EFFECTS OF CLOVE AND TEA TREE OILS ON ESCHERICHIACOLI O157:H7 IN BLANCHED SPINACH AND MINCED

COOKED BEEF

MARÍA R. MOREIRA1–3, ALEJANDRA G. PONCE2, CARLOS E. DEL VALLE1

and SARA I. ROURA1,2

1Grupo de Investigación en Ingeniería en AlimentosFacultad de Ingeniería

Universidad Nacional de Mar del PlataMar del Plata, Argentina

2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos Aires, Argentina

Accepted for Publication April 9, 2007

ABSTRACT

The effectiveness of essential oil application in foods is the result of factorassociations such as composition and storage temperatures. The aim was toevaluate the effectiveness of clove and tea tree essential oils to control Escheri-chia coli O157:H7 on blanched spinach and minced cooked beef exposed at 8and 20C For both essential oils, the highest concentrations (three and fourtimes minimal inhibitory concentrations) were needed to restrict O157:H7populations in blanched spinach and minced cooked beef, respectively. Theantimicrobial action of these essential oils was dependent on the oil concen-tration, the food composition and the storage temperature. The inhibition ofpathogen and native microorganisms was more important at high tempera-tures than at low temperatures. Preliminary findings in actual foods suggestthat clove and tea tree oils could be used as potential biopreservatives capableof controlling foodborne pathogens when foods are exposed to abusivetemperatures.

PRACTICAL APPLICATIONS

The exploration of naturally occurring antimicrobials in food preserva-tion receives increasing attention due to consumer awareness of natural food

3 Corresponding author. TEL: +54-0223-481-6600; FAX: +54-0223-481-0046; EMAIL: moreira_maria@fi.mdp.edu.ar

Journal of Food Processing and Preservation 31 (2007) 379–391. All Rights Reserved.© 2007, Universidad Nacional de Mar del PlataJournal compilation © 2007, Blackwell Publishing

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products. Biopreservatives, a wide range of natural products from both plantsand microorganisms, are useful in extending the shelf life of foods, reducing oreliminating pathogenic bacteria and increasing overall quality of food prod-ucts. The use of clove and tea tree essential oils allow controlling E. coligrowth on actual food system exposed to abusive refrigeration temperature.The oil concentrations required to produce a certain level of inhibition inactual foods were questionable due to the organoleptic impact.

However, these novel natural preservatives in combination with otherfactors in obstacles technologies are an alternative to control the pathogengrowth minimizing undesirable changes in organoleptic characteristics.

INTRODUCTION

The demand for safe foods, coupled with the preference by consumers forfoods free of synthetic additives, has increased the interest for natural preser-vatives in recent years (Roller 2004). Great emphasis has recently beenfocused on the utilization of spices and their essential oils as natural agents forfood preservation (Bruni et al. 2004). Biopreservatives, a wide range of naturalproducts from both plants and microorganisms, can be useful in extending theshelf life of foods, reducing or eliminating pathogenic bacteria, and increasingoverall quality of food products (Draughon 2004). Most studies have evaluatedthe antimicrobial activity of naturally occurring botanicals on in vitro experi-ments. In a previous work, Moreira et al. (2005) reported in an in vitro studythat the essential oils from clove and tea tree had inhibitory effects onO157:H7 using the agar diffusion method. Other in vitro studies have demon-strated antibacterial activity of essential oils against Listeria monocytogenes,Salmonella, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereusand Staphylococcus aureus (Hammer et al. 1999; Elgayyar et al. 2001;Friedman et al. 2002; Burt 2004; Dadalioglu and Evrendilek 2004; Draughon2004). However, the information of their effect in actual food systems is stillinsufficient. Higher concentrations are needed to achieve the same effects infood systems (Shelef 1983). The greater availability of nutrients in foodscompared to laboratory media may enable bacteria to repair damaged cellsfaster (Gill et al. 2002). The studies on actual foods show that some botanicalshave the potential to be effective biopreservatives, although product develop-ment to optimize food functionality and flavor will be challenging and morestudies are needed on the topic of botanical applications to fully understandhow best to optimize their use and provide the fundamentals needed by thefood industry (Draughon 2004). The future of naturally occurring antimicro-bials seems promising, as new preservation methods are rapidly incorporatedin a variety of foods once their effectiveness and safety have been well

380 M.R. MOREIRA ET AL.

documented. The effectiveness of essential oil application in actual foodsystems is the result of multiple factor associations such as the food compo-sition or the storage temperatures (Smith-Palmer et al. 2001; Gill et al. 2002;Burt 2004; Canillac and Mourey 2004; Ponce et al. 2004; Tsegaye et al. 2004).The presence of surfactants and organic substances that interact with activesites of the antimicrobial substances also plays an important role (Hammeret al. 1999). In addition, the mode that essential oils were applied on foods isfundamental to antibacterial and antioxidant activity (Pandit and Shelef 1994).

