have not been included in the table.
to suspend EOs (Friedman et al., 2002). The perfor- However well EOs perform in antibacterial assays
mance of the most frequently used solvents, ethanol in vitro, it has generally been found that a greater
andTween-80,hasbeencomparedwiththatofagarfor concentration of EO is needed to achieve the same
thestabilisation oforeganoandcloveoils.Theuseof effectinfoods(Shelef,1983;SmidandGorris,1999).
agar (0.2%) was found to produce as homogenous a The ratio has been recorded to be approximately
dispersion as a true solution in absolute ethanol twofold in semi-skimmed milk (Karatzas et al.,
(Remmal et al., 1993b). Furthermore, the MICs of 2001), 10-fold in pork liver sausage (Pandit and
oregano and clove EOs were significantly lower in Shelef, 1994), 50-fold in soup (Ultee and Smid,
thepresenceofagarthaninthepresenceofTween-80 2001) and 25- to 100-fold in soft cheese (Mendoza-
or ethanol. It was concluded that solvents and deter- Yepesetal.,1997).Anexceptiontothisphenomenon
gents could decrease the antibacterial effect of EOs isAeromonashydrophila;nogreaterproportionofEO
(Remmaletal.,1993a,b).Thisissupportedbythefact wasneededtoinhibitthisspeciesoncookedporkand
thatTween-80hasbeenrecommendedasaneutraliser on lettuce incomparison totests invitro (Stecchiniet
of phenolic disinfectants (Cremieux et al., 1981) and al., 1993; Wan et al., 1998). Several studies have
thishasbeenconfirmedinworkontheactionofthyme recorded the effect of foodstuffs on microbial resis-
oil against Salmonella typhimurium (Juven et al., tancetoEOsbutnoneappearstohavequantifieditor
1994). Tween-80 has also been shown to protect to have explained the mechanism, although sugges-
Listeriamonocytogenesfromtheantibacterialactivity tions have been made as to the possible causes. The
of an EO component during freeze–thaw cycles greater availability of nutrients in foods compared to
(Cressy et al., 2003). A further disadvantage of the laboratory media may enable bacteria to repair dam-
use of Tween-80 to dissolve EOs is the fact that the aged cells faster (Gill et al., 2002). Not only are the
turbidityoftheresultingdispersioncanhampervisual intrinsic properties of the food (fat/protein/water con-
observations and OD measurements (Carson et al., tent, antioxidants, preservatives, pH, salt and other
1995b). additives)relevantinthisrespect—theextrinsicdeter-
A selection of MICs for EOs and EO components minants (temperature, packaging in vacuum/gas/air,
tested in vitro against food borne pathogens is pre- characteristics of microorganisms) can also influence