Disinfectant action of Cymbopogon sp.

essential oils in different phases

of biolm formation by Listeria monocytogenes on stainless steel surface
Mara Maciel Mattos de Oliveira
a,
*
, Danilo Florisvaldo Brugnera
a
, Maria das Graas Cardoso
b
,
Eduardo Alves
c
, Roberta Hilsdorf Piccoli
a
a
Departamento de Cincia dos Alimentos, Universidade Federal de Lavras (UFLA), Lavras, MG, CEP 37200-000, CP 3037, Brazil
b
Departamento de Qumica, UFLA, Lavras, MG, CEP 37200-000, CP 3037, Brazil
c
Departamento de Fitopatologia, UFLA, Lavras, MG, CEP 37200-000, CP 3037, Brazil
a r t i c l e i n f o
Article history:
Received 23 June 2009
Received in revised form 10 August 2009
Accepted 17 August 2009
Keywords:
Natural disinfectants
Cymbopogon citratus
Cymbopogon nardus
a b s t r a c t
Disinfectant solutions based on the essential oils of Cymbopogon citratus (D.C.) Stapf. and Cymbopogon
nardus (L.) Rendle, applied alone or in combination, and a control disinfectant solution were tested in
two phases (3 and 240 h) of biolm formation by Listeria monocytogenes ATCC 19117 on AISI 304 (#4)
stainless steel surface. Disinfectant solutions based on essential oils have effectively reduced the number
of surface-adhered cells, especially after 60 min of contact. The disinfectant solutions based on a
combination of essential oils were capable of reducing 100% (5.64 Log CFU cm
2
) the number of
surface-adhered cells after 60 min of contact, at 240 h of biolm formation. Essential oils of C. citratus
and C. nardus, alone or in combination, are new alternatives for disinfection of industrial stainless steel
surfaces contaminated by L. monocytogenes.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Bacterial adhesion, with subsequent biolm formation at indus-
trial processing environments, is a potential source of contamina-
tion that can lead to food deterioration or transmission of
foodborne diseases. At food processing industries, surfaces of stain-
less steel equipments and utensils are known to be major sites of
bacterial adhesion and biolm formation (Chmielewski & Frank,
2003; Wong, 1998).
In recent years, priority has been given to efcient cleaning and
disinfection programs that eliminate the risk of food contamina-
tion by microorganisms present on industrial surfaces (Gibson,
Taylor, Hall, & Holah, 1999). Several studies aiming to nd effective
strategies have been published (Gandhi & Chikindas, 2007). Search
for new disinfectants has become a new research area. Growing
negative consumer perception against synthetic compounds has
led to the development of natural alternatives (Davidson, 1997;
Roller, 1995). Thus, essential oils of condiment, aromatic and
medicinal plants, which are potent natural antibacterial agents
(Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004;
Oussalah, Caillet, Saucier, & Lacroix, 2007), have been recently
evaluated for their antibacterial activity on biolms, aiming at
the possibility of using these secondary metabolites or their con-
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 still
limited, pointing to the need of further studies.
Innumerous microorganisms are capable of participating in the
process of adhesion and biolm formation. In the food industry,
they can be divided into spoilage and pathogenic microorganisms.
With respect to the latter, Listeria monocytogenes is known for its
resistance to heat and high concentration of salts, survival under
refrigeration 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 bacterium
difcult to be controlled in food processing environments, thus it is
commonly found in meat and dairy processing industries (Chambel
et al., 2007; Chasseignaux et al., 2002; Cruz et al., 2008; Lpez
et al., 2008; Senczek, Stephan, & Untermann, 2000).
Poaceae belong to one of the largest plant families comprising
around 500 genera and approximately 8000 species, being
essentially 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 Cymbopogon
citratus (D.C.) Stapf. (lemongrass) and Cymbopogon nardus L. Rendle
0956-7135/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2009.08.003
* Corresponding author. Tel.: +55 35 3829 1392; fax: +55 35 3829 1401.
E-mail address: mmacielmattos@yahoo.com.br (Mara Maciel Mattos de Oli-
veira).
Food Control 21 (2010) 549553
Contents lists available at ScienceDirect
Food Control
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodcont
(citronella) was demonstrated in vitro against L. monocytogenes
(Oussalah et al., 2007).