When foods are kept at proper refrigeration temperatures, the growth ofmicrobial populations is arrested. However, in some food-handling operationssuch as food-catering services and self-serve restaurants, food items may beaccidentally exposed to abusive temperatures. Therefore, biopreservatives thatwould be capable of controlling foodborne pathogens when foods are exposedto abusive temperatures could make an important contribution to such opera-tions. The aim of this work was to evaluate the effectiveness of clove and teatree essential oils to control E. coli O157:H7 on actual food systems (blanchedspinach and minced cooked beef) exposed to abusive refrigeration (8–10C)and environmental (20–22C) temperatures.

MATERIALS AND METHODS

Sample Preparation

Raw spinach leaves were washed, steam blanched for 2 min and shred-ded. The final composition was water contents 910 g/kg, proteins 29 g/kg andtotal fats 4 g/kg. Raw minced beef was pan fried under a low fire for 5 min.The final composition was water contents 520–560 g/kg, protein 250 g/kg andtotal fats 190 g/kg.

Culture Maintenance

E. coli strain O157:H7, ATCC 25518 (American Type Culture Collection,provided from CERELA [Centro de Referencia de Lactobacilos, Tucumán,Argentina]) was used. A stock culture was maintained on tryptic soy broth(Britania, Buenos Aires, Argentina) at 4C. Before it was used, the O157:H7was cultured in brain–heart infusion broth (BHI; Britania) for 24 h at 37C.Approximately 0.1 mL of culture was transferred to 9.0 mL of BHI at twoconsecutive 24-h intervals immediately before each experiment.

Essential Oils Application

The essential oils (Nelson and Russell, London, England) utilized in thiswork were tea tree (Melaleuca alternifolia) and clove (Syzygium aromaticum)

381EFFECTS OF CLOVE AND TEA TREE OILS ON E. COLI

oils obtained from steam distillates. Clove essential oil was applied to the foodsystems (blanched spinach or minced cooked beef) at one, two and three timesthe minimal inhibitory concentration (MIC). Tea tree essential oil was appliedto samples at two, three and four times the MIC. MIC is defined as the oilconcentration that produces a 90% “in vitro” inhibition of O157:H7 (14). TheMICs utilized for both essential oils were 2.5 mL/L for clove and 5.0 mL/L fortea tree (Moreira et al. 2005).

One hundred grams of food was thoroughly mixed with the essential oilsand immediately inoculated with active cultures of O157:H7 reaching a patho-gen concentration of approximately 106 cfu/mL. The active culture and thefood samples were mixed together. This pathogen inoculum represents a highcontamination level. Control samples free of essential oils were also preparedin order to evaluate its effectiveness on O157:H7 growth. Another controlsample free of essential oils and O157:H7 inoculum was used to account forthe possible growth of native E. coli strains. In both blanched spinach andminced cooked beef, the growth of native E. coli strains was not detectedduring storage.

Inoculated food samples were stored for 24 h at 8–10C (representing aninadequate refrigeration storage) and at 20–22C (representing an inadequateroom temperature).

Enumeration of Total Microflora and O157:H7

The survival and growth of O157:H7 and total background microflora onsamples were examined at regular intervals during the storage period. Sam-pling for viable cells was carried out at 0, 3, 7, 10, 15, 18 and 24 h. To allowfor an adequate contact between the essential oils and microorganisms,samples reported as corresponding to time 0, actually had 5 min of contact(Ponce et al. 2004). O157:H7 was counted by surface plating on eosin meth-ylene blue (EMB; Britania) after incubation at 37C for 24 h in air. EMB beinga selective medium that allows the characterization of typical E. coli colonies,only those greenish with metallic brightness were taking into account. Totalmicroflora was counted by surface plating on plate count agar (Britania) afterincubation at 37C for 24–48 h in air.

Statistical Analysis

Each assay was performed in duplicate on three separate experimentalruns. Differences among samples were tested by analysis of variance (Boxet al. 1978). Wherever differences are reported as significant, a 99.9% confi-dence level was used.

