Ribeiro, D. S. et al.
Vol. 4, N.1: pp. 1-9, February 2013
ISSN: 2179-4804
1
Journal of Biotechnology
and Biodiversity
Evaluation rosemary essential oil in the control of
multidrug-resistant Escherichia coli in Coalho cheese.
Daniele Silva Ribeiro1,*, Felix G. Siqueira2,3, Eudes da Silva Velozo4, Alaíse Gil Guimarães1.
ABSTRACT
The use of essential oils from plants with known antimicrobial potential is a promising alternative to prevent the
proliferation of microorganisms carried by food. We aimed to evaluate the antimicrobial potential of rosemary
essential oil in the control of a strain of multidrug-resistant Escherichia coli that was used to inoculate Coalho
cheese. The rosemary essential oil (REO) was obtained by hydro-distillation, and its minimum inhibitory
concentration (MIC) against the test strain was determined by the agar diffusion method, yielding a MIC of 20%
(v/v). The E. coli strain EC16 was used to inoculate two samples of commercial cheese. One sample, designated as
the test sample, was inoculated with 20% (v/v) REO, and another sample was used without the addition of REO as a
control. Both samples were the same size and were refrigerated. During seven days of refrigeration, the number of
E. coli present in each sample was determined. There was a 2.3-log reduction in the first 24 hours in the test
sample. The results showed that the rosemary essential oil has inhibitory activity against the E. coli strain EC16
within a food matrix. Thus, rosemary essential oil could be used in the control of pathogenic bacteria. However, its
use as a preservative in food should also be based on sensory characteristics.
Keywords: Rosmarinus officinalis L. essential oil, natural antimicrobial, bacterial control, Coalho cheese.
Avaliação do óleo essencial de alecrim no controle de
Escherichia coli multirresistente em queijo Coalho
RESUMO
O uso de óleos essenciais de plantas, de conhecido potencial antimicrobiano, é uma alternativa promissora na
inibição da proliferação de microrganismos veiculados por alimentos. No presente estudo, objetivou-se avaliar o
potencial antimicrobiano do óleo essencial de alecrim (Rosmarinus officinalis L.) no controle de uma cepa de
Escherichia coli multirresistente inoculada em queijo de coalho. O óleo essencial de alecrim (OEA) foi obtido
através de hidrodestilação, sua concentração inibitória mínima - CIM frente à cepa teste, determinada pela técnica
de difusão em ágar, foi de 20% (v/v). A cepa de E. coli - EC 16 utilizada foi inoculada em duas amostras de queijo
de coalho comercial. Em uma das amostras foi adicionado OEA a 20% (v/v), sendo considerada amostra Teste e a
outra amostra, sem adição de OEA, amostra Controle. Ambas foram porcionadas e submetidas à refrigeração, para
serem avaliadas, durante sete dias, através da contagem de E. coli. Houve uma redução 2,3 ciclos logarítmicos, nas
primeiras 24 horas, na amostra Teste. Os resultados mostraram que o óleo essencial de alecrim demonstrou possuir
propriedades inibitórias frente à cepa de E. coli – EC16 dentro da matriz alimentícia avaliada. Infere-se que o
mesmo pode ser usado no controle de bactérias patogênicas. Todavia, a sua utilização como conservante em
alimentos deverá ser pautada em análises sensoriais.
Keywords: Óleo essencial de Rosmarinus officinalis L., antimicrobianos naturais, controle de bactérias, queijo
coalho.
*Author for correspondence.
1
Graduate Program in Food Science, Federal University of Bahia, Faculty of Pharmacy, P.O Box 40170-175,
Ondina, Salvador – BA, Brazil. *[email protected], [email protected].
2
Federal University of Bahia, Multidisciplinary Institute on Health, Vitória da Conquista – BA, Brazil.
3
Embrapa Agroenergia, Brasília, Brazil. [email protected].