The objective of this study was to evaluate the disinfectant
action of C. citratus and C. nardus essential oils, alone and in com-
bination, in two phases of biolm formation by L. monocytogenes
ATCC 19117 on AISI 304 (#4) stainless steel surface.
2. Materials and methods
2.1. Microorganism used, standardization, inoculum preparation and
storage
The microorganism used was L. monocytogenes ATCC 19117, ac-
quired from the Culture Collection Section of the Medical Biology
Division of the Adolfo Lutz Institute (So Paulo SP, Brazil). To
standardize the number of cells, the strain was initially inoculated
in an Erlenmeyer ask 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 in
saline solution [NaCl 0.9% (p/v)]. Then, from the saline solution,
and using Trypic Soy Agar (TSA) as culture medium, spread plating
methodology 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 of
distilled water, with the nal pH adjusted to 7.27.4). For strain
reactivation and use, an aliquot of the freezing culture medium
was transferred to test tubes containing TSB, with two subcultures
at 37 C for 24 h. The culture was striated in TSA added to Petri
dishes and incubated at 37 C for 24 h. Of the colonies formed on
the TSA surface, some were removed and transferred into an Erlen-
meyer ask containing 150 mL of TSB, which was incubated at
37 C until reaching the number of cells necessary for the experi-
ment, approximately 9.17 Log CFU mL
1
(OD
600 nm
= 0.895).
2.2. Biolm formation experimental model
The experimental model of biolm formation by L. monocytoge-
nes was elaborated based on a system rst used by Bagge, Hjelm,
Johansen, Huber, and Gram (2001); Gram, Bagge-Ravn, Ng,
Gymoese, and Vogel (2007), with modications. In the present
study, 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 magnetic
agitator to allow the free circulation of the substrate inside the
beaker. The beaker was sealed with a Petri dish and plastic lm.
AISI 304 (#4) stainless steel was chosen for being the most utilized
in the food industry.
2.3. Preparation of the coupons and stainless steel base
In order to initiate the bacterial cell adhesion stage, the coupons
and stainless steel base were previously hygienized and sterilized.
First they were cleaned with acetone 100%, washed by immersion
in alkaline detergent [NaOH 1% (w/v), pH 13.2] for 1 h, rinsed with
sterilized distilled water, dried and cleaned with alcohol 70% (v/v).
After the hygienization, they were washed with sterilized distilled
water, 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 TSB
containing the bacterial culture were added to the beaker contain-
ing the magnetic bar, at a nal concentration of approximately
8 Log CFU mL
1
.The stainless steel base containing the coupons
was placed inside the beaker, which was sealed and incubated at
37 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 new
sterilized base, which was immersed in 1000 mL of TSB in a beaker
containing a magnetic bar. Both the TSB and the beaker with the
magnetic bar had been also previously sterilized. The system was
sealed and incubated at 37 C under 50 rpm agitation. This proce-
dure was repeated every 48 h, completing 240 h of incubation, to
form a mature biolm.
2.5. Essential oils
2.5.1. Extraction
Fresh leaves of C. citratus and C. nardus (2.000 g) were collected
from Medicinal Plant Nursery of the Federal University of Lavras in
Minas Gerais, Brazil. Essential oils were extracted by hydrodistilla-
tion using a modied Clevenger apparatus. Fresh C. citratus and C.
nardus leaves were chopped and placed with water in a 4 L volu-
metric ask. The ask was coupled to the modied Clevenger appa-
ratus and the extraction was performed for 2.5 h with the
temperature maintained at approximately 100 C. The hydrolate
obtained was centrifuged at 321.8g for 5 min, with the essential
oil being removed with a Pasteur pipette and stored at refrigera-
tion temperature in glass asks wrapped in aluminum foil (Gui-
mares et al., 2008).
