Food Control 21 (2010) 549–553

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Food Control

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Disinfectant action of Cymbopogon sp. essential oils in different phasesof biofilm formation by Listeria monocytogenes on stainless steel surface

Maíra Maciel Mattos de Oliveira a,*, Danilo Florisvaldo Brugnera a, Maria das Graças Cardoso b,Eduardo Alves c, Roberta Hilsdorf Piccoli a

a Departamento de Ciência dos Alimentos, Universidade Federal de Lavras (UFLA), Lavras, MG, CEP 37200-000, CP 3037, Brazilb Departamento de Química, UFLA, Lavras, MG, CEP 37200-000, CP 3037, Brazilc Departamento de Fitopatologia, UFLA, Lavras, MG, CEP 37200-000, CP 3037, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 June 2009Received in revised form 10 August 2009Accepted 17 August 2009

Keywords:Natural disinfectantsCymbopogon citratusCymbopogon nardus

0956-7135/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodcont.2009.08.003

* Corresponding author. Tel.: +55 35 3829 1392; faE-mail address: mmacielmattos@yahoo.com.br (M

veira).

Disinfectant solutions based on the essential oils of Cymbopogon citratus (D.C.) Stapf. and Cymbopogonnardus (L.) Rendle, applied alone or in combination, and a control disinfectant solution were tested intwo phases (3 and 240 h) of biofilm formation by Listeria monocytogenes ATCC 19117 on AISI 304 (#4)stainless steel surface. Disinfectant solutions based on essential oils have effectively reduced the numberof surface-adhered cells, especially after 60 min of contact. The disinfectant solutions based on acombination of essential oils were capable of reducing 100% (5.64 Log CFU cm�2) the number ofsurface-adhered cells after 60 min of contact, at 240 h of biofilm formation. Essential oils of C. citratusand C. nardus, alone or in combination, are new alternatives for disinfection of industrial stainless steelsurfaces contaminated by L. monocytogenes.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Bacterial adhesion, with subsequent biofilm formation at indus-trial processing environments, is a potential source of contamina-tion that can lead to food deterioration or transmission offoodborne diseases. At food processing industries, surfaces of stain-less steel equipments and utensils are known to be major sites ofbacterial adhesion and biofilm formation (Chmielewski & Frank,2003; Wong, 1998).

In recent years, priority has been given to efficient cleaning anddisinfection programs that eliminate the risk of food contamina-tion by microorganisms present on industrial surfaces (Gibson,Taylor, Hall, & Holah, 1999). Several studies aiming to find effectivestrategies have been published (Gandhi & Chikindas, 2007). Searchfor new disinfectants has become a new research area. Growingnegative consumer perception against synthetic compounds hasled to the development of natural alternatives (Davidson, 1997;Roller, 1995). Thus, essential oils of condiment, aromatic andmedicinal plants, which are potent natural antibacterial agents(Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004;Oussalah, Caillet, Saucier, & Lacroix, 2007), have been recentlyevaluated for their antibacterial activity on biofilms, aiming atthe possibility of using these secondary metabolites or their con-

ll rights reserved.

x: +55 35 3829 1401.aíra Maciel Mattos de Oli-

stituents as disinfectants in the food industry (Chorianopoulos,Giaouris, Skandamis, Haroutounian, & Nychas, 2008; Knowles,Roller, Murray, & Naidu, 2005; Lebert, Leroy, & Talon, 2007; Sand-asi, Leonard, & Viljoen, 2008). The results obtained are promising,yet divergent. According to Chorianopoulos et al. (2008), the infor-mation available on the use of essential oils as disinfectants is stilllimited, pointing to the need of further studies.

Innumerous microorganisms are capable of participating in theprocess of adhesion and biofilm formation. In the food industry,they can be divided into spoilage and pathogenic microorganisms.With respect to the latter, Listeria monocytogenes is known for itsresistance to heat and high concentration of salts, survival underrefrigeration temperatures, capacity to colonize surfaces and dis-semination of severe foodborne infections (Gandhi & Chikindas,2007; Pan, Breidt Junior, & Kathariou, 2006; Torres, Sierra, Poutou,Carrascal, & Mercado, 2005). Such properties make this bacteriumdifficult to be controlled in food processing environments, thus it iscommonly found in meat and dairy processing industries (Chambelet al., 2007; Chasseignaux et al., 2002; Cruz et al., 2008; Lópezet al., 2008; Senczek, Stephan, & Untermann, 2000).