4
Federal University of Bahia, Medicines Department, Faculty of Pharmacy, Salvador – BA, Brazil,
[email protected].
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
INTRODUCTION
The preservation of foods has become
increasingly complex because consumers are
demanding more natural foods with low levels
of chemical additives and with the convenience
of a long-shelf life (Leuschner & Zamparini,
2002; Del Campo et al., 2003). Consequently,
the search for natural additives, especially of
plant origin, has increased notably in recent
years (Fernandez-Lopez et al., 2004; Souza et
al., 2008). This search is especially important
given the antibiotic resistance that many
microorganisms have acquired because of
widespread antibiotic use, making it necessary to
develop alternative food preservation methods.
Many compounds found naturally in herbs and
spices and their essential oils have been shown
to possess antimicrobial activities against
foodborne pathogens, and these activities have
been widely studied (Souza et al., 2004).
According to Rahaman & Kang (2009), essential
oils are natural antimicrobials with promising
potential applications in the food and
pharmaceutical industry for the control of
pathogenic bacteria.
Studies have shown that plants of the Lamiaceae
family, especially rosemary, oregano, sage,
thyme and basil, have activity against many
foodborne pathogens (Santoyo et al., 2005;
Bozin et al., 2007; Gachkar et al., 2007; Zago et
al., 2009; Luqman et al., 2007; Barbosa, 2010).
Rosmarinus officinalis L., popularly known as
rosemary, is used as to flavor food, and it is a
powerful antibacterial, antimutagenic and
chemopreventive agent (Oluwatuyi, Kaatz &
Gibbons, 2004). Moreno et al. (2006) have
reported that rosemary is a rich source of
phenolic compounds with high antimicrobial
activity against gram-positive and gram-negative
bacteria. Compounds such as these may be
useful in the control of bacteria in food.
Foods with high protein and water contents,
such as cheese, provide a suitable environment
for the development of microorganisms. In
particular, Coalho cheese often has high levels
of bacterial contamination, as reported in several
studies (Florentino & Martins, 1999; Mendes et
al., 1999; Leite Júnior et al., 2000; Leite et al.,
2002; Borges et al., 2003; Feitosa et al., 2003;
Duarte et al., 2005a; Oliveira et al., 2010).
The microorganisms generally involved in these
processes are coliform bacteria, Staphylococcus
coagulase-positive bacteria and Salmonella spp.,
2
the presence of which suggests contamination
due to poor hygiene during production and/or
the use of raw materials of poor quality. Foods
contaminated with these microorganisms may
expose consumers to foodborne illnesses
(Nascimento et al., 2001; Duarte et al., 2005a).
The use of essential oils is a promising
alternative for the control of pathogens in
Coalho cheese. These oils can also be used to
prolong the shelf life of foods and to prevent
foodborne illnesses. The present study was
undertaken to evaluate the effect of essential oils
obtained from fresh leaves of rosemary
(Rosmarinus officinalis L.) in the control of
multidrug-resistant Escherichia coli in Coalho
cheese.
MATERIALS AND METHODS
Preparation of the plant material and
extraction of the rosemary essential oil
Approximately 936g of fresh leaves of
Rosmarinus officinalis Linn were collected in
the region of Coração de Maria (SE) This plant
material was sent to the Herbarium Alexandre
Leal Costa of Biology Institute of Federal
University of Bahia, where the specimen was
identified and cataloged (voucher specimen
number 96109).
The REO was extracted using classical hydrodistillation in a modified Clevenger apparatus
for three hours according to Brazilian
Pharmacopea. Then, approximately 950 mg of
essential oil was collected and stored in a sterile
container protected from the presence of light
and temperatures below 0°C.
Strain Test
The strain of Escherichia coli – EC 16, isolated
from in natura Coalho cheese and resistant to
ampicillin (10μg/disc), cephalothin (30mg/disc),
gentamicin (10 μg/disc) and chloramphenicol
(30 μg/disc), was grown for 24 hours in
Typticase Soy Agar (TSA) at 37ºC. The standard
suspension was made in saline (0.85% NaCl)
and was standardized to the 0.5 McFarland
standard, corresponding to a concentration of
approximately 108 CFU mL-1.