2.5.2. Identication and quantication of chemical constituents
The essential oil components were identied by gas chromatog-
raphy coupled to mass spectrometry (GCMS). A Shimadzu gas
chromatograph (model GC 17A) equipped with a mass selective
detector (model QP 5050A), was operated under the following con-
ditions: fused silica capillary column (30 m 0.25 mm) coated
with DB-5 MS stationary phase; ion source temperature of
280 C; column temperature programmed at an initial temperature
of 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 nally 300 C for 10 min; helium car-
rier gas (1 mL min
1
); initial column pressure of 100.2 kPa; split
ratio 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 of
1.000, decomposition interval of 0.50 and fragments of 40 Da and
550 Da decomposed. A mixture of linear hydrocarbons (C
9
H
20
to
C
26
H
54
) was injected under identical conditions. The mass spectra
obtained were compared to those of the database (Wiley 229)
and the Kovats retention index (KI) calculated for each peak was
compared to the values, according to Adams (2001).
Quantication of the essential oil constituents was carried out
using a Shimadzu gas chromatograph (model GC 17A) equipped
with a ame ionization detector (FID) under the following condi-
tions: DB5 capillary column; column temperature programmed
from 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, nalizing at a temperature
of 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 ratio
of 1:10; volume injected of 1 lL (1% solution in dichloromethane)
and column pressure of 115 kPa. Quantication of each constituent
was 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 essential
oils) (Table 1), the following proportions and dilutions suggested
550 M.M.M. de Oliveira et al. / Food Control 21 (2010) 549553
by Chorianopoulos et al. (2008) were used, with modication: dis-
tilled water was substituted by saline solution with 0.5% (v/v) of
Tween 80. The saline solution was used to provide osmotic concen-
tration adequate to the bacterial cell so that the bactericide effect
would be attributed only to the essential oils. Tween 80 was used,
as well as ethanol, to dilute the essential oils. The essential oils
were initially diluted with ethanol followed by the addition of
the saline solution with 0.5% (v/v) of Tween 80. The amount of
essential oil used in each disinfectant solution was based on previ-
ous studies about the bacteriostatic effect on L. monocytogenes
planktonic 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 bacterial
cells adhered to the stainless steel coupon surface was evaluated
in two different biolm formation phases: 3 and 240 h. Thus, the
stainless steel coupons were previously immersed three times in
saline solution to remove the planktonic cells, followed by immer-
sion in 4 mL of the disinfectant solutions containing essential oils
and in the control disinfectant solution for 15 and 60 min at
28 C, under static conditions. Contact time was based on previous
studies where the time of bactericidal action was determined un-
der planktonic cells (data not shown). In these studies, a contact
time of 15 min was the highest time needed for total elimination
of the L. monocytogenes planktonic cells. In this study, a 60 min
contact 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 was
determined after treatments using the disinfectant solutions based
on essential oils and the control solution at 3 and 240 h of biolm
formation. Aiming to obtain the number of adhered cells reduced
after each disinfectant treatment, coupons were also computed at
3 and 240 h without any disinfectant treatment. Initially, the cou-
pons were immersed three times in saline solution, removing any
disinfectant solution residue, as well as planktonic cells. This was
followed 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 followed
by 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 containing
TSA, utilizing the spread plate technique. After this procedure, the
Petri dishes were incubated at 37 C for 24 h.
2.8. Experimental design and statistical analysis
The two biolm 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 block
design. Three blocks were used and each treatment was repeated
twice inside each block. When signicant differences were de-
tected through variance analysis, the means were compared by
the 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 by
the counts obtained after using them to treat the stainless steel
coupons containing L. monocytogenes sessile cells. The actions of
the disinfectant solutions based on essential oils and control disin-
fectant solution differed between the two phases of biolm forma-
tion. At 3 h of biolm formation, the disinfectant solution based on
C. nardus essential oil was more effective, following by the disinfec-
tant solution based on a combination of C. citratus and C. nardus
essential oils (P < 0.05). At this phase, the disinfectant solution
based on C. citratus essential oil proved to be less effective and
did not differ from the control disinfectant solution (P > 0.05)
(Table 2). On the other hand, at 240 h, all solutions based on the
essential oils were more effective than the control disinfectant
solution (P < 0.05). The disinfectant solution based on a combina-
tion of C. citratus and C. nardus essential oils was more effective
than the disinfectant solution based on C. citratus essential oil
(P < 0.05), however, did not differ from the disinfectant solution
based on C. nardus essential oil (P > 0.05). The efcacy of the disin-
fectant solution based on C. nardus essential oil did not differ from
that based on C. citratus essential oil (P > 0.05) (Table 3).