382 M.R. MOREIRA ET AL.

RESULTS AND DISCUSSION

Effect of Clove Oil on Food Samples

Blanching reduced the initial number of microorganisms in freshspinach by two magnitude orders to log10 values of about 4.9. After inocula-tion, the total microbial load reached values in the range of 6.7 to 6.9 (log10).This would indicate that the proportion of inoculated microorganisms tomicroorganisms surviving the blanching operation was about 100:1.Figure 1A–D shows the evolution of O157:H7 populations and total micro-flora on blanched spinach treated with different clove oil concentrations at twostorage temperatures. During storage, both O157:H7 and total microorganismcounts were similar. This would indicate that the populations of O157:H7predominated and that no other microorganism groups presented a differentialrate of growth under the experimental storage conditions. The levels at whichthe samples were inoculated with O157:H7 in this study were very high andthe native microflora surviving blanching would not have been able to compete(Fig. 1A–D).

The initial O157:H7 counts were not significantly (P < 0.01) different incontrol samples and samples treated with clove essential oil at one or twoMICs. The use of essential oil at three MICs, however, resulted in a significant2-log cycle reduction in the O157:H7 counts, exerting a bactericidal effect(Fig. 1A). During storage at room temperature (20–22C), O157:H7 popula-tions in control samples remained at their initial levels for about 15 h andincreased thereafter; this would represent the beginning of the exponentialphase. Samples treated with the essential oil at three MICs remained at thelevels attained after the oil application and also began to increase after 15 h ofstorage, although they remained 2 log cycles below the populations in thecontrol samples. Samples treated with the essential oil at one and two MICspresented a decrease in the O157:H7 counts after the third hour of storagecompared with control sample, and even though the populations also began torecover after 15 h of storage, they remained below the populations of thecontrol samples until the end of the experiment. A possible explanation wouldbe that at these concentrations (one and two MICs), the essential oil requireshigher contact time with respect to three MICs, to produce an effect on theO157:H7 population.

In samples stored at 8–10C (Fig. 1C), differences in O157:H7 countsbetween control and samples treated with clove at one and two MICs werenot significant throughout 24 h of storage. During that time, the microbialcounts did not increase significantly. This temperature level would be lowenough to restrict the growth of pathogen and total microflora (Thomas et al.1999). Because the samples had been subjected to a blanching operation, the

383EFFECTS OF CLOVE AND TEA TREE OILS ON E. COLI

psychotropic bacteria would probably have been eliminated or reduced tovery low levels. As it happened with samples stored at 20–22C, clove oilapplication at three MICs produced reductions in the O157:H7 counts ofabout 2 log cycles. Between 10 and 15 h of storage, the populations ofO157:H7 in these samples increased to levels comparable to samples treatedat one and two MICs. O157:H7 recovery could be attributed to the associa-tion of several factors, such as the lack of other competitive microorganisms

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FIG. 1. TOTAL MICROFLORA AND O157:H7 SURVIVAL IN BLANCHED SPINACHTREATED WITH CLOVE ESSENTIAL OIL DURING 24 H OF STORAGE AT TWO ABUSIVE

TEMPERATURES(A) O157:H7 counts at 20–22C, (B) total microbial counts at 20–22C, (C) O157:H7 counts at

8–10C and (D) total microbial counts at 8–10C. Control samples ( ), minimal inhibitory

concentration (MIC) ( ), two MICs ( ) and three MICs ( ). Each point represents the mean of

duplicate on three separate experimental runs. SD bars are not shown because they are masked by

the graph symbol. SD range for control samples: 0.04–0.70; MIC samples: 0.03–0.30; two MIC

samples: 0.06–0.80; three MIC samples: 0.05–0.90.

384 M.R. MOREIRA ET AL.

that could restrict the pathogen growth and the possibility that O157:H7 mayelicit resistance mechanisms as stress response against nonproteinaceouscompounds often present in herbs and spices (Brul and Coote 1999). Anothercontributing factor could be that the stress response to essential oils hadinfluence on over the stress tolerance to low temperature. Lou and Yousef(1996) observed that L. monocytogenes became more tolerant to mild heat(56C) after being stressed by the presence of ethanol, hydrogen peroxide andlow pH.

Cooking of beef reduced the initial microbial count in fresh beef by3 log10 cycles to final values in the range of 3.0 to 3.1 log10. After pathogeninoculation, the total microbial load reached log10 values of 6.2 to 6.3. Thiswould indicate that the proportion of inoculated microorganisms to microor-ganisms surviving the cooking operation was about 1,000:1.

Figure 2A,B shows the evolution of O157:H7 populations on cooked beeftreated with different concentrations of clove oil at two storage temperatures.As it happened with blanched spinach, during storage, both O157:H7 and totalbacteria counts were similar so, only the O157:H7 profile is presented inFig. 2.