Screening
The screening was performed using the agar
diffusion technique with sterile filter paper discs
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
(Bauer et al., 1966). The filter paper discs (6 mm
in diameter) were individually impregnated with
15 µL of the rosemary essential oil, arranged on
disposable Petri dishes containing Mueller
Hinton agar inoculated with strain EC16 in
suspension at the concentration of 108 CFU mL1
. The plates were incubated at 37°C±1°C for 24
hours. After incubation, the diameters (mm) of
the inhibition zones were measured. Samples
were considered positive for antimicrobial
activity when the inhibition zones of bacterial
growth had diameters of equal or greater than 10
mm (Souza et al., 2005). The test was performed
in triplicate. In parallel, the same experiment
was performed using dilutions of the rosemary
essential oil in dimethylsulfoxide (DMSO) to
determine
the
minimum
inhibitory
concentration.
Determination of the minimum inhibitory
concentration – MIC
To determine the MIC, we used the well
diffusion technique adapted from Bauer et al.
(1966).
In Falcon tubes, 20 mL of Mueller Hinton agar,
previously melted at a temperature of 45°C, was
inoculated with 2 mL of the strain test
suspension, which had a concentration of 108
CFU mL-1. The agar was then poured into sterile
Petri dishes containing a thin layer of 1%
bacteriological agar, and sterile tips were place
upside down in the agar to create wells. After
solidification of the medium in a laminar flow
hood, the tips were removed from the agar, thus
forming wells (6 mm in diameter). Thirtymicroliter aliquots of rosemary essential oil
diluted in DMSO at concentrations of 500, 400,
300, 200 and 100 µL mL-1 were added to the
wells. After incubation at 37°C for 24 hours, the
inhibition zones were analyzed. The MIC was
defined as the lowest concentration for which
the inhibition zone had a diameter equal or
greater than 10 mm (Souza et al., 2005). The
assays were performed in triplicate.
Preparation of the sample
The Coalho cheese used in this study was
purchased from the local market. It was vacuumpacked and free of preservatives, as indicated on
the label, with validity period of three months
from the date of manufacture and five days after
opening. The packaged product was exposed to
ultraviolet radiation in a laminar flow hood for
3
15 minutes before opening. The cheese was then
portioned into 10g samples for use in the
experiment. These 10g samples were packaged
in sterile bags and again submitted to ultraviolet
radiation for 20 minutes to minimize possible
contamination.
After these procedures, a sample of the Coalho
cheese was examined microbiologically using
the following tests: a standard plate count using
Plate Count Agar (PCA), enumeration of
Escherichia coli using Chromo Cult agar (Merck
®), Staphylococcus coagulase activity using
Baird Parker Agar and a Salmonella spp. test
using the methods of Silva (2007a).
Evaluation of rosemary essential oil for the
control of E. coli EC16 in Coalho cheese
To test the antimicrobial activity of REO in a
food matrix, the bacterial suspension was
serially diluted in 0.1% peptone water to the
final concentration of 105 CFU/mL.
In a sterile environment, the 10 g portions of
Coalho cheese packed in sterile bags were
inoculated with 500 μL of REO at the minimum
inhibitory concentration (20%, v/v) previously
determined and with 500 μL of the test strain at
105 CFU mL-1. Still packed, the portions of
cheese were massaged and macerated manually
and left to stand for two hours for better
adhesion of the inoculum. After this incubation,
the bags containing the cheese were stored under
refrigeration (8°C ± 1°C) and analyzed at
intervals of 0, 24, 48, 72, 96, 120, 144 and 168
hours, a total of eight treatments over a period of
time equivalent to seven days of storage
(adapted from Souza, 2006; Silva, 2007b;
Bíscola, 2007). The same procedure was used in
the control experiment, except that no REO was
used.