Poaceae belong to one of the largest plant families comprisingaround 500 genera and approximately 8000 species, beingessentially herbaceous and generically known as grasses. Cymbo-pogon genus is the most representative for its essential oil produc-tion, since few grasses are aromatic (Ortiz, Marrero, & Navarro,2002). Antimicrobial activity of the essential oils of Cymbopogoncitratus (D.C.) Stapf. (lemongrass) and Cymbopogon nardus L. Rendle

550 M.M.M. de Oliveira et al. / Food Control 21 (2010) 549–553

(citronella) was demonstrated in vitro against L. monocytogenes(Oussalah et al., 2007).

The objective of this study was to evaluate the disinfectantaction of C. citratus and C. nardus essential oils, alone and in com-bination, in two phases of biofilm formation by L. monocytogenesATCC 19117 on AISI 304 (#4) stainless steel surface.

2. Materials and methods

2.1. Microorganism used, standardization, inoculum preparation andstorage

The microorganism used was L. monocytogenes ATCC 19117, ac-quired from the Culture Collection Section of the Medical BiologyDivision of the Adolfo Lutz Institute (São Paulo – SP, Brazil). Tostandardize the number of cells, the strain was initially inoculatedin an Erlenmeyer flask containing 150 mL of Trypic Soy Broth (TSB),incubated at 37 �C. The growth curve was determined by perform-ing periodic absorbance readings (600 nm) and serial dilutions insaline solution [NaCl 0.9% (p/v)]. Then, from the saline solution,and using Trypic Soy Agar (TSA) as culture medium, spread platingmethodology was improved to determine the Log CFU mL�1.Throughout the experiment, the strain was stored under refrigera-tion in freezing culture medium (15 mL glycerol, 0.5 g bacteriolog-ical peptone, 0.3 of yeast extract and 0.5 g NaCl, per 100 mL ofdistilled water, with the final pH adjusted to 7.2–7.4). For strainreactivation and use, an aliquot of the freezing culture mediumwas transferred to test tubes containing TSB, with two subculturesat 37 �C for 24 h. The culture was striated in TSA added to Petridishes and incubated at 37 �C for 24 h. Of the colonies formed onthe TSA surface, some were removed and transferred into an Erlen-meyer flask containing 150 mL of TSB, which was incubated at37 �C until reaching the number of cells necessary for the experi-ment, approximately 9.17 Log CFU mL�1 (OD600 nm = 0.895).

2.2. Biofilm formation experimental model

The experimental model of biofilm formation by L. monocytoge-nes was elaborated based on a system first used by Bagge, Hjelm,Johansen, Huber, and Gram (2001); Gram, Bagge-Ravn, Ng,Gymoese, and Vogel (2007), with modifications. In the presentstudy, the experimental model consisted of the following items:AISI 304 (#4) stainless steel base, with four divisions, each sup-porting 21 AISI 304 (#4) stainless steel coupons (1 � 8 � 18 mm),vertically displaced; 1000 mL beaker; magnetic bar and magneticagitator to allow the free circulation of the substrate inside thebeaker. The beaker was sealed with a Petri dish and plastic film.AISI 304 (#4) stainless steel was chosen for being the most utilizedin the food industry.

2.3. Preparation of the coupons and stainless steel base

In order to initiate the bacterial cell adhesion stage, the couponsand stainless steel base were previously hygienized and sterilized.First they were cleaned with acetone 100%, washed by immersionin alkaline detergent [NaOH 1% (w/v), pH 13.2] for 1 h, rinsed withsterilized distilled water, dried and cleaned with alcohol 70% (v/v).After the hygienization, they were washed with sterilized distilledwater, dried for 2 h at 60 �C and autoclaved at 121 �C for 15 min(Rossoni & Gaylarde, 2000).

