Antimicrobial Activity of Plant Essential Oils Using Food Model Media: Efficacy, Synergistic Potential and Interaction with Food Components

Transcription

1 Dublin Institute of Technology Articles School of Food Science nd Environmentl Helth Antimicrobil Activity of Plnt Essentil Oils Using Food Model Medi: Efficcy, Synergistic Potentil nd Interction with Food Components Jorge Gutierrez Dublin Institute of Technology, Ctherine Brry-Ryn Dublin Institute of Technology, Pul Bourke Dublin Institute of Technology, Follow this nd dditionl works t: Prt of the Food Microbiology Commons Recommended Cittion Gutierrez, J., Brry-Ryn, C. & Bourke, P. (0) Antimicrobil ctivity of plnt essentil oils using food model medi: efficcy, synergistic potentil nd interction with food components. Food Microbiology, Vol., issue, April 0, pp.-0. doi:.1/ j.fm This Article is brought to you for free nd open ccess by the School of Food Science nd Environmentl Helth t ARROW@DIT. It hs been ccepted for inclusion in Articles by n uthorized dministrtor of ARROW@DIT. For more informtion, plese contct yvonne.desmond@dit.ie, rrow.dmin@dit.ie, brin.widdis@dit.ie.

2 Title: Antimicrobil ctivity of plnt essentil oils using food model medi: efficcy, synergistic potentil nd interctions with food components J. Gutierrez, C. Brry-Ryn, P. Bourke* School of Food Science nd Environmentl Helth, Dublin Institute of Technology, Cthl Brugh Street, Dublin 1, Irelnd * Corresponding uthor. Tel: +-0; Fx: +-0; E-mil: pul.bourke@dit.ie Running Title: Antibcteril efficcy of plnt EO s in food model medi Key words: essentil oils, ntimicrobil, synergy, food model, food composition 1

3 Abstrct The im of this study ws to optimize the ntimicrobil efficcy of plnt essentil oils (EO s) for control of Listeri spp. nd spoilge bcteri using food model medi bsed on lettuce, met nd milk. The EO s evluted were lemon blm, mrjorm, oregno nd thyme nd their minimum inhibitory concentrtions (MIC) were determined ginst Enterobcter spp., Listeri spp., Lctobcillus spp., nd Pseudomons spp. using the gr dilution method nd/or the bsorbnce bsed microplte ssy. MICs were significntly lower in lettuce nd beef medi thn in TSB. Listeri strins were more sensitive thn spoilge bcteri, nd oregno nd thyme were the most ctive EO s. EO combintions were investigted using the checkerbord method nd Oregno combined with thyme hd dditive effects ginst spoilge orgnisms. Combining lemon blm with thyme yielded dditive ctivity ginst Listeri strins. The effect of simple sugrs nd ph on ntimicrobil efficcy of oregno nd thyme ws ssessed in beef extrct nd tomto serum model medi. EO s retined greter efficcy t ph nd.% sugr, but sugr concentrtions bove % did not negtively impct EO efficcy. In ddition to proven ntimicrobil efficcy, creful selection nd investigtion of EO s pproprite to the sensory profile of foods nd composition of the food system is required. This work shows tht EO s might be more effective ginst food-borne pthogens nd spoilge bcteri when pplied to foods contining high protein level t cidic ph, s well s moderte levels of simple sugrs. 1

4 Introduction Illnesses cused due to the consumption of foods contminted with pthogens such s Listeri monocytogenes hs wide economic nd public helth impct worldwide (Gndhi nd Chikinds 0). L. monocytogenes cn dpt to survive nd grow in wide rnge of environmentl conditions s well s in lrge vriety of rw nd processed foods, including milk nd diry products, vrious mets nd met products or fresh produce. Food spoilge includes physicl dmge, chemicl chnges, such s oxidtion, color chnges, or ppernce of off-flvors nd off-odors resulting from microbil growth nd metbolism in the product (Grm et l. 0). The spoilge of refrigerted met is cused in prt by Pseudomons species which re responsible for the off-odors, offflvors, discolortion, gs production nd slime production (Ousslh et l. 0). In some cses, chnge in tmosphere by vcuum-pcking inhibits the erobic pseudomonds in mets cusing shift in the microflor to lctic cid bcteri (LAB) nd Enterobctericee (Grm et l. 0). The pseudomonds re lso found in psteurized milk nd re generlly from post-process contmintion (Eneroth et l. 00). The spoilge microflor ssocited with fresh vegetbles includes Pseudomons spp. s well s other Grm-negtive bcteri, such s Enterobcteri (Rgert et l. 0). Current technologies for preservtion nd shelf life extension of food include chemicl preservtives, het processing, modified tmosphere pckging (MAP), vcuum pckging (VP) or refrigertion. Unfortuntely, these steps do not eliminte undesirble pthogens such s L. monocytogenes from these products or dely microbil spoilge entirely. Alterntive preservtion techniques such s novel non-therml technologies nd

5 nturlly derived ntimicrobil ingredients re under investigtion for their ppliction to food products. Greter consumer wreness nd concern regrding synthetic chemicl dditives hs led reserchers nd food processors to look for nturl food dditives with brod spectrum of ntimicrobil ctivity (Mrino et l. 01). In this context, plnt essentil oils re gining interest for their potentil s preservtive ingredients or decontminting tretments, s they hve GRAS sttus nd wide cceptnce from consumers (Burt et l. 0). The ntimicrobil components re commonly found in the essentil oil frctions nd it is well estblished tht mny hve wide spectrum of ntimicrobil ctivity, with potentil for control of L. monocytogenes nd spoilge bcteri within food systems (Smith-Plmer et l., Hmmer et l., Elgyyr et l. 01, Dormn nd Dens 0, Moreir et l. 0, Ousslh et l. 0b, Gutierrez et l., 0). Oregno (Orignum vulgre) nd thyme (Thymus vulgris) re mongst the most ctive EO s, while lemon blm (Meliss officinlis) nd mrjorm (Orignum mjorn) disply good ntimicrobil ctivity ginst Grm-positive nd Grm-negtive bcteri, respectively. Recently, some reserchers hve reported the efficcy of plnt EO s s ntimicrobil gents ginst food borne pthogens nd spoilge microflor in met (Bustt et l., 0; Crrmiñn et l., 0). Although some studies hve shown tht plnt extrcts re useful for reduction of pthogens ssocited with met (Mytle et l. 0, Ahn et l. 0), others reported very low ntimicrobil ctivity or no effect ginst L. monocytogenes or Slmonell when EO s were pplied to beef or chicken (Uhrt et l. 0, Firouzi et l. 0). Thus, the ppliction of plnt EO s for control of food-borne pthogens nd food spoilge bcteri requires the evlution of efficcy