At the appointed times, samples of Coalho
cheese were ten-fold serially diluted (10-1 to 107
) in sterile peptone water 0.1%, followed by
plating on ChromoCult ® agar to determine the
total coliform and E. coli counts. The plates
were incubated at 35-37°C for 24 hours. This
experiment was performed in triplicate, and the
results were converted in to log CFU g-1 of
Coalho cheese (adapted from Souza, 2006;
Silva, 2007b; Bíscola, 2007; Barbosa, 2010).
Statistical analysis
The comparison between different treatments
was performed by Turkey's test adopting a 95%
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
confidence level. Statistical analysis was carried
out using statistical ASSISTAT software version
7.6 beta (2011), free software.
4
However, Angioni et al. (2004), Lopez et al.
(2005) and Hentz and Santin (2007) showed that
the inhibitory activity of rosemary essential oil is
weak or absent against gram-negative bacteria,
in contrast to the activity against gram-positive
bacteria and the activity of the other oils.
According to Lambert (2002), Bezic et al.
(2003) and Harris (2003), the structure of the
cell wall of gram-negative bacteria is primarily
made up of a lipopolysaccharide that blocks the
penetration of hydrophobic oils and prevents the
accumulation of essential oils in the membrane
of target cells, conferring a greater resistance to
these bacteria. In this study, the assessment of
antimicrobial activity and the determination of
the MIC demonstrated the susceptibility of the
EC16 strain of Escherichia coli, which is
resistant to several antibiotics, to the oil.
According to Burt (2004), given the large
number of different groups of compounds
present in essential oils, the antibacterial activity
of REO is most likely not attributable to a
specific mechanism but to several mechanisms
related to various targets in the cell.
RESULTS AND DISCUSSION
Antimicrobial Activity and Determination of
the MIC
The Escherichia coli EC16 test strain was
susceptible to the action of the pure rosemary
essential oil, which resulted in an inhibition zone
of 21 mm in diameter. There was not
antimicrobial activity of DMSO against strain,
characterizing its viability as solvent of the
REO.
The MIC of Rosmarinus officinalis L. essential
oil able to inhibit the growth of the EC16 strain
of Escherichia coli was 200 µL mL-1, equivalent
to an inhibition zone of 11 mm in diameter.
Several studies have demonstrated the
antimicrobial activity of rosemary essential oil
against different microorganisms (Santoyo et al.,
2005; Bozin et al., 2007; Gachkar et al., 2007;
Zago et al., 2009; Luqman et al., 2007).
Table 1: Average Escherichia coli EC16 counts in coalho cheese - controls and samples treated with
rosemary essential oil (REO).
Counts of EC16 X Time
Coalho
cheese
0h
24 h
48 h
72 h
96 h
120 h
144 h
168 h
Controls
3.0x105b 3.8x105a
5
3
9.1 x105a 5.1 x107a 6.1 x108a 3.2 x109a
3
3
3
3
1.7 x109a
3
4.3x10 a 2.6 x10 b 2.0 x10 b 2.1 x10 b 4.0 x10 b 6.8 x10 b 7.2 x10 b
Treatments
Values followed by the same letter in the same row were not significantly different (p≤0.05).
The antimicrobial activity and, consequently, the
minimum inhibitory concentration of essential oils
can be influenced by the growing region of the
plant, the extraction method used, the plant part
used (leaf or whole plant), the method of
preparation of the raw material (fresh or dry), the
type of organism, the cultivation conditions
(incubation time, temperature, oxygen), the culture
medium, the concentration of the test substance
and the solvents used to dilute the oil, among other
factors (Hammer, Larson & Riley, 1999;
Dellacassa et al., 1999; Burt, 2004; Duarte et al.,
2005b; Ríos & Recio, 2005; Bertini et al., 2005;
Nascimento et al., 2007; Celiktas et al., 2007).