2.4. Bacterial cell adhesion to stainless steel coupon surface

Initially, 1000 mL of TSB previously sterilized and 70 mL of TSBcontaining the bacterial culture were added to the beaker contain-

ing the magnetic bar, at a final concentration of approximately8 Log CFU mL�1.The stainless steel base containing the couponswas placed inside the beaker, which was sealed and incubated at37 �C under 50 rpm agitation. Every 48 h, the coupons were re-moved from the base and immersed three times into a saline solu-tion to remove the planktonic cells, and again placed in a newsterilized base, which was immersed in 1000 mL of TSB in a beakercontaining a magnetic bar. Both the TSB and the beaker with themagnetic bar had been also previously sterilized. The system wassealed and incubated at 37 �C under 50 rpm agitation. This proce-dure was repeated every 48 h, completing 240 h of incubation, toform a mature biofilm.

2.5. Essential oils

2.5.1. ExtractionFresh leaves of C. citratus and C. nardus (2.000 g) were collected

from Medicinal Plant Nursery of the Federal University of Lavras inMinas Gerais, Brazil. Essential oils were extracted by hydrodistilla-tion using a modified Clevenger apparatus. Fresh C. citratus and C.nardus leaves were chopped and placed with water in a 4 L volu-metric flask. The flask was coupled to the modified Clevenger appa-ratus and the extraction was performed for 2.5 h with thetemperature maintained at approximately 100 �C. The hydrolateobtained was centrifuged at 321.8g for 5 min, with the essentialoil being removed with a Pasteur pipette and stored at refrigera-tion temperature in glass flasks wrapped in aluminum foil (Gui-marães et al., 2008).

2.5.2. Identification and quantification of chemical constituentsThe essential oil components were identified by gas chromatog-

raphy coupled to mass spectrometry (GC–MS). A Shimadzu gaschromatograph (model GC 17A) equipped with a mass selectivedetector (model QP 5050A), was operated under the following con-ditions: fused silica capillary column (30 m � 0.25 mm) coatedwith DB-5 MS stationary phase; ion source temperature of280 �C; column temperature programmed at an initial temperatureof 50 �C for 2 min, and increase of 4 �C min�1 up to 200 �C,10 �C min�1 up to 300 �C and finally 300 �C for 10 min; helium car-rier gas (1 mL min�1); initial column pressure of 100.2 kPa; splitratio of 1:83 and volume injected of 0.5 lL (1% solution in dichlo-romethane). The following conditions were used for the mass spec-trometer (MS): impact energy of 70 eV; decomposition velocity of1.000, decomposition interval of 0.50 and fragments of 40 Da and550 Da decomposed. A mixture of linear hydrocarbons (C9H20 toC26H54) was injected under identical conditions. The mass spectraobtained were compared to those of the database (Wiley 229)and the Kovats retention index (KI) calculated for each peak wascompared to the values, according to Adams (2001).

Quantification of the essential oil constituents was carried outusing a Shimadzu gas chromatograph (model GC 17A) equippedwith a flame ionization detector (FID) under the following condi-tions: DB5 capillary column; column temperature programmedfrom an initial temperature of 50 �C for 2 min, raised to 4 �C min�1

up to 200 �C, 10 �C min�1 up to 300 �C, finalizing at a temperatureof 300 �C for 10 min; injector temperature of 220 �C; detector tem-perature of 240 �C; nitrogen carrier gas (2.2 mL min�1); split ratioof 1:10; volume injected of 1 lL (1% solution in dichloromethane)and column pressure of 115 kPa. Quantification of each constituentwas obtained by means of area normalization (%).

2.6. Stainless steel coupon treatment using disinfectant solutions

For the elaboration of the disinfectant solutions based on essen-tial oils and control disinfectant solution (without the essentialoils) (Table 1), the following proportions and dilutions suggested

Table 1Compositions of the disinfectant solutions based on essential oils and controldisinfectant solution.

Disinfectantsolutions

Composition (%)

Essentialoil

Ethanol Saline solution with 0.5% (v/v) ofTween 80

C. nardus 3.12 16.88 80.00C. citratus 1.56 18.44 80.00Combinationa 1.56 18.44 80.00Control 0.00 20.00 80.00

a Combination of C. citratus and C. nardus essential oils at 1:1 ratio.

Table 2Number of Listeria monocytogenes cells adhered to AISI 304 (#4) stainless steelsurface, in Log CFU cm�2, at 3 h of biofilm formation, after treatment with disinfectantsolutions based on essential oils and control disinfectant solution.