6 within food products or in model systems tht closely simulte food composition. In generl, the efficcy of mny dded nd nturlly occurring ntimicrobils my be reduced by certin food components (Glss nd Johnson 0). Therefore, to successfully pply EO s in food systems, primry studies in representtive food model medi should be employed to determine potentil interctions between EO s nd food components tht could impct on their ntimicrobil efficcy. Another spect for the optimized ppliction of EO s in foods is the impct on sensory cceptbility. If high concentrtions re required to chieve useful EO ntimicrobil ctivity, uncceptble levels of inpproprite flvours nd odours my result. We previously reported tht lettuce smples treted with thyme nd lemon blm t concentrtions of 00 nd 1,000 ppm, respectively, were rejected by pnelists s they perceived strong chemicl odors from these smples (Gutierrez et l. 0). Therefore, reserch in this re should be focused on optimizing EO combintions nd pplictions to obtin effective ntimicrobil ctivity t sufficiently low concentrtions so s not to dversely ffect the orgnoleptic cceptbility of foods. Furthermore, the use of ntimicrobils cn reduce or eliminte trget microorgnisms but it my lso produce fvorble conditions for other microorgnisms (Dvidson nd Brnen 0). It is recognized tht this sitution is less likely to develop towrds substnces tht hve more thn one mode of ction (Ippolito nd Nigro 0). It is suggested tht the ntimicrobil ctivity of EO s is ttributed to more thn one mechnism (Burt 0, Moreir 0). Thus, combining EO s could led to useful efficcy ginst both spoilge nd pthogenic trget orgnisms. Whole plnt extrcts hve higher ntimicrobil ctivity thn when

7 mjor components re mixed, nd minor components of plnt EO s my be criticl to ctivity with potentiting influence or synergistic effects (Burt 0). Thus, the min objectives of this work were: (i) to evlute the ntimicrobil ctivity of plnt essentil oils (EO s) ginst Listeri spp. nd spoilge bcteri in food model medi, in order to optimize product ppliction, (ii) to ssess the efficcy of EO s in combintion ginst selected bcteri to determine potentil for their synergistic ppliction t low doses; nd (iii) to monitor nd quntify the effect of food components on the EO efficcy. The sensitivity of different ntimicrobil ssys ws lso ssessed nd compred in order to select those tht were the most suitble to clculte MICs.. Mteril nd methods Essentil oils The essentil oils (EO s) used in this study were lemon blm (Meliss officinlis), mrjorm (Orignum mjorn), oregno (Orignum vulgre) nd thyme (Thymus vulgris). They were selected bsed on previously reported efficcy (Gutierrez et l. 0), nd were obtined from Guinness Chemicl Ltd. (Portloise, Irelnd) s CO soluble supercriticl fluid extrcts. 1.. Bcteri The bcteri used in this study re listed in Tble 1. All cultures were mintined t -0ºC in % glycerol nd grown in Tryptic Soy Broth (TSB, ph., Schrlu Chemie) for hours t 0ºC, except for the Listeri strins, which were incubted t ºC, in order to

8 obtin sub-cultures. Working cultures were prepred in selected model medi from subcultures nd grown under optiml conditions for ech bcterium for hours. Working cultures were djusted to the required concentrtion of CFU/ml using the McFrlnd stndrd (Biomerieux Inc.) Food model medi Lettuce lef model medi (L) ws prepred s described by Frncis et l. () but with some modifictions. 0g of iceberg lettuce (Lctuc stiv sp.) were dded to 0ml of sterile deionized wter nd shken for 1 min. The suspension ws filtered using 1. cm Whtmn filters nd ph ws djusted from. to. by mixing two prts of lettuce medi with one prt 0.M potssium phosphte buffer, giving finl concentrtion of 0.1M phosphte buffer, ph.. The buffered medium ws then utoclved t ºC for 1 min. To investigte the EO efficcy in met-bsed model medi, experiments were performed with utoclved beef extrct (BE, 1% protein, Schrlu Chemie). Milk model medi (M) ws mde mixing skimmed milk powder (Schrlu Chemie) with gr solution (Schrlu Chemie), both utoclved seprtely, in order to obtin finl solid medi solution with 1.% gr. Beef extrct nd milk model medi were djusted to ph. to seprte ph effects. 1.. Kinetic nlyses Bcteri which were grown in TSB, lettuce lef model medi or beef extrct (Tble 1), were monitored in microplte spectrophotometer (PowerWve, Biotek) t 00 nm over h t 0 min intervls. Growth curves were nlyzed using Gen softwre (Biotek) nd

9 the increse in lg phse (λ) nd the mximum specific growth rte (µmx) were clculted. Sttisticl nlysis ws performed using SPSS 1.0 (SPSS Inc., Chicgo, U.S.A). Dt represent the mens of experiments performed in duplicte nd replicted t lest twice. Differences between bcteri were nlyzed by ANOVA followed by LSD (p < 0.0). Differences between control medi (TSB) nd model medi were exmined using pired smple t-tests (p < 0.0) Antimicrobil ssys The Agr-well Diffusion Test (ADT), Agr Dilution Method (ADM) nd bsorbnce bsed Microtitre Plte Assy (MPA) were used to determine the MICs of selected EO s. MICs were considered s the lowest concentrtion of the EO resulting in complete inhibition of growth nd were obtined from t lest different experiments nd expressed in ppm. Differences between ntimicrobil ssys were nlyzed by ANOVA followed by LSD (p < 0.0) Agr-Well Diffusion Test (ADT) The ADT ws performed s previously described (Bgmboul et l. 0, Schelz et l. 0) but with some modifictions. ml of Tryptic Soy Agr (TSA, ph., Schrlu Chemie) were inoculted with CFU/ml of the indictor strin nd then poured onto Petri dish nd llowed to solidify. Wells of.-mm dimeter were septiclly bored into the gr, nd 0 µl of serilly-diluted EO solutions in ethnol, were dded to the wells. The pltes were kept t o C for h to llow dispersl nd subsequently incubted under optiml conditions for growth of the trget strins. The ntimicrobil ctivity ws visully

10 pprised s inhibition zones surrounding the wells. Ethnol ws used s negtive control nd the indictor strins were L. innocu NCTC nd P. fluorescens Agr Dilution Method (ADM) The ADM ws performed s described by Hmmer et l. () nd Ousslh et l. (0b), but with some modifictions. TSA or Milk Model Medi were inoculted with the pproprite EO nd serilly diluted using the sme model medi to the pproprite concentrtions, poured onto Petri dish nd llowed to solidify. Pltes were then seeded with CFU of the trget microorgnism, nd incubted t the pproprite temperture. The positive control consisted of TSA or Milk Model Medi inoculted with the sme mount of cells but without ny EO, while uninoculted pltes contining the EO served s negtive control. Trget microorgnisms were previously grown in TSB or liquid model medi to llow the cells to dpt to the food environment. L. innocu NCTC nd L. monocytogenes NCTC1 were the trget Listeri strins seeded into TSA nd the milk model medi, respectively. P. fluorescens ws selected s trget in both medi. Pltes were evluted for the presence or the bsence of colonies fter hours of incubtion t conditions optiml for ech bcterium Absorbnce bsed Microtitre Plte Assy (MPA) Ninety-six well microtitre pltes were used (Srstedt Ltd) to perform the MPA. This ssy ws bsed on previous work (Schelz et l. 0) but with the following modifictions, where liquots of EO solutions in growth medi (0 µl) were dded into the first row of microtitre plte. The reminder of the wells were filled with 0 µl of the pproprite