Effect of rosemary essential oil in the control of
E. coli EC16 in Coalho cheese.
1.1 x109a
7.1 x103b
The results of the microbiological tests using
samples of Coalho cheese before inoculation with
the strain of E. coli demonstrated the absence
and/or very low counts of microorganisms. These
tests were conducted to control for the interference
of other bacteria in the sample, especially E. coli,
because the presence of this and other organisms
prior to inoculation with strain EC16 could
compromise the results of the experiment.
According to the results presented in Figure 1 and
Table 1, REO added to the Coalho cheese at the
minimum inhibitory concentration (200 µL/mL),
resulted in a reduction of 2.3 logarithmic cycles in
the bacterial population in the first 24 hours; this
value was practically stable during the whole
experiment, which lasted 168 hours. However, our
result contradicts those of Mendoza-Yepes et al.
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
(1997), Porte & Godoy (2001), Burt (2004) and
Kruger (2006), who reported that to achieve the
same antimicrobial activity in vitro, the
concentration of the essential oil added to food
should be considerably higher due to the
interference of food ingredients in the action of oil.
Figure 1: Antimicrobial activity of rosemary essential
oil against strain Escherichia coli EC16 in coalho
cheese over 168 hours (p≤0.05).
Over 168 hours (seven days), there was not a total
reduction in the microbial population in the sample
because of the oil concentration tested (MIC) and
because of interference by the food matrix. In this
particular case, there is a high content of lipids in
the product - in the amount total fat solids in
Coalho cheese ranges from 35.0% to 60.0%
(Brasil, 2001a). In a similar experiment carried out
by Kruger (2006) in which oregano essential oil
was used in the control of Listeria monocytogenes
in sausages, the microbial reduction was not stable
during storage of the product, and the pathogen
population recovered. The author suggests that
such recovery was possible due to the strong
interference that the food matrix has with the
antimicrobial activity of the oil.
Although not well understood, fat in food can form
a protective barrier around bacteria and/or the lipid
fraction can absorb the essential oil, partially or
totally inhibiting the action of this oil against
bacteria in the aqueous phase (Pandit & Shelef,
1994; Tassou et al., 1995; Lambert et al., 2001;
Mejlholm & Dalgaard, 2002; Silva, 2007b). This
behavior could be observed in the results presented
by Smith-Palmer et al. (2001), who tested essential
oils of clove and cinnamon against Salmonella
Enteritidis in cheese with low (16%) and high
5
(30%) fat contents; they observed the best action
for cheese with low fat contents for both oils.
Several studies have reported high levels of fecal
coliforms and confirmed the presence of the
indicator organism of this group, E. coli, in Coalho
cheese (Florentino & Martins, 1999; Mendes et al.,
1999; Leite Júnior et al., 2000; Leite et al., 2002;
Borges et al., 2003; Feitosa et al., 2003; Duarte et
al., 2005a; Oliveira et al., 2010). This fact
indicates that this bacterium, which is positive for
lactose fermentation, easily adapts to foods rich in
lactose. This adaptation was demonstrated by
analyzing the control sample without the addition
of REO (Figure 1), in which there was an increase
of 4 logarithmic cycles during 120 hours of
storage.
The use of essential oils can improve food safety,
especially with respect to microbiological
continuation. Rahman & Kang (2009) recommend
applying essential oils or their compounds as part
of a barrier system; i.e., they suggest using these
oils as an antimicrobial component along with
other preservation techniques. Porte & Godoy
(2001) also concluded that rosemary extracts,
combined with other agents, can help control
bacterial growth and prevent rancidity in foods.
However, according to Del Campo et al. (2000),
the use of essential oils as antimicrobial agents in
foods may be limited by the oil’s sensory
attributes. This research was not aimed at
evaluating the sensory characteristics of the
product; however, sensory analysis should be
included future studies, given that the use of high
concentrations of essential oil could interfere with
the acceptance of the product.