C. citratus and C. nardus essential oils presented monoterpenes
as 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. nardus
essential 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 mainly
toxic effects on the structure and function of the cell membrane. As
a result of their lipophilic character, the monoterpenes will prefer-
ably dislocate from the aqueous phase towards the membrane
structures (Sikkema, de Bont, & Poolman, 1995). Accumulation of
the essential oil constituents in the lipid double layer of the cyto-
plasm membrane will confer a characteristic of permeability. In
bacteria, cytoplasmic membrane permeabilization is associated
to dissipation of the proton motive force, regarding reduction of
the ATP pool, internal pH and electric potential, and loss of ions,
such as potassium and phosphate ions (Bakkali et al., 2008).
Table 1
Compositions of the disinfectant solutions based on essential oils and control
disinfectant solution.
Disinfectant
solutions
Composition (%)
Essential
oil
Ethanol Saline solution with 0.5% (v/v) of
Tween 80
C. nardus 3.12 16.88 80.00
C. citratus 1.56 18.44 80.00
Combination
a
1.56 18.44 80.00
Control 0.00 20.00 80.00
a
Combination of C. citratus and C. nardus essential oils at 1:1 ratio.
Table 2
Number of Listeria monocytogenes cells adhered to AISI 304 (#4) stainless steel
surface, in Log CFU cm
2
, at 3 h of biolm formation, after treatment with disinfectant
solutions based on essential oils and control disinfectant solution.
Disinfectant solutions Contact times Averages
15 min 60 min
Control 4.20 2.88 3.54
A
C. citratus 2.92 2.71 2.82
AB
Combination 2.81 2.48 2.64
B
C. nardus 2.04 0.58 1.31
C
Means 2.99
A
2.16
B
Averages followed by same letter in the column for disinfectants and in the row for
contact times, do not differ by the t test (LSD), at 5% probability (minimum sig-
nicant difference = 0.85 for disinfectants and 0.60 for contact times).
M.M.M. de Oliveira et al. / Food Control 21 (2010) 549553 551
The difference found between the actions of disinfectant solu-
tions based on essential oils within each biolm formation phase
analyzed (Tables 2 and 3) may be attributed mainly to their dis-
tinct chemical composition. Differences in the antibacterial activity
existing between essential oils of different plants are due to eco-
logical and growth factors and are related to the concentration
and nature of their chemical constituents, such as composition,
functional groups, structural conguration of the components
and possible synergistic interactions (Chang, Chen, & Chang, 2001).
Efcacy of the control disinfectant solution at 3 h of biolm 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 the
majority of the L. monocytogenes sessil cells are still at the revers-
ible adhesion phase. Bacteria adhesion to the surface occurs in two
phases: reversible adhesion followed by irreversible adhesion
(Mittelman, 1998). During reversible adhesion, bacteria are easily
removed by application of minimum forces (Chmielewski & Frank,
2003; Watnick & Kolter, 2000), accountable for the reduced sessil
cell count after the control disinfectant solution treatment 3 h after
biolm formation. On the other hand, removal of irreversible ad-
hered cells is difcult, requiring application of a strong mechanical
force or chemical interruption of the adherence force by applying
enzymes, detergents, surfactants, disinfectants, or heat (Sinde &
Carballo, 2000).
With respect to contact times utilized, it was observed that the
action of the disinfectant solutions based on essential oils and of
the control solution was more effective after 60 min (P < 0.05),
for the two phases analyzed (3 and 240 h) (Tables 2 and 3). Since
the 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 the
longer the contact time is between the microorganism and the
essential oil solution, the greater the loss of the intercellular com-
ponents will be. According to Denyer and Hugo (1991), despite the
fact that some loss in the amount of cell content is tolerated by the
bacteria without loss of their viability, extensive loss of cell content
or of essential molecules and ions will lead to cell death. This fact
could explain the higher efciency of a 60 min contact time, as
compared to a 15 min contact time.