11 medium. The EO s were then diluted two fold long ech column. Finlly, 0 µl of medi contining x CFU/ml of the indictor strin ws dded to ll wells. Positive controls contined growth medi inoculted with the orgnism under investigtion. Negtive controls contined EO s nd sterile growth medi only. The pltes were then plced in the Biotek microplte spectrophotometer set t the pproprite temperture for ech test orgnism. The bsorbnce ws recorded t 00 nm every 0 minutes over hour incubtion period Synergy studies: checkerbord method The checkerbord method ws performed using -well microtitre pltes (Schelz et l., 0) to obtin the frctionl inhibitory concentrtion (FIC) index of EO combintions EO s in the lettuce lef model medi. Pltes consisted of columns contining 0 µl of EO A diluted twofold in lettuce model medi long the x xis s well s rows with the sme mount of EO B diluted twofold in the sme medi long the y xis. Subsequently, 0 µl of the lettuce medi contining x CFU/ml of the indictor strin were dded to ll wells. Pltes were then incubted t o C for h. The FIC indices were clculted s FIC A + FIC B, where FIC A = (MIC A combintion / MIC A lone) nd FIC B = (MIC B combintion / MIC B lone). The results were interpreted s synergy (FIC < 0.), ddition (0. FIC 1), indifference (1 < FIC ) or ntgonism (FIC > ). Experiments were performed in triplicte. Combintions of oregno, thyme, bsil nd mrjorm were tested ginst spoilge bcteri, wheres mixtures of oregno, thyme, lemon blm nd sge were tested ginst the Listeri strins. Concentrtions used for the combintions were bsed on MIC vlues

12 obtined in lettuce lef model medi nd ssys were performed in duplicte nd then replicted Interctive effects of food ingredients nd ph in beef extrct nd tomto serum medi (BE-TS) The effect of food ingredients nd ph on the ntimicrobil efficcy of EO s ws performed using rnge of model medi consisting of beef extrct mixed with tomto serum (Schrlu Chemie) t different rtios (Tble ). The concentrtions of protein, ft nd slt were suitble for optiml EO efficcy (Gutierrez et l., 0b), while percentge of crbohydrtes, minly composed of glucose nd fructose, incresed from 0 to.% nd the ph rnge ws from.0 to.. L. monocytogenes NCTC, L. skei ATCC nd P. putid were chosen s trget microorgnisms. The growth of selected bcteri in ech model medium with EO ws monitored using the well-micropltes, which were performed nd ssessed in the Biotek microplte spectrophotometer. A second btch of experiments ws performed in the sme model medi but djusted to ph.. The effect of food components on EO efficcy ws evluted considering the MIC nd the growth prmeters of trget bcteri, s described in sections. nd.., respectively. Positive controls contined model medi inoculted with the orgnism under investigtion. Negtive controls contined EO s nd sterile model medi only. 1. Results.1. Kinetic nlysis in food model medi

13 The lg phse nd µmx of bcteri grown in TSB, lettuce medi or BE re shown in Tble. Bcteril growth ws function of the medi used. The lg phse nd µmx of ll bcteri grown in lettuce medi ws longer nd lower respectively, thn in TSB or BE (p < 0.0). In generl, no significnt differences were observed between lg phse nd growth rtes vlues of bcteri grown in TSB nd BE (p < 0.0). Growth rte of the reference strin L. monocytogenes NCTC ws significntly lower (p < 0.0) in BE thn in TSB. In lettuce medi, the lg phse of spoilge bcteri ws considerbly shorter thn tht of Listeri spp. (p < 0.0). Growth rtes of ll bcteri cultured in lettuce medi were similr, wheres in TSB growth rtes of spoilge orgnisms were lower thn those for Listeri strins (p < 0.0) Sensitivity of ntimicrobil ssys When MICs of selected EO s were compred using different ntimicrobil ssys (MPA, ADM nd ADT), no significnt differences were observed between MICs of oregno, thyme or lemon blm tested by MPA nd ADM (Tble ). Furthermore, the MICs of oregno nd thyme ginst both trget microorgnisms s well s those of lemon blm ginst the Listeri strin, were significntly lower (p< 0.0) using MPA or ADM thn those recorded by ADT. When indictor strins were exposed to mrjorm only, the MICs clculted by ADT were the sme s those observed by MPA or ADM. Therefore, ADM nd MPA protocols were selected s most pproprite for clculting MICs in solid nd liquid food model medi, respectively... Antimicrobil efficcy in food model medi (MPA method) 1

14 The MIC vlues obtined for ech EO in TSB, lettuce lef model medi nd beef extrct re presented in Tble. The verge efficcy of EO s ginst Listeri spp. ws in the following order: oregno thyme > lemon blm, while the efficcy order of EO s ginst the spoilge bcteri ws: oregno thyme > mrjorm. When P. fluorescens nd the Listeri spp. were exposed to the EO s in lettuce medi, the MIC vlues were pproximtely fold lower thn in TSB for ll EO s. However, when E. cloce ws exposed to EO s within TSB or vegetble model medi, the MIC vlues were comprble. E. cloce ws more susceptible to the EO s thn P. fluorescens in TSB. In BE, MICs of EO s ginst Listeri spp. were significntly lower (P < 0.0) thn in TSB. MICs of lemon blm ginst the food-borne pthogen in BE were comprble to those observed in the vegetble medi. MICs of oregno nd thyme ginst Pseudomons spp. in BE were similr to those found in TSB, whilst the MIC of mrjorm ginst the sme spoilge bcteri ws significntly lower (p<0.0) in BE thn in TSB. Listeri strins were lwys more sensitive thn the spoilge bcteri. Furthermore, when L. monocytogenes NCTC1 nd P. fluorescens were exposed to oregno or thyme on milk model medi (M), it ws observed tht the MICs of these EO s were pproximtely fold higher thn those obtined on the control medi TSA. MICs of oregno nd thyme ginst the Listeri strin on the milk model medi were 1,000 nd,000 ppm respectively. P. fluorescens ws more resistnt to both oregno nd thyme on sme food model medi, with corresponding MICs of,000 nd,000 ppm, respectively... Synergy studies 1