Disinfectant solutions Contact times Averages

15 min 60 min

Control 4.20 2.88 3.54A

C. citratus 2.92 2.71 2.82AB

Combination 2.81 2.48 2.64B

C. nardus 2.04 0.58 1.31C

Means 2.99A 2.16B

Averages followed by same letter in the column for disinfectants and in the row forcontact times, do not differ by the t test (LSD), at 5% probability (minimum sig-nificant difference = 0.85 for disinfectants and 0.60 for contact times).

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by Chorianopoulos et al. (2008) were used, with modification: dis-tilled water was substituted by saline solution with 0.5% (v/v) ofTween 80. The saline solution was used to provide osmotic concen-tration adequate to the bacterial cell so that the bactericide effectwould be attributed only to the essential oils. Tween 80 was used,as well as ethanol, to dilute the essential oils. The essential oilswere initially diluted with ethanol followed by the addition ofthe saline solution with 0.5% (v/v) of Tween 80. The amount ofessential oil used in each disinfectant solution was based on previ-ous studies about the bacteriostatic effect on L. monocytogenesplanktonic cells (data not shown). For each disinfectant solution,the amount of essential oil used was a minimum inhibitory con-centration (MIC).

The disinfectant action of each solution against the bacterialcells adhered to the stainless steel coupon surface was evaluatedin two different biofilm formation phases: 3 and 240 h. Thus, thestainless steel coupons were previously immersed three times insaline solution to remove the planktonic cells, followed by immer-sion in 4 mL of the disinfectant solutions containing essential oilsand in the control disinfectant solution for 15 and 60 min at28 �C, under static conditions. Contact time was based on previousstudies where the time of bactericidal action was determined un-der planktonic cells (data not shown). In these studies, a contacttime of 15 min was the highest time needed for total eliminationof the L. monocytogenes planktonic cells. In this study, a 60 mincontact time was chosen considering the usefulness of these solu-tions in the process of hygienization at food industries.

2.7. Enumeration of adhered bacterial cells

The number of cells adhered to the stainless steel coupons wasdetermined after treatments using the disinfectant solutions basedon essential oils and the control solution at 3 and 240 h of biofilmformation. Aiming to obtain the number of adhered cells reducedafter each disinfectant treatment, coupons were also computed at3 and 240 h without any disinfectant treatment. Initially, the cou-pons were immersed three times in saline solution, removing anydisinfectant solution residue, as well as planktonic cells. This wasfollowed by removal of the adhered cells using previously steril-ized standard swabs (15 mm � 25 mm). The swabs were trans-ferred to test tubes containing 10 mL of saline solution followedby vortex agitation for 1 min. Serial dilutions up to 10�6 were car-ried out in test tubes containing 9 mL of saline solution. Aliquots(100 lL) of each dilution were inoculated in Petri dishes containingTSA, utilizing the spread plate technique. After this procedure, thePetri dishes were incubated at 37 �C for 24 h.

2.8. Experimental design and statistical analysis

The two biofilm formation phases (3 and 240 h) were individu-ally analyzed. In each experiment, a 4 � 2 factorial scheme (disin-fectants � contact time) was used arranged in a randomized blockdesign. Three blocks were used and each treatment was repeated

twice inside each block. When significant differences were de-tected through variance analysis, the means were compared bythe t test (LSD) at 5% probability. The statistical analyses were con-ducted utilizing the Sisvar program version 4.6 (Ferreira, 2003).

3. Results and discussion

The effectiveness of the disinfectant solutions can be shown bythe counts obtained after using them to treat the stainless steelcoupons containing L. monocytogenes sessile cells. The actions ofthe disinfectant solutions based on essential oils and control disin-fectant solution differed between the two phases of biofilm forma-tion. At 3 h of biofilm formation, the disinfectant solution based onC. nardus essential oil was more effective, following by the disinfec-tant solution based on a combination of C. citratus and C. nardusessential oils (P < 0.05). At this phase, the disinfectant solutionbased on C. citratus essential oil proved to be less effective anddid not differ from the control disinfectant solution (P > 0.05)(Table 2). On the other hand, at 240 h, all solutions based on theessential oils were more effective than the control disinfectantsolution (P < 0.05). The disinfectant solution based on a combina-tion of C. citratus and C. nardus essential oils was more effectivethan the disinfectant solution based on C. citratus essential oil(P < 0.05), however, did not differ from the disinfectant solutionbased on C. nardus essential oil (P > 0.05). The efficacy of the disin-fectant solution based on C. nardus essential oil did not differ fromthat based on C. citratus essential oil (P > 0.05) (Table 3).