The effectiveness of disinfectants is frequently determined by
the number of surface-adhered cells they are capable to reduce, ob-
tained by standard plate count. Count obtained from the stainless
steel coupons without any disinfectant treatment at 3 and 240 h
after biolm formation was used to determine reduction in the
number of stainless steel surface-adhered L. monocytogenes cells
after treatments with the essential oil solutions and control solu-
tion. All the treatments using disinfectant solutions based on
essential oils were effective in reducing the number of L. monocyt-
ogenes cells adhered to the surface. However, two disinfectant
solutions were the most outstanding: disinfectant solution based
on C. nardus essential oil, that at 3 h of biolm formation and a
60 min contact time, was capable of reducing 88.13% of the ad-
hered cells (4.89 Log CFU cm
2
), and disinfectant solution based
on the combination of C. nardus and C. citratus essential oils, that
after 240 h of biolm formation with a 60 min contact time was
capable 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 recommendation
by the American Public Health Association (American Public Health
Association (APHA), 1992), physical or chemical disinfectants
should eliminate pathogenic bacteria and reduce the number of
deteriorating 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 the
combination of C. nardus and C. citratus essential oils was efcient
and in agreement with the proposed recommendations.
It was also observed that the total reduction in the number of
surface-adhered cells presented by the disinfectant solution based
on the combination of C. nardus and C. citratus essential oils at
240 h of biolm formation (Table 4) emphasizes the synergistic ac-
tion of the essential oils utilized. The term synergism can be de-
ned as increase in the activity of compounds or factors when
applied together, compared to their individual activity (Ceylan &
Fung, 2004; Williamson, 2001). The study on synergism resulting
from the combination of essential oils of different plant species
was 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 has
been found on the synergistic action of a combination of essential
oils against surface-adhered bacteria.
In conclusion, our ndings suggest that C. citratus and C. nardus
essential oils are new alternatives to sanitize industrial stainless
steel surfaces contaminated by L. monocytogenes. Their synergistic
effect 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 and
Scientic Development (CNPq) for the rst authors scholarship,
and the Research Support Foundation of the State of Minas Gerais
(FAPEMIG) for the nancial support.
Table 4
Reduction 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 biolm formation, after
treatment with disinfectant solutions based on essential oils and control disinfectant
solution.
Disinfectant solutions 15 min 60 min
Log CFU cm
2a
% Log CFU cm
2a
%
3 h
Control 0.69 14.11 2.01 41.11
C. citratus 1.97 40.28 2.18 44.58
Combination 2.08 42.53 2.41 49.28
C. nardus 2.85 58.28 4.31 88.13
240 h
Control 0.59 10.46 1.16 20.56
C. citratus 1.50 26.59 4.09 72.51
C. nardus 3.28 58.15 4.46 79.07
Combination 3.87 68.61 5.64 100.00
a
Values obtained by subtracting the number of cells adhered to stainless steel
coupons treated with disinfectant solutions from the number of cells adhered to
stainless steel coupons without any disinfectant treatment (4.89 Log CFU cm
2
at
3 h and 5.64 Log CFU cm
2
at 240 h).
Table 3
Number of Listeria monocytogenes cells adhered to AISI 304 (#4) stainless steel
surface, in Log CFU cm
2
, at 240 h of biolm formation, after treatment with the
disinfectant solutions based on essential oils and control disinfectant solution.
Disinfectant solutions Contact times Averages
15 min 60 min
Control 5.05 4.48 4.76
A
C. citratus 4.14 1.55 2.84
B
C. nardus 2.36 1.18 1.77
BC
Combination 1.77 0.00 0.88
C
Averages 3.33
A
1.80
B
Means followed by same letter in the column for disinfectants and in the row for
contact times, do not differ by the t test (LSD), at 5% probability (minimum sig-
nicant difference = 1.25 for disinfectants and 0.88 for contact times).
552 M.M.M. de Oliveira et al. / Food Control 21 (2010) 549553
References
Adams, R. P. (2001). Identication of essential oils components by gas chromatography/
mass spectroscopy. Carol Stream: Allured.
Al-Bayati, F. A. (2008). Synergistic antibacterial activity between Thymus vulgaris
and Pimpinella anisum essential oils and methanol extracts. Journal of
Ethnopharmacology, 116, 403406.
American Public Health Association (APHA) (1992). Compendium of methods for the
microbiological examination of foods. Hanover: EPS Group.
Bagge, D., Hjelm, M., Johansen, C., Huber, I., & Gram, L. (2001). Shewanella
putrefaciens adhesion and biolm formation on food processing surfaces.