The application of clove essential oil at one and two MICs did not resultin significant differences (P < 0.01) in O157:H7 counts over control samples,neither after application nor during storage at 8–10C or 20–22C (Fig. 2A,B).After 3 h of storage at 20–22C with clove at two and three MICs, a slightreduction (1 log cycle) on pathogen counts was observed (Fig. 2A).

O157:H7 populations increased at both storage temperatures but theirnumbers after 24 h of storage were 2 log10 cycles higher in samples stored at20–22C than in samples stored at 8–10C (Fig. 2A,B). This would be the effectof different temperature levels on the O157:H7 capacity to proliferate. Nev-ertheless, unlike what happened on blanched spinach, O157:H7 countsincreased after 24 h of storage at 8–10C. A possible explanation would be thatbeef provides a greater availability of nutrients for microbial growth thanspinach does.

The fact that clove oil presented less inhibitory effects on cooked beefthan on blanched spinach could be due to various factors. Menon and Garg(2001) found that clove oil did not result in a decline in L. monocytogenespopulations in cooked meat and postulated that the food could either exertsome protective effect on the microorganisms or absorb the antimicrobialcompounds in clove oil. Burt (2004) found that the antibacterial activity oforegano oil against Salmonella Typhimurium was limited by the physicalstructure of the food. This author also indicates that fats, proteins, watercontent, antioxidants, preservatives, pH, salt and other additives are relevant inthe bacterial sensitivity to biopreservatives. The diffusion rate of oil activeprinciples and their low vapor pressures can also limit the microorganisms

385EFFECTS OF CLOVE AND TEA TREE OILS ON E. COLI

exposure (Ponce et al. 2004). Smith-Palmer et al. (2001) reported that theeffectiveness of essential oils on L. monocytogenes was affected by cheesecomposition. Likewise, Canillac and Mourey (2004) reported that the intro-duction of sodium fat into the test medium decreased the bactericidal effects ofessential oils. It is generally accepted that high levels of fat and/or protein in

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FIG. 2. O157:H7 SURVIVAL IN MINCED COOKED MEAT TREATED WITH CLOVEESSENTIAL OIL DURING 24 H OF STORAGE AT TWO ABUSIVE TEMPERATURES

(A) O157:H7 counts at 20–22C and (B) O157:H7 counts at 8–10C. Control samples ( ), minimal

inhibitory concentration (MIC) ( ), two MICs ( ) and three MICs ( ). Each point represents the

mean of duplicate on three separate experimental runs. SD bars are not shown because they are

masked by the graph symbol. SD range for control samples: 0.02–0.20; MIC samples: 0.04–0.45;

two MIC samples: 0.04–0.26; three MIC samples: 0.02–0.45.

386 M.R. MOREIRA ET AL.

foodstuffs protect the bacteria from the action of essential oils. The essentialoils would be dissolved in the lipid phase of the food and would be lessavailable to act on bacteria that are present in the aqueous phase (Aureli et al.1992; Mejlholm and Dalgaard 2002). Thus, the differences between beef andspinach in structure and composition, and especially in the fat, protein andwater contents, could explain the differences in the inhibitory effects of cloveoil.

Effect of Tea Tree Oil on Food Samples

At 8–10C and 20–22C, O157:H7 and total microflora counts on spinachsamples were not immediately affected by the application of tea tree oil at two,three and four MICs (5.8–6.0 log10). In a previous work, Ponce et al. (2004)found that the application of tea tree oil did not affect immediately the nativemicrobial counts of Swiss chard leaves, although differences may be producedlater on during storage. After 3 h of exposure, significant differences(P < 0.01) were obtained between control and oil-treated samples (6.9 and5.8 log10, respectively). At both storage temperatures, no significant differ-ences were observed between samples treated with three and four MICs. Forsamples stored at 20–22C, the differences between controls and oil-treatedsamples (2.0 to 2.5 log10 cycles) remained during the 24 h of storage. Insamples stored at 8–10C, the O157:H7 populations in oil-treated samples weremaintained 1 log10 cycle below those of control samples.

Figure 3A,B shows the evolution of O157:H7 counts on minced cookedmeat at both storage temperatures. Like what happened previously, similarprofiles were obtained for both populations; so only O157:H7 evolution ispresented. O157:H7 populations on cooked minced beef were not immediatelyaffected by the application of tea tree oil. However, after 3 h of storage,samples kept at 20–22C (Fig. 3A) and treated with tea tree oil at three and fourMICs, presented a slightly reduction of about 1 log10 cycle, with respect tocontrol counts. These differences remained during the 24-h storage (Fig. 3A).