15 The FIC indices for the EO combintions in lettuce lef model medi re shown in Tble. With reference to the FIC scle, no synergistic effect (< 0.) ws found, but ddition occurred with number of combintions. More incidences of dditive effects were found with EO combintions ginst Listeri strins. Combintions of oregno with thyme or lemon blm were more effective ginst L. monocytogenes. The combintion of thyme with lemon blm hd greter efficcy ginst L. innocu. Only one combintion (oregno with thyme) hd dditive effects ginst both spoilge microorgnisms. No ntgonism ws observed for ny of the combintions evluted... Influence of BE-TS model medi composition on bcteril growth As shown in figure 1, the µmx of L. monocytogenes nd P. putid incresed significntly (p < 0.0) when grown in medium B (ph.0, 1.1% sugrs, see Tble ) thn in medium A (ph.0; 0% sugrs). A similr trend ws observed when L. skei grew in medium C (ph.;.% sugrs), by comprison with medium B. On the contrry, the µmx vlues of L. monocytogenes nd L. skei grown in medium D (ph.;.0% sugrs) were significntly lower (p < 0.0) thn those obtined in medium C. Considering the lg phse of selected bcteri, no significnt differences were observed between medi A nd B. However, the lg phse vlues of L. monocytogenes nd L. skei grown in medi D nd C, respectively, were significntly longer (p < 0.0) thn those obtined in medium C, for L. monocytogenes, or medium B, for L. skei. The opposite ws observed for P. putid since its lg phse ws significntly reduced (p < 0.0) in medium C, compred to medi B or A. None of the trget micro orgnisms were cpble of growing in model medium E (ph.;.% sugrs). P. putid ws lso unble to grow in medium D.

16 Influence of BE-TS model medi composition on EO efficcy The EO efficcy incresed significntly (p < 0.0) in BE-TS model medi contining mjor percentge of sugrs s well s more cidic ph vlues (Tble ). However, the MICs of oregno or thyme ginst P. putid were the sme (p < 0.0) when tested in the different food model medi. Growth experiments with the selected bcteri were lso performed in the sme BE-TS model medi but djusted to ph., in order to investigte the effect of sugrs on the EO ntimicrobil ctivity. In generl, the µmx of the cultures exposed to oregno or thyme decresed when the percentge of sugrs incresed (Fig. ). Moreover, when L. monocytogenes ws grown in medium B (1.1% sugrs) contining the EO s, the growth rte vlues incresed, by comprison with those recorded in medium A (0.00% sugrs). Similr trends were observed with controls. However, the µmx of Listeri cultures in medium C (.% sugrs) with oregno or thyme ws lower (p < 0.0) thn tht observed in medium B with the sme EO s. The growth rte vlues of Listeri control cultures in medi B nd C were not significntly different (p < 0.0). When L. skei nd P. putid were exposed to thyme in medi B nd C, respectively, the µmx decresed (p < 0.0) compred to those obtined in medium A, for L. skei, nd medium B, for P. putid. With respect to the control cultures, there ws no significnt difference in the growth rte of L. skei in medi A nd B s well s tht of P. putid in medi B nd C (p < 0.0). In generl, the lg phse of cultures grown in neutrlized model medi regrdless of presence or bsence of EO s incresed significntly (p < 0.0) in medium E (Fig. ). Furthermore, inclusion of oregno or thyme led to significntly 1

17 longer lg phse with 0 to.% of sugrs, by comprison with control (p < 0.0). The lg phse of P. putid grown in model medium C contining oregno ws longer thn in medium B. When the sme bcterium ws exposed to thyme, the lg phse incresed significntly in medium B, by comprison with medium A. In the control cultures, no significnt differences were observed between lg phse vlues in medi A, B nd C Discussion Most reserchers currently use gr or broth dilution series to ssess ntimicrobil ctivity of spices, herbs nd their EO s, nd in some cses both ssys for comprtive purposes becuse ntimicrobil performnce in the two systems cn vry (Holley nd Ptel 0). In this work, no significnt differences were observed between MIC vlues using the Microplte Assy (MPA) or the Agr Dilution Method (ADM). Furthermore, these methods proved to be more sensitive thn the Agr well-diffusion Test (ADT). Although tube mcrodilution nd diffusion from inhibitor-impregnted pper discs on gr surfces re still used, there is hevy relince on microwell plte systems contining inhibitors nd trget microorgnisms in broth. Some uthors hve suggested tht the gr well/disk diffusion tests might only be used s selection method when lrge numbers of EO s nd or bcteril isoltes hve to be screened, since the comprison of published dt re not fesible (Dormn nd Dens 00, Burt 0). The hydrophobicity of EO components is known to limit the vlue of these diffusion tests for estimting ntimicrobil potency ccurtely (Holley nd Ptel 0). Although severl substnces hve been used to dissolve the EO or to stbilize it in wter-bsed culture medi, such s ethnol, methnol, Tween-, Tween-0, cetone, polyethylene glycol, propylene glycol, n-hexne, dimethyl sulfoxide or 1

18 gr (Burt 0), we did not find ny improvement on the EO efficcy by using some of these substnces, in greement with other reserchers, such s Smith-Plmer et l. (), Dormn nd Dens (00) or Elgyyr et l. (01) Over the lst decde mny tests hve been crried out in synthetic growth medi in order to evlute the EO ntimicrobil ctivity ginst spoilge nd food-borne pthogens ssocited with met, milk nd vegetbles. However, results obtined in model medi my be more useful prior to further studies on rel food, rther thn those observed using stndrd lbortory medi, since these product liquid models my ssist in the optimised finl ppliction of EO s nd would lso reflect the nutrient vilbility nd composition of food produce. In this respect, some uthors hve lredy used fruit nd vegetble model medi to investigte EO efficcy (Cerrutti nd Alzmor, Del Cmpo et l. 00, Hsieh et l. 01, Ultee nd Smid 01, Vlero nd Slmeron, 0). In most of these cses the plnt extrcts efficcy decresed in the food model medi, by comprison with the in vitro control medi In this study, the ntimicrobil efficcy of plnt EO s ws evluted in different food model medi nd compred to tht observed in lb control medi (TSB) using their MIC vlues ginst spoilge bcteri nd Listeri spp Since food system composition is known to impct on the ntimicrobil efficcy of EO s, Burt (0) suggested tht the low ft content of vegetbles my contribute to the success of EO s in fresh produce. In most cses the efficcy of EO s in lettuce model medi ws fold times higher thn tht in TSB (Tble ). The fct tht the lg phse nd the growth rte of ll bcteri in lettuce medi ws longer nd lower respectively, by comprison to those observed within TSB (Tble ), my hve contributed to the higher 1