C. citratus and C. nardus essential oils presented monoterpenesas major chemical constituents. For C. citratus essential oil, the ma-jor constituents found were: geranial (42.91%) and neral (30.90%),which isomerically form citral. Minor components were 2-undeca-none (4.12%), linalol (1.51%), myrcene (1.36%), geraniol (1.17%),(E)-b-ocimene (0.20%) and (Z)-b-ocimene (0.14%). For C. nardusessential oil, citronellal (34.60%), geraniol (23.17%) and citronellol(12.09%) were the dominant components followed by b-elemene(3.28%), c-cadinene (2.93%), d-cadinene (2.63%), citronellyl acetate(2.06%), germacrene D (1.78%), a-muurolene (1.10%), limonene(1.08%), geranial (0.57%), linalool (0.50%) and neo-isopulegol(0.25%).

The mechanism of action of the monoterpenes involves mainlytoxic effects on the structure and function of the cell membrane. Asa result of their lipophilic character, the monoterpenes will prefer-ably dislocate from the aqueous phase towards the membranestructures (Sikkema, de Bont, & Poolman, 1995). Accumulation ofthe essential oil constituents in the lipid double layer of the cyto-plasm membrane will confer a characteristic of permeability. Inbacteria, cytoplasmic membrane permeabilization is associatedto dissipation of the proton motive force, regarding reduction ofthe ATP pool, internal pH and electric potential, and loss of ions,such as potassium and phosphate ions (Bakkali et al., 2008).

Table 4Reduction in the number of Listeria monocytogenes cells adhered to AISI 304 (#4)stainless steel surface, in Log CFU cm�2, at 3 and 240 h of biofilm formation, aftertreatment with disinfectant solutions based on essential oils and control disinfectantsolution.

Disinfectant solutions 15 min 60 min

Log CFU cm�2a % Log CFU cm�2a %

3 hControl 0.69 14.11 2.01 41.11C. citratus 1.97 40.28 2.18 44.58Combination 2.08 42.53 2.41 49.28C. nardus 2.85 58.28 4.31 88.13

240 hControl 0.59 10.46 1.16 20.56C. citratus 1.50 26.59 4.09 72.51C. nardus 3.28 58.15 4.46 79.07Combination 3.87 68.61 5.64 100.00

a Values obtained by subtracting the number of cells adhered to stainless steelcoupons treated with disinfectant solutions from the number of cells adhered tostainless steel coupons without any disinfectant treatment (4.89 Log CFU cm�2 at3 h and 5.64 Log CFU cm�2 at 240 h).

Table 3Number of Listeria monocytogenes cells adhered to AISI 304 (#4) stainless steelsurface, in Log CFU cm�2, at 240 h of biofilm formation, after treatment with thedisinfectant solutions based on essential oils and control disinfectant solution.

Disinfectant solutions Contact times Averages

15 min 60 min

Control 5.05 4.48 4.76A

C. citratus 4.14 1.55 2.84B

C. nardus 2.36 1.18 1.77BC

Combination 1.77 0.00 0.88C

Averages 3.33A 1.80B

Means followed by same letter in the column for disinfectants and in the row forcontact times, do not differ by the t test (LSD), at 5% probability (minimum sig-nificant difference = 1.25 for disinfectants and 0.88 for contact times).

552 M.M.M. de Oliveira et al. / Food Control 21 (2010) 549–553

The difference found between the actions of disinfectant solu-tions based on essential oils within each biofilm formation phaseanalyzed (Tables 2 and 3) may be attributed mainly to their dis-tinct chemical composition. Differences in the antibacterial activityexisting between essential oils of different plants are due to eco-logical and growth factors and are related to the concentrationand nature of their chemical constituents, such as composition,functional groups, structural configuration of the componentsand possible synergistic interactions (Chang, Chen, & Chang, 2001).