Applied and Environmental Microbiology, 67, 23192325.
Bakkali, F., Averbeck, S., Averbeck, D., & Idaomar, M. (2008). Biological effects of
essential oils: A review. Food and Chemical Toxicology, 46, 446475.
Burt, S. (2004). Essential oils: Their antibacterial properties and potential
applications in foods: A review. International Journal of Food Microbiology, 94,
223253.
Ceylan, E., & Fung, D. Y. C. (2004). Antimicrobial activity of spices. Journal of Rapid
Methods and Automation in Microbiology, 12, 155.
Chambel, L., Sol, M., Fernandes, I., Barbosa, M., Zilho, I., Barata, B., et al. (2007).
Occurrence and persistence of Listeria spp. in the environment of ewe and cows
milk cheese dairies in Portugal unveiled by an integrated analysis of
identication, typing and spatialtemporal mapping along production cycle.
International Journal of Food Microbiology, 116, 5263.
Chang, S. T., Chen, P. F., & Chang, S. C. (2001). Antibacterial activity of leaf essential
oils and their constituents from Cinnamomum osmophloeum. Journal of
Ethnopharmacology, 77, 123127.
Chasseignaux, E., Gerault, P., Toquin, M., Salvat, G., Colin, P., & Ermel, G. (2002).
Ecology of Listeria monocytogenes in the environment of raw poultry meat and
raw pork meat processing plants. FEMS Microbiology Letters, 210, 271275.
Chmielewski, R. A. N., & Frank, J. F. (2003). Biolm formation and control in food
processing facilities. International Journal of FoodScience and Technology, 2, 2232.
Chorianopoulos, N. G., Giaouris, E. D., Skandamis, P. M., Haroutounian, S. A., &
Nychas, G. J. E. (2008). Disinfectant test against monoculture and mixed-culture
biolms composed of technological, spoilage and pathogenic bacteria:
Bactericidal effect of essential oil and hydrosol of Satureja thymbra and
comparison with standard acidbase sanitizers. Journal of Applied
Microbiology, 104, 15861696.
Cruz, C. D., Silvestre, F. A., Kinoshita, E. M., Landgraf, M., Franco, B. D. G. M., & Destro,
M. T. (2008). Epidemiological survey of Listeria monocytogenes in a gravlax
salmon processing line. Brazilian Journal of Microbiology, 39, 375383.
Davidson, P. M. (1997). Chemical preservatives and natural antimicrobial
compounds. In M. P. Doyle, L. R. Beuchat, & T. J. Montville (Eds.), Food
microbiology fundamentals and frontiers (pp. 520556). New York: ASM.
Delaquis, P. J., Stanich, K., Girard, B., & Mazza, G. (2002). Antimicrobial activity of
individual and mixed fractions of dill, cilantro, coriander and eucalyptus
essential oils. International Journal of Food Microbiology, 74, 101109.
Denyer, S. P., & Hugo, W. B. (1991). Biocide-induced damage to the bacterial
cytoplasmic membrane. In S. P. Denyer & W. B. Hugo (Eds.), Mechanisms of
action of chemical biocides: Their study and exploitation (pp. 171188). Oxford:
Blackwell Scientic Publications.
Ferreira, D. F. (2003). SISVAR Sistema de anlise de varincia para dados
balanceados: Programa de anlises estatsticas e planejamento de experimentos.
Verso 4.6. Software. Lavras: DEX/UFLA.
Fu, Y. J., Zu, Y. G., Chen, L. Y., Shi, X. G., Wang, Z., Sun, S., et al. (2007). Antimicrobial
activity of clove and rosemary essential oils alone and in combination.
Phytotherapy Research, 21, 989994.
Gandhi, M., & Chikindas, M. L. (2007). Listeria: A foodborne pathogen that knows
how to survive. International Journal of Food Microbiology, 113, 115.
Gibson, H., Taylor, J. H., Hall, K. E., & Holah, J. T. (1999). Effectiveness of cleaning
techniques used in the food industry in terms of the removal of bacterial
biolms. Journal of Applied Microbiology, 87, 4149.
Gram, L., Bagge-Ravn, D., Ng, Y. Y., Gymoese, P., & Vogel, B. F. (2007). Inuence of
food soiling matrix on cleaning and disinfection efciency on surface attached
Listeria monocytogenes. Food Control, 18, 11651171.