19 efficcy of EO s in the vegetble medi. The rich nutrients in TSB compred to lettuce medi my enble bcteri to repir dmged cells fster, s suggested by Gill et l. (0). However, the EO s were more effective in BE thn in TSB nd the MIC of lemon blm in the met bsed model medi ws comprble to tht obtined in lettuce medi. Gutierrez et l. (0b) observed tht the presence of high concentrtions of protein in BE promoted the growth of L. monocytogenes, but the efficcy of oregno nd thyme ws lso greter t these higher concentrtions of protein. These uthors explined tht peptones with hydrophobic properties might disply interctions with EO s to fcilitte their dissolution in BE. Brnuskien et l. (0) reported tht proteins usully possess high binding cpcity for flvor voltile compounds. Recently, some studies hve recorded the EO ntimicrobil efficcy, lone or in combintion with other preservtion methods, ginst spoilge nd food-borne pthogens when pplied to met (Mytle et l. 0, Ahn et l. 0, Ghlfi et l. 0, Solomkos et l. 0) or milk (Cv et l. 0). Prticulrly, Creg et l. (0) observed tht chilli extrcts (Cpsicum nnuum) hd bcteriosttic effect ginst P. eruginos t concentrtions of,000 ppm. In this study the MICs of oregno nd thyme ginst the Pseudomons strins were 1,00 nd,00 ppm, respectively, in BE. When Cv et l. (0) ssessed the ntimicrobil ctivity of EO s of cinnmon brk, cinnmon lef, nd clove ginst L. monocytogenes in semi skimmed milk incubted t C for dys, they observed tht the MIC ws 00 ppm for cinnmon brk EO nd,000 ppm for the cinnmon lef nd clove EO s. Concentrtions incresed to 1,000 ppm for cinnmon brk EO,,00 ppm for clove EO, nd,000 ppm for cinnmon lef EO when the semi skimmed milk ws incubted t C for h. The ntimicrobil efficcy of oregno nd 1

20 thyme ginst L. monocytogenes in the milk model medi used in this work ws very similr, with corresponding MICs of 1,000 ppm nd,000 ppm, respectively. The EO s possessing the strongest ntibcteril properties re usully composed of phenolic compounds, such s eugenol (clove, cinnmon lef), cinnmic cid (chilli, cinnmon brk), crvcrol (oregno) or thymol (thyme) (Burt 0, Holley nd Ptel 0), thus it seems resonble tht their mechnism of ction nd ntimicrobil efficcy would be similr Oregno nd thyme were the most effective EO s for inhibition of Listeri nd spoilge orgnisms in ll the food model medi (Tble ). Mrjorm lso displyed high ntimicrobil ctivity ginst the Grm-negtive bcteri, while lemon blm hd good efficcy ginst the Grm-positive Listeri spp. (Tble ). The high ntimicrobil ctivity of mrjorm ginst Grm-negtive bcteri might be due to the presence of hydroxyl groups in EO compounds, s described previously (Elgyyr et l. 01, Burt 0, Ousslh et l. 0b). Longry Delmre et l. (0) ttributed the strong ctivity of sge ginst Grm-positive bcteri to the presence of β-cryophyllene, compound tht is found in the composition of the lemon blm EO s used in this study Plnt EO s re generlly more ctive ginst grm-positive bcteri thn grmnegtive bcteri (Burt 0). Some uthors suggest tht the outer membrne surrounding the cell wll of grm-negtive bcteri my restrict diffusion of hydrophobic compounds through its lipopolyscchride covering (Vr, Dvidson nd Brnen 0). In the current work, grm-negtive strins, P. fluorescens, P. putid nd E. cloce were more resistnt to the ction of the EO s thn the Grm-positive Listeri spp.

21 (Tble ). As the lg phse vlues of both Listeri strins in lettuce medi were much longer thn those obtined for spoilge orgnisms (Tble 1), this my hve lso promoted the efficcy of the EO s. However, the MIC vlues for oregno nd thyme ginst Listeri spp. were similr to those observed with the sme EO s ginst E. cloce (Tble ) in TSB. The growth rte of E. cloce in TSB ws pproximtely fold lower thn those ttined by the Listeri strins. Although the growth rte of P. fluorescens ws not significntly different to tht for E. cloce, the Pseudomons strins were the most resistnt to oregno nd thyme in TSB (Tble 1). Pseudomons spp. re known to show consistently high resistnce to plnt ntimicrobils (Hmmer et l., Holley nd Ptel 0). However, both E. cloce nd P. fluorescens hd the sme sensitivity to the EO s in the lettuce model medi (Tble ). Both of these spoilge orgnisms were isolted from lettuce nd the lg phse for E. cloce within the vegetble medi ws shorter thn tht of P. fluorescens (Tble ). Combintions of EO s were ssessed for synergistic ctivity t lower concentrtions in order to reduce undesirble impcts on orgnoleptic properties of food (Tble ). No synergy s described by FIC indices ws observed in lettuce model medi but n importnt number of combintions displyed dditive effects t very low concentrtions, such s oregno combined with thyme ginst spoilge bcteri nd thyme in combintion with lemon blm ginst L. innocu. Some studies hve concluded tht whole EO s hve greter ntibcteril ctivity thn the mjor components mixed (Gill et l. 0, Mourey nd Cnillc 0). Burt (0) suggested tht the minor components present in the EO s extrcts re more criticl to the ctivity thn EO min components mixed, nd my hve synergistic effects or potentiting influence. As mny plnt EO s

22 possess compounds with similr structures, their combintions my exhibit dditive rther thn synergistic effects. Furthermore, s the EO efficcy lso depends on lipophilic properties, potency of functionl groups or their queous solubility (Dormn nd Dens 00), the mixture of compounds within whole EO s my contribute to tht dditive effect. Furthermore, since nother importnt spect for the optimised ppliction of EO s in food is the evlution of interction with food ingredients, five different model medi were prepred using beef extrct nd tomto serum in order to ssess nd quntify the effect of ph nd sugrs on the ntimicrobil efficcy of oregno nd thyme. In generl, the ntimicrobil ctivity of these EO s incresed when the ph decresed. Previously, it ws lso observed tht the inhibitory effect of plnt extrcts ws greter t cidic ph vlues (Del Cmpo et l. 00, Hsieh et l. 01). The susceptibility of bcteri to EO s ppers to increse with lower ph vlues since the hydrophobicity of EO s increses t low ph, consequently enbling esier dissolution in the lipids of the cell membrne of trget bcteri (Juven et l. ). The mjor efficcy of EO s t ph. or. ws confirmed with the lg phse nd growth rte results t these phs, which were longer nd lower, respectively, thn t higher ph levels. As the ph ws reduced, the lg phse incresed nd the growth rte declined for Listeri nd L. skei, nd consequently, the ddition of either oregno or thyme enhnced the EO efficcy. However, no significnt differences were observed between lg phse nd growth rte vlues of L. skei t ph.0 or.0 but the MICs of selected EO s decresed t more reduced ph. The sme trend ws observed with P. putid lthough mximum specific growth rte nd lg phse 1

23 vlues incresed nd decresed, respectively, t more cidic ph. Thus, EO efficcy my lso hve been promoted by the presence of sugrs. The increse of sugrs percentge up to.% seemed to improve the ntimicrobil efficcy of oregno nd thyme. Moreover, the presence of high concentrtions of crbohydrtes (.0 or.%) did not hve ny negtive impct on the EO efficcy, in greement with the generl observtion tht crbohydrtes in foods do not protect bcteri from the ction of EO s s much s ft nd protein do (Shelef et l. ). However, Gutierrez et l. (0b) reported protective effect of crbohydrte for bcteri where strch t or % hd negtive impct on the ntimicrobil ctivity of oregno nd thyme. Therefore, EO ppliction should be orientted to food products contining more simple sugrs thn complex crbohydrtes This work shows method for the evlution of the ntimicrobil ctivity of EO s in food model medi prior to optimised further ppliction in rel food, s well s link between orgnoleptic impct, food composition nd EO efficcy. Both gr nd broth dilution ntimicrobil ssys were suitble to clculte MICs of selected EO s ginst Listeri nd spoilge bcteri in vegetble, met or milk bsed model medi, which might be the first step in order to pproch optimising EO efficcy when pplied to food. On the other hnd, oregno nd thyme nd their combintion could hve potentil for controlling spoilge bcteri in fresh product chllenge studies. Combintions of lemon blm with thyme might be useful to reduce the presence of or control Listeri spp. in finl products. Our results show tht EO combintions cted ginst pthogens nd nturl spoilge microflor nd therefore hve potentil for use t combined low concentrtions to ssist in reduction of the sensory impct ssocited with high concentrtions of EO s in food.