Efficacy of the control disinfectant solution at 3 h of biofilm for-mation did not differ from that of the solution containing C. citratus(P > 0.05) (Table 2), and can be attributed to the fact that themajority of the L. monocytogenes sessil cells are still at the revers-ible adhesion phase. Bacteria adhesion to the surface occurs in twophases: reversible adhesion followed by irreversible adhesion(Mittelman, 1998). During reversible adhesion, bacteria are easilyremoved by application of minimum forces (Chmielewski & Frank,2003; Watnick & Kolter, 2000), accountable for the reduced sessilcell count after the control disinfectant solution treatment 3 h afterbiofilm formation. On the other hand, removal of irreversible ad-hered cells is difficult, requiring application of a strong mechanicalforce or chemical interruption of the adherence force by applyingenzymes, detergents, surfactants, disinfectants, or heat (Sinde &Carballo, 2000).

With respect to contact times utilized, it was observed that theaction of the disinfectant solutions based on essential oils and ofthe control solution was more effective after 60 min (P < 0.05),for the two phases analyzed (3 and 240 h) (Tables 2 and 3). Sincethe antibacterial action of essential oils starts, mainly, through in-creased permeability of the cytoplasmic membrane, with conse-quent leaking of the cell contents, it was observed that thelonger the contact time is between the microorganism and theessential oil solution, the greater the loss of the intercellular com-ponents will be. According to Denyer and Hugo (1991), despite thefact that some loss in the amount of cell content is tolerated by thebacteria without loss of their viability, extensive loss of cell contentor of essential molecules and ions will lead to cell death. This factcould explain the higher efficiency of a 60 min contact time, ascompared to a 15 min contact time.

The effectiveness of disinfectants is frequently determined bythe number of surface-adhered cells they are capable to reduce, ob-tained by standard plate count. Count obtained from the stainlesssteel coupons without any disinfectant treatment at 3 and 240 hafter biofilm formation was used to determine reduction in thenumber of stainless steel surface-adhered L. monocytogenes cellsafter treatments with the essential oil solutions and control solu-tion. All the treatments using disinfectant solutions based onessential oils were effective in reducing the number of L. monocyt-ogenes cells adhered to the surface. However, two disinfectantsolutions were the most outstanding: disinfectant solution based

on C. nardus essential oil, that at 3 h of biofilm formation and a60 min contact time, was capable of reducing 88.13% of the ad-hered cells (4.89 Log CFU cm�2), and disinfectant solution basedon the combination of C. nardus and C. citratus essential oils, thatafter 240 h of biofilm formation with a 60 min contact time wascapable of reducing 100% of the adhered cells (5.64 Log CFU cm�2).The lowest reductions were observed after treatment with the con-trol disinfectant solution (Table 4). According to recommendationby the American Public Health Association (American Public HealthAssociation (APHA), 1992), physical or chemical disinfectantsshould eliminate pathogenic bacteria and reduce the number ofdeteriorating microorganisms at acceptable levels, i.e.,0.3 Log CFU cm�2 of mesophilic aerobic microorganism for stain-less steel surfaces at the end of the hygienization process. Thus,it can be concluded that the disinfecting solution based on thecombination of C. nardus and C. citratus essential oils was efficientand in agreement with the proposed recommendations.

It was also observed that the total reduction in the number ofsurface-adhered cells presented by the disinfectant solution basedon the combination of C. nardus and C. citratus essential oils at240 h of biofilm formation (Table 4) emphasizes the synergistic ac-tion of the essential oils utilized. The term synergism can be de-fined as increase in the activity of compounds or factors whenapplied together, compared to their individual activity (Ceylan &Fung, 2004; Williamson, 2001). The study on synergism resultingfrom the combination of essential oils of different plant specieswas carried out in vitro, presenting promising results (Al-Bayati,2008; Delaquis, Stanich, Girard, & Mazza, 2002; Fu et al., 2007;Gutierrez, Barry-Ryan, & Bourke, 2008). However, no report hasbeen found on the synergistic action of a combination of essentialoils against surface-adhered bacteria.

In conclusion, our findings suggest that C. citratus and C. nardusessential oils are new alternatives to sanitize industrial stainlesssteel surfaces contaminated by L. monocytogenes. Their synergisticeffect must not be ignored, as it can enhance the individual anti-bacterial activity of these compounds.

Acknowledgements

The authors thank the National Counsel of Technological andScientific Development (CNPq) for the first author’s scholarship,and the Research Support Foundation of the State of Minas Gerais(FAPEMIG) for the financial support.

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