Guimares, L. G. de L., Cardoso, M. das G., Zacaroni, L. M., Lima, R. K. de, Pimentel, F.
A., & Morais, A. R. de (2008). Inuence of light and temperature on the oxidation
of the essential oil of lemongrass (Cymbopogon citratus (D.C.) Stapf). Qumica
Nova, 31, 14761480.
Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2008). The antimicrobial efcacy of plant
essential oil combinations and interactions with food ingredients. International
Journal of Food Microbiology, 124, 9197.
Knowles, J. R., Roller, S., Murray, D. B., & Naidu, A. S. (2005). Antimicrobial action of
carvacrol at different stages of dual-species biolm development by
Staphylococcus aureus and Salmonella enterica Serovar Typhimurium. Applied
and Environmental Microbiology, 71, 797803.
Lebert, I., Leroy, S., & Talon, R. (2007). Effect of industrial and natural biocides on
spoilage, pathogenic and technological strains grown in biolm. Food
Microbiology, 24, 281287.
Lpez, V., Villatoro, D., Ortiz, S., Lpez, P., Navas, J., Dvila, J. C., et al. (2008).
Molecular tracking of Listeria monocytogenes in an Iberian pig abattoir and
processing plant. Meat Science, 78, 130134.
Mittelman, M. W. (1998). Structure and functional characteristics of bacterial
biolms in uid processing operations. Journal of Dairy Science, 81,
27602764.
Ortiz, R. S., Marrero, G. V., & Navarro, A. L. T. (2002). Instructivo tcnico para el
cultivo de Cymbopogon citratus (D.C) Stapf (caa santa). Revista Cubana de
Plantas Medicinales, 7, 8995.
Oussalah, M., Caillet, S., Saucier, L., & Lacroix, M. (2007). Inhibitory effects of
selected plant essential oils on the growth of four pathogenic bacteria: E. coli
O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria
monocytogenes. Food Control, 18, 414420.
Pan, Y., Breidt Junior, F., & Kathariou, S. (2006). Resistance of Listeria monocytogenes
biolms to sanitizing agents in a simulated food processing environment.
Applied and Environmental Microbiology, 72, 77117717.
Roller, S. (1995). The quest for natural antimicrobials as novel means of food
preservation: Status report on a European research project. International
Biodeterioration and Biodegradation, 36, 333345.
Rossoni, E. M. M., & Gaylarde, C. C. (2000). Comparison of sodium hypochlorite and
peracetic acid as sanitizing agents for stainless steel food processing surfaces
using epiuorescence microscopy. International Journal of Food Microbiology, 61,
8185.
Sandasi, M., Leonard, C. M., & Viljoen, A. M. (2008). The effect of ve common
essential oil components on Listeria monocytogenes biolms. Food Control, 19,
10701075.
Senczek, D., Stephan, R., & Untermann, F. (2000). Pulsed-eld gel
electrophoresis (PFGE) typing of Listeria strains isolated from a meat
processing plant over a 2-year period. International Journal of Food
Microbiology, 62, 155159.
Sikkema, J., de Bont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane
toxicity of hydrocarbons. Microbiological Reviews, 59, 201222.
Sinde, E., & Carballo, J. (2000). Attachment of Salmonella spp. and Listeria
monocytogenes to stainless steel, rubber and polytetrauoroethylene: The
inuence of free energy and the effect of commercial sanitizers. Food
Microbiology, 17, 439447.
Torres, K., Sierra, S., Poutou, R., Carrascal, A., & Mercado, M. (2005). Pathogenesis of
Listeria monocytogenes, microorganism zoonotic emergent. Revista MVZ Crdoba,
10, 511543.
Watnick, P., & Kolter, R. (2000). Minireview: Biolm, city of microbes. Journal of
Bacteriology, 182, 26757679.
Williamson, E. M. (2001). Synergy and other interactions in phytomedicines.
Phytomedicine, 8, 401409.
Wong, A. C. L. (1998). Biolms in food processing environments. Journal of Dairy
Science, 81, 27652770.
M.M.M. de Oliveira et al. / Food Control 21 (2010) 549553 553