24 1 1 Thus, potentil combintions tht my ddress spoilge, shelf life s well s sfety concerns ssocited with redy to use foods should be evluted using product chllenge studies. These should incorporte stndrd processing steps to ensure their efficcy in rel systems s well s concurrent sensory nlysis. Furthermore, the ntimicrobil efficcy of the EO s in this study ws found to be function of ingredient mnipultion. The ntimicrobil ctivity of oregno nd thyme ws incresed t high concentrtions of protein nd cidic ph conditions. Concentrtions bove % of sugrs did not reduced EO efficcy. Therefore, the ppliction of EO s should be further investigted for control of microbil sfety nd spoilge concerns in proteinceous foods nd/or foods contining simple sugrs with low ph vlues, which my promote the ntibcteril efficcy of EO s. The retention of nti-microbil efficcy of EO s within suitble food systems should be evluted lone s well s tking hurdle effects of other preservtion methods into ccount Acknowledgments This work ws supported by funding from Irish Deprtment of Agriculture nd Food s prt of the Ntionl Development Pln References Ahn, J., Grün, I. U., Mustph, A., 0. Effects of plnt extrcts on microbil growth, color chnge, nd lipid oxidtion in cooked beef. Food Microbiol.,

25 Bgmboul, C. F., Uyttendele, M. nd Debevere J., 0. Inhibitory effect of thyme nd bsil essentil oils, crvcrol, thymol, estrgol, linlool nd p-cymene towrds Shigell sonnei nd S. flexneri. Food Microbiol. 1(1), -. Brnuskien, R., Venskutonis, P. R., Dewettinck, K., Verhe, R., 0. Properties of oregno (Orignum vulgre L.), citronell (Cymbopogon nrdus G.) nd mrjorm (Mjorn hortensis L.) flvors encpsulted into milk protein-bsed mtrices. Food Res. Int., 1. Burt, S. 0. Essentil oils: their ntibcteril properties nd potentil pplictions in foods review. Int. J. Food Microbiol. (), -. Bustt, C., Vidl, R. S, Popiolski, A. S., Mossi, A. J., Driv, C., Rodrigues, M. R., Corzz, F.C., Corzz, M. L., Vldimir Oliveir, J., Cnsin, R. L., 0. Appliction of Orignum mjorn L. essentil oil s n ntimicrobil gent in susge. Food Microbiol. (1): -. Creg, M., Fernndez, E., Dorntes, L., Mot, L., Jrmillo, M. E., Hernndez- Snchez, H., 0. Antibcteril ctivity of Cpsicum extrct ginst Slmonell typhimurium nd Pseudomons eruginos inoculted in rw beef met. Int. J. Food Microbiol., 1. Crrmiñn, J. J., Rot, C., Burillo, J., Herrer, A., 0. Antibcteril efficiency of Spnish Sturej montn essentil oil ginst Listeri monocytogenes mong nturl flor in minced pork. J Food Prot. 1(), 0-0. Cv, R., Nowk, E., Tbod, A., Mrin-Iniest, F., 0. Antimicrobil ctivity of clove nd cinnmon essentil oils ginst Listeri monocytogenes in psteurized milk. J. Food Prot. 0(1), -.

26 Cerrutti, P, Alzmor, S. M.,. Inhibitory effects of vnillin on some food spoilge yests in lbortory medi nd fruit purées. Int. J. Food Microbiol. (-), -. Dvidson, P. M nd Brnen, A. L., 0. Food ntimicrobils An introduction. In: Dvidson, P. M, Sofos, J. N., Brnen, A. L. (Eds.), Antimicrobil in Food. CRC Press, Tylor nd Frncis Group ed., pp. 1-. Del Cmpo, J., Amiot, M. J., Nguyen-The, C., 00. Antimicrobil effect of rosemry extrcts. J. Food Prot. (), -1. Dormn, H. J. D., Dens, S. G., 00. Antimicrobil gents from plnts: ntibcteril ctivity of plnt voltile oils. J. Appl. Microbiol., 0-1. Elgyyr, M., Drughon F.A., Golden, D.A., Mount, J.R., 01. Antimicrobil ctivity of essentil oils from plnts ginst selected pthogenic nd sprophytic microorgnisms. J. Food Prot. (), -. Eneroth, A., Ahrne, S., Molin, G., 00. Contmintion routes of Grm-negtive spoilge bcteri in the production of psteurized milk, evluted by rndomly mplified polymorphic DNA (RAPD). Int. Diry J., 1. Firouzi, R., Shekrforoush, S. S., Nzer, A. H., Borumnd, Z., Jooyndeh, A. R., 0. Effects of essentil oils of oregno nd nutmeg on growth nd survivl of Yersini enterocolitic nd Listeri monocytogenes in brbecued chicken. J. Food Prot. 0(), -0. Frncis, G. A. nd O Beirne, D.,. Effects of storge tmosphere on Listeri monocytogenes nd competing microflor using surfce model system. Int. J. Food Sci. Technol., -.

27 Gill, A.O., Delquis, P., Russo, P., Holley, R.A., 0. Evlution of ntilisteril ction of cilntro oil on vcuum pcked hm. Int. J. Food Microbiol.,. Ghlfi, H., Benkerroum, N., Doguiet, D. D., Bensid, M., Thonrt, P., 0. Effectiveness of cell-dsorbed bcteriocin produced by Lctobcillus curvtus CWBI-B nd selected essentil oils to control Listeri monocytogenes in pork met during cold storge. Lett. Appl. Microbiol. (), -. Gndhi, M., Chikinds, M. L., 0. Listeri: A foodborne pthogen tht knows how to survive. Int. J. Food Microbiol. (1): 1-1. Glss, K. A. nd Johnson, E. A., 0. Antgonistic effect of ft on the ntibotulinl ctivity of food preservtives nd ftty cids. Food Microbiol. 1,. Grm, L., Rvn, L., Rsch, M., Bruhn, J. B., Christensen, A. B., Michel, G., 0. Food spoilge interctions between food spoilge bcteri. Int. J. Food Microbiol.,. Gutierrez, J., Rodriguez, G., Brry-Ryn, C. nd Bourke, P., 0. Efficcy of plnt essentil oils ginst food-borne pthogens nd spoilge bcteri ssocited with redy to et vegetbles: ntimicrobil nd sensory screening. J. Food Prot. 1(), 1-1. Gutierrez, J., Brry-Ryn, C., Bourke, P., 0b. The nti-microbil efficcy of plnt essentil oil combintions nd interctions with food ingredients. Int. J. Food Microbiol. (1), 1-. Hmmer, K. A., Crson, C. F. nd Riley, T.V.,. Antimicrobil ctivity of essentil oils nd other plnt extrcts. J. Appl. Microbiol., -0.