Using the minimum inhibitory concentration
determined in this study, the essential oil of the
leaves of R. officinalis was able to reduce the
population of the EC16 strain of E.coli by 2.3
logarithmic cycles and kept the product practically
stable during the 168 hours observation period.
This reduction was significant, and the bacterial
load approached the maximum acceptable value
for fecal coliform under the law RDC No. 12/2001
(Brasil, 2001b); this limit is 5x102 CFU/g for
Coalho cheese.
Essential oils are substances that are GRAS generally recognized as safe (Al-Reza, Rahman &
Kang, 2009), and E. coli bacteria can serve as an
indicator of the sanitary quality of food. It is
vitally important that new techniques of
conservation and preservation of food be
investigated to contribute to food security.
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
CONCLUSION
Based on the results obtained in this study, it can
be concluded that the REO has an inhibitory effect
against the EC16 strain of E. coli in vitro and in
the food matrix, such that this oil can be used to
control this microorganism. However, the use of
REO as a preservative in foods should also be
based on sensory analysis that evaluates the
acceptance of REO as an additive in food. The use
of essential oils should also be combined with
other preservation techniques.
REFERENCES
ANGIONI, A., BARRA, A., CERETI, E., BARILE, D.,
COISSON, J.D., ARLORIO, M., DESSI, S.,
CORONEO, V., CABRAS, P. Chemical composition,
plant genetic differences, antimicrobial and antifungal
activity investigation of the essential oil of Rosmarinus
officinalis L. Journal of Agricultural and Food
Chemistry, v. 52, n. 11, p. 3530 – 3535, 2004.
AL-REZA, S. M., RAHMAN, A., KANG, S.C.
Chemical composition and inhibitory effect of essential
oil and organic extracts of Cestrum nocturnum L. on
food-borne pathogens. International Journal of Food
Science and Technology, v. 44, n. 6, p. 1176–1182,
2009.
BARBOSA, L. N. Propriedade antimicrobiana de
óleos essenciais de plantas condimentares com
potencial de uso como conservante em carne e
hambúrguer bovino e testes de aceitação. – São
Paulo, Botucatu, Dissertação (mestrado) – Universidade
Estadual Paulista, Instituto de Biociências, Botucatu, p.
121, 2010.
BAUER, A.W.M.M., KIRBY, J.C., TURCK, M.
Antibiotic susceptibility testing by a standardized single
disk method. American Journal of Clinical Pathology,
v. 45, n. 4, p. 493-496, 1966.
BEZIC, N., SKOCIBUCIC, M., DUNKIC, V.,
RADONIC, A. Composition and antimicrobial activity
of Achillea clavennae L. essential oil. Phytotherapy
Researcher, v. 17, n. 9, p. 1037-1040, 2003.
6
nisina contra Listeria monocytogenes: avaliação em
modelos cárneos. São Paulo. Dissertação (mestrado) Faculdade de Ciências Farmacêuticas da Universidade
de São Paulo. Departamento de Alimentos e Nutrição
Experimental, p. 74, 2007.
BORGES, M.F., FEITOSA, T., NASSU, R.T., MUNIZ,
C.R., AZEVEDO, E.H.F., FIGUEIREDO, E.A.T.
Microrganismos patogênicos e indicadores em queijo
coalho produzido no Estado do Ceará, Brasil. Boletim
do Centro
de Pesquisa e Processamento de
Alimentos, v. 21, n. 1, p. 31-40, 2003.
BOZIN, B., MIMICA-DUKIC, N., SAMOJLIK. I.,
JOVIN, E. Antimicrobial and antioxidant properties of
rosemary and sage (Rosmarinus officinalis L. and
Salvia officinalis L., Lamiaceae) essential oils. Journal
of Agricultural and Food Chemistry, v. 55, n. 19, p.
7879–7885, 2007.
BRASIL. Ministério da Agricultura, Pecuária e
Abastecimento Secretaria de Defesa Agropecuária.