On the other hand, in samples stored at 8–10C, only those treated withfour MICs showed some effect on O157:H7 populations and the differences inmicrobial counts (about 1 log10 cycle) manifested themselves only after 7 h ofstorage (Fig. 3B). The larger delay in the time needed for tea tree oil to affectthe O157:H7 populations in samples stored at 8–10C, as compared to samplesstored at 20–22C, would be the result of the effect of temperature on diffusioncoefficients.

Two important variables appear when essential oils are applied either invivo or in vitro assays; one is the contact time for oils to exert their effect andthe other is the concentration required to achieve the same degree of inhibition,and these two variables would be interacting. The lower water content of foods

387EFFECTS OF CLOVE AND TEA TREE OILS ON E. COLI

compared to laboratory media may hamper the progress of antibacterial agentsto the target site in the bacteria cell (Smith-Palmer et al. 2001). Furthermore,in in vitro assays, microorganisms and essential oils come into close contact,but in vivo, the food matrix has cell membranes that act as physical barriersinterfering oil and microorganism contact. Moreira et al. (2005) reported that

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FIG. 3. O157:H7 SURVIVAL IN MINCED COOKED MEAT TREATED WITH TEA TREEESSENTIAL OIL DURING 24 H OF STORAGE AT TWO ABUSIVE TEMPERATURES

(A) O157:H7 counts at 20–22C and (B) O157:H7 counts at 8–10C. Control samples ( ), minimal

inhibitory concentration (MIC) ( ), two MICs ( ) and three MICs ( ). Each point represents the

mean of duplicate on three separate experimental runs. SD bars are not shown because they are

masked by the graph symbol. SD range for control samples: 0.01–0.27; two MIC samples:

0.05–0.15; three MIC samples: 0.05–0.20; four MIC samples: 0.04–0.65.

388 M.R. MOREIRA ET AL.

in in vitro assays, the maximum O157:H7 reductions (3.5 log) exerted by cloveoil required 15 min of contact time and that longer contact times did not resultin higher O157:H7 reductions.

However, from the data presented in Figs. 1–3, it can be observed thatonly clove oil at three MICs, applied on blanched spinach, produced signifi-cant inhibition of O157:H7 within the first 5 min of application. In all the otherexperimental conditions where the oils exerted an inhibitory effect, the actionof the oil made itself evident at least 3 h after application. The effects of cloveapplication at MIC over O157:H7 growth when comparing in vivo (blanchedspinach) and in vitro assays at two contact times (3 and 20 h), indicated thatO157:H7 reductions observed by in vivo assays were significantly lower(0.75–1 log10) than in vitro (3.5 log10) assays.

The fact that microorganisms were neither eliminated nor completelyinhibited could be attributed to the high initial cell number O157:H7 inocula-tion. Bacteriostatic effects were exerted at short storage times using concen-trations higher than the MIC; however, at the end of the abusive temperaturestorage (24 h), although pathogen load exceeded the levels allowed for foodsafety, they were significantly lower (P < 0.01) than control samples.

It has generally been found that a greater concentration of essential oils isneeded to achieve the same effect in actual foods than in in vitro assays (Smidand Gorris 1999). Karatzas et al. (2001) reported that greater concentration ofessential oil (approximately twofold) was needed to reach the same effects insemiskimmed milk. The greater availability of nutrients in foods compared tolaboratory media may enable bacteria to repair damaged cells faster (Gill et al.2002).

The oil concentrations required to produce a certain level of inhibition inactual foods could be questionable due to the organoleptic impact. Blanchedspinach and minced cooked beef treated with higher concentration of cloveand tea tree oils presented a strong off-flavor. In this way, the oil concentra-tion required to reach an effective O157:H7 control produced in both foodsystems undesirables changes in organoleptic characteristics. Synergisticeffects would have to be exploited to maximize the antibacterial activity ofessential oils and to minimize the concentrations required to achieve a par-ticular antibacterial affect without adversely affecting the sensorial accept-ability. A number of potential synergic factors have been suggested for usewith essential oils such as reducing pH, adding organic acids, low reducingoxygen tension using modified atmospheres, applying mild heat shocks andso on (Burt 2004).

The potential use of some of the novel “natural” preservatives in combi-nation with other factors in obstacle technologies, requires further research andmay lead to the use of essential oils at lower concentration to reach the desiredantibacterial effects without undesirable changes in the flavor or aroma.

389EFFECTS OF CLOVE AND TEA TREE OILS ON E. COLI

ACKNOWLEDGMENTS

This work was supported by Consejo Nacional de Investigaciones Cientí-ficas y Técnicas (CONICET) and Universidad Nacional de Mar del Plata(UNMDP).

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