28 Holley, R. A., Ptel, D., 0. Improvement in shelf-life nd sfety of perishble foods by plnt essentil oils nd smoke ntimicrobils. Food Microbiol. : -. Hsieh, P-C., Mu, J-L., Hung, S-H., 01. Antimicrobil effect of vrious combintions of plnt extrcts. Food Microbiol. 1, -. Ippolito, A. nd Nigro, F., 0. Nturl ntimicrobils in posthrvest storge of fresh fruits nd vegetbles. In Roller, S. (Ed.), Nturl Antimicrobils for the Miniml Processing of Foods. Woodhed Publishing LTD, pp. 1-. Juven, B.J., Knner, J., Schved, F., Weisslowicz, H.,. Fctors tht interct with the ntibcteril ction of thyme essentil oil nd its ctive constituents. J. Appl. Bcteriol., 1 Mytle, N., Anderson, G.L., Doyle, M.P., Smith, M.A., 0. Antimicrobil ctivity of clove (Syzgium romticum) oil in inhibiting Listeri monocytogenes on chicken frnkfurters. Food Control 1,. Mrino, M., Bersni, C., Comi. G., 01. Impednce mesurement to study ntimicrobil ctivity of essentil oils from Lmicee nd Composite. Int. J. Food Microbiol., 1-. Moreir, M. R., Ponce, A. G., Del Vlle C. E. nd Rour, S.I., 0. Inhibitory prmeters of essentil oils to reduce foodborne pthogen. LWT-Food Sci. Tech. (), -0. Mourey, A., Cnillc, N., 0. Anti-Listeri monocytogenes ctivity of essentil oils components of conifers. Food Control 1,.

29 Ousslh, M., Cillet, S., Sucier, L. nd Lcroix, M., 0. Antimicrobil effects of selected plnt essentil oils on the growth f Pseudomons putid strin isolted from met. Met Sci.,. Ousslh, M., Cillet, S., Sucier, L. nd Lcroix, M., 0b. Inhibitory effects of selected plnt essentil oils on the growth of four pthogenic bcteri: E. coli O1:H, Slmonell Typhimurium, Stphylococcus ureus nd Listeri monocytogenes. Food Control 1(): -. Rgert, P., Devlieghere, F., Debevere, J., 0. Role of microbiologicl nd physiologicl spoilge mechnisms during storge of minimlly processed vegetbles. Posthrvest Biol. Technol. (), 1-. Schelz, A., Molnr, J. nd Hohmnn, J., 0. Antimicrobil nd ntiplsmid ctivities of essentil oils. Fitoterpi (), -. Shelef, L.A., Jyothi, E.K., Bulgrelli, M.A.,. Growth of enteropthogenic nd spoilge bcteri in sge-contining broth nd foods. J. Food Sci. ( 0), 0. Smith-Plmer, A., Stewrt, J., Fyfe., L.,. Antimicrobil properties of plnt essentil oils nd essences ginst five importnt food-borne pthogens. Lett. Appl. Microbiol., -1. Solomkos, N., Govris, A., Koidis, P., Botsoglou, N., 0. The ntimicrobil effect of thyme essentil oil, nisin, nd their combintion ginst Listeri monocytogenes in minced beef during refrigerted storge. Food Microbiol. (1):1-1.

30 Uhrt, M., Mks, N., Rvishnkr, S., 0. Effect of spices on growth nd survivl of Slmonell typhimurium DT in ground beef stored t nd C. J. Food Sfety (), 1. Ultee A., Smid, E. J., 01. Influence of crvcrol on growth nd toxin production by Bcillus cereus. Int. J. Food Microbiol., Vlero, M., Slmeron, M. C., 0. Antibcteril ctivity of essentil oils ginst Bcillus cereus in tyndllized crrot broth. Int. J. Food Microbiol.,

31 Figures legend Fig.1. Mximum specific growth (µmx) rte nd lg phse (λ) of L. monocytogenes NCTC, Lb. skei ATCC nd Ps. putid grown in beef extrct nd tomto serum model medi A (, ph.0), B (, ph.0), C (, ph.), D (, ph.), nd E ( (p<0.0)., ph.). Different letters signify sttisticl differences between vlues 1 1 Fig.. Mximum specific growth rte (µmx) of L. monocytogenes (i), Lb. skei (ii) nd Ps. putid (iii) in neutrlized beef extrct nd tomto serum model medi A (, 0.00% sugrs), B (, 1.1% sugrs), C (,.% sugrs), D (,.0% sugrs), nd E (,.0% sugrs) contining oregno (1. ppm) or thyme (. ppm). Different letters signify sttisticl differences between vlues (p<0.0) Fig.. Lg phse (λ) of L. monocytogenes (i), Lb. skei (ii) nd Ps. putid (iii) in neutrlized beef extrct nd tomto serum model medi A (, 0.00% sugrs), B (, 1.1% sugrs), C (,.% sugrs), D (,.0% sugrs), nd E (,.0% sugrs) contining oregno (1. ppm) or thyme (. ppm). Different letters signify sttisticl differences between vlues (p<0.0). 1 0

32 0. µmx (h -1 ) b c f c c λ (h) bg b d c e d d d d d L. monocytogenes Lb. skei Ps. putid e f g g h Fig.1. Mximum specific growth (µmx) rte nd lg phse (λ) of L. monocytogenes NCTC, Lb. skei ATCC nd Ps. putid grown in beef extrct nd tomto serum model medi A (, ph.0), B (, ph.0), C (, ph.), D (, ph.), nd E (, ph.). Different letters signify sttisticl differences between vlues (p<0.0). 1

33 0. 0. b c (i) c g g c µmx (h -1 ) d e e d f (ii) µmx (h -1 ) b b c d ef f e g d d h i j 0 0. (iii) 0. f f f µmx (h -1 ) d e e 0.0 b bg g 0 c c Fig.. Mximum specific growth rte (µmx) of L. monocytogenes (i), Lb. skei (ii) nd Ps. putid (iii) in neutrlized beef extrct nd tomto serum model medi A (, 0.00% sugrs), B (,1.1% sugrs), C (,.% sugrs), D (,.0% sugrs), nd E (,.0% sugrs) contining oregno (1. ppm) or thyme (. ppm). Different letters signify sttisticl differences between vlues (p<0.0).