Regulamento técnico de identidade e qualidade de
queijo de coalho - Instrução Normativo Nº 30, de 26
de junho de 2001. Anexo II, 5-8, 2001a.
BRASIL. Ministério da Saúde. Resolução da Diretoria
Colegiada nº 12 de 02 de janeiro de 2001.
Regulamento
Técnico
sobre
Padrões
Microbiológicos para Alimentos. Diário Oficial [da]
República Federativa do Brasil, Brasília, 02 de janeiro
de 2001, seção I, 45-53, 2001b.
BURT, S. Essential oils: Their antibacterial properties
and potential applications in foods—a review.
International Journal of Food Microbiology, v. 94, n.
3, p. 223–253, 2004.
CELIKTAS, O.Y., KOCABAS, E.E.H., BEDIR, E.,
SUKAN, F.V., OZEK, T., BASER, K.H.C.
Antimicrobial activities of methanol extracts and
essential oils of Rosmarinus oficinalis, depending on
location and seasonal variations. Food Chemistry. v.
100, n. 2, p. 553-559, 2007.
DEL CAMPO, J., AMIOT, M.J., NGUYEN-THE, C.
Antimicrobial effect of rosemary extracts. Journal of
Food Protection, v. 63, n. 10, p. 1359-1368, 2000.
BERTINI, L. M., PEREIRA, A. F., OLIVEIRA, C. L.
L., MENEZES, E. A., MORAIS, S. M., CUNHA, F. A.,
CAVALCANTI, E. S. B. Perfil de sensibilidade de
bactérias frente a óleos essenciais de algumas plantas
do nordeste do Brasil. Infarma, v. 17, n. 3/4, p. 80-83,
2005.
DEL CAMPO, J., AMIOT, M.J., NGUYEN-THE, C.
Determination of most bioactive phenolic compounds
from rosemary against Listeria monocytogenes:
influence of concentration, pH and NaCl. Journal of
Food Science, v. 68, n. 6, p. 2066-2071, 2003.
BÍSCOLA, V. Influencia da matriz alimentar no
efeito antimicrobiano de óleo essencial de orégano e
DELLACASSA, E., LORENZO, D., MOYNA, P.,
FRIZZO, C.D., ATTI-SERAFINI, L., DUGO, P.
Rosmarinus officinalis L. (Labiatae) essential oils from
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
7
the South of Brazil and Uruguay. Journal of Essential
Oils Research, v. 11, n. 1, p. 27-30, 1999.
Microbiologia de alimentos. Porto Alegre: Artmed, cap.
22, p. 454-489, 2005.
DUARTE, D.M.S., SCHUSH, D.M.T., SANTOS, S.B.,
RIBEIRO, A.R., VASCONSELOS, A.M.M., SILVA,
J.V.D., MOTA, R.A. Pesquisa de Listeria
monocytogenes
e
microrganismos
indicadores
higiênico-sanitários em queijo de coalho produzido e
comercializado no estado de Pernambuco. Arquivos do
Instituto Biológico, v. 72, n. 3, p. 297-302, 2005a.
KRUGER, M.F. Controle de Listeria monocytogenes
em lingüiça frescal refrigerada através do uso de óleo
essencial de orégano e nisina. (Master’s thesis).
Retrieved
from
http://www.teses.usp.br/teses/disponiveis/9/9131/tde13092006-110306/ , 2006.
DUARTE,
M.C.T.,
FIGUEIRA,
G.M.,
SARTORATTO,
A.,
REHDER,
V.L.G,
DELARMELINA, C. Anti-candida activity of Brazilian
medicinal plants. Journal Ethnopharmacology. v. 97,
n. 2, p. 305-311, 2005b.
FEITOSA, T., BORGES, M.F., NASSU, R.T.,
AZEVEDO, E.H.F., MUNIZ, C. R. Pesquisa de
Salmonella sp., Listeria sp. e microrganismos