34 (i) e 1 b b bc c b λ (h) d d d d f fg g h 0 1 b c c c (ii) c c c λ (h) d d d b 0 b bc c (iii) c bc c d d λ (h) 1 e e 0 Fig.. Lg phse (λ) of L. monocytogenes (i), Lb. skei (ii) nd Ps. putid (iii) in neutrlized beef extrct nd tomto serum model medi A (, 0.00% sugrs), B (,1.1% sugrs), C (,.% sugrs), D (,.0% sugrs), nd E (,.0% sugrs) contining oregno (1. ppm) or thyme (. ppm). Different letters signify sttisticl differences between vlues (p<0.0).

35 Tble 1 Microorgnisms used in this study Strin Reference Origin Food model medi b Enterobcter cloce * Iceberg lettuce TSB, L Pseudomons fluorescens * Iceberg lettuce TSB, L, M, BE Pseudomons putid * Iceberg lettuce TSB, BE Lctobcillus skei ATCC Fermented drink TSB, BE Listeri innocu NCTC Cow brin TSB, L, BE Listeri monocytogenes NCTC 1 Cheese TSB, M, BE Listeri monocytogenes IL * Iceberg lettuce TSB, L Strins indicted with n sterisk were provided by the Deprtment of Life Sciences, University of Limerick, Irelnd b Bcteri were grown in control medi (TSB), lettuce lef model medi (L), milk (M) or beef extrct (BE).

36 Tble Composition of the food model medi contining beef extrct nd tomto serum t different rtios Food model medi Ingredients (%) Protein Ft Crbohydrtes Slt ph (A) BE-TS 0:0 c (B) BE-TS b : (C) BE-TS 0: (D) BE-TS : (E) BE-TS 0: BE: Beef extrct b TS: Tomto serum c Rtios re expressed in percentge

37 Tble Lg phse nd mximum specific growth rte of selected bcteri in TSB, lettuce lef model medi nd beef extrct Microorgnism TSB Lettuce medi Beef extrct λ µmx b λ µmx λ µmx E.. erogenes. ± 1.01 c 0.1 ± ± ± 0.00 ND ND L. innocu NCTC. ± ± ± ± ± ± 0.01 L. monocytogenes IL. ± ± ± ± 0.00 ND ND L. monocytogenes NCTC. ± ± 0.0 ND d ND. ± ± 0.01 P. fluorescens. ± ± 0.0. ± ± ± ± 0.00 P. putid.01 ± ± 0.0 ND ND.0 ± ± 0.00 Lg phse is expressed in hours. b Mximum specific growth rte is expressed in hours -1 c Stndrd devition d ND, not determined Dt represent the mens of experiments performed in duplicte nd replicted t lest twice

38 Tble MICs of selected EO s compring the Microplte Assy (MPA), the Agr Dilution Method (ADM) nd the Agr well-diffusion Test (ADT) Microorgnism Oregno Thyme Lemon blm Mrjorm L. innocu NCTC MPA 0 ± 0 1 ± 0 1, ± 0,000 ± 0 ADM ± 0 ± 1,0 ± 0,000 ±, ADT ± b 1,0 ± b,000 ± 0 b,000 ± 0 P. fluorescens MPA 1, ± 00 1,00 ±,000 ±,00,00 ±, ADM ± 1,0 ± 0,000 ± 0 b,000 ± 0 b ADT,00 ± 0 b,0 ± 1, b,000 ± 0 b 1,00 ±,0 b MICs re expressed in ppm. For ech microorgnism, mens in the sme column followed by different letters re significntly different (p<0.0). All experiments were performed in duplicte nd replicted t lest three times.

39 Tble MIC of EO s used in this study ginst the selected bcteri in TSB (A), lettuce lef model medi (B) or beef extrct (C). Microorgnism Oregno Thyme Mrjorm Lemon Blm (A) E. cloce 00 00,000 ND L. innocu NCTC 0 0 ND,00 L. monocytogenes IL 0 0 ND,00 L. monocytogenes NCTC 0 0 ND,00 P. fluorescens,000,000 0,000 ND P. putid,000,000 0,000 ND (B) E. cloce,000 ND L. innocu NCTC 0 ND L. monocytogenes IL 0 ND P. fluorescens,000 ND (C) L. innocu NCTC 0 1 ND 00 L. monocytogenes NCTC 0 1 ND 00 P. fluorescens 1,00,00 1,00 ND P. putid 1,00,00 1,00 ND ND, not determined All experiments were performed in duplicte nd replicted t lest three times.

40 Tble FIC vlues of EO combintions in lettuce lef model medi EO combintions E. cloce P. fluorescens L. innocu NCTC L. monocytogenes IL FIC Std Dev.* FIC Std Dev.* FIC Std Dev.* FIC Std Dev.* Oregno + Mrjorm 1. (I) ± (I) ± 0.00 ND ND Oregno + Lemon blm ND ND 1.0 (I) ± (I) ± 0. Oregno + Thyme 0. (A) ± (A) ± (A) ± (I) ± 0.0 Thyme + Mrjorm 1.00 (A) ± (I) ± 0.0 ND ND Thyme + Lemon blm ND ND 0. (A) ± (I) ± 0. Results re interpreted s synergy (S, FIC < 0.), ddition (A, 0. FIC 1), indifference (I, 1 < FIC ) or ntgonism (AN, FIC > ) ND, not determined

41 Tble MICs of selected EO s in the beef extrct nd tomto serum model medi t different rtios Strin Medi A (0:0, ph.0) Beef extrct nd Tomto Serum Model Medi (BE-TS) Medi B (:, ph.0) Medi C (0:, ph.) Medi D (:, ph.) Medi E (0:0, ph.) L. monocytogenes NCTC Oregno.0 ± ± 0.00 b 1. ± 0.00 c.1 ± 0.00 d NG Thyme 1.00 ± ±.0 b 0.1 ±. b 1. ± 0.00 c NG Lemon blm ± ±. b.00 ± 0.00 c. ± 1. d NG Mrjorm,1.00 ± 0.00,. ± 0. b 1,.0 ± 0.00 c 1. ± 0.00 d NG L. skei ATCC Oregno 1.0 ± ± ± 0.00 b.0 ± 0.00 c NG Thyme ± ± ± 0.00 b 1.00 ± 0.00 c NG Lemon blm, ± 0.00, ± 0.00, ± 0.00 b 1,.0 ±.00 c NG Mrjorm,.0 ± 1,0.,1.00 ± 0.00 b,. ± 0. bc 1,11. ± 1.0 cd NG P. putid Oregno 1,.0 ±.00 1,.00 ± ,.00 ± 0.00 NG NG Thyme,00.00 ± 0.00,00.00 ± 0.00,00.00 ± 0.00 NG NG Lemon blm,00.00 ±, , ± 0.00 b 1,.00 ± 1,00.00 b NG NG Mrjorm 1,00.00 ± 0.00,1.0 ±,1.00 b,.00 ± 0.00 c NG NG NG, No growth ws observed in control medi without ny EO MICs re expressed in ppm. Mens in the sme row followed by different letters re significntly different for ech bcteril popultion (p<0.0). All experiments were performed in duplicte nd replicted t lest three times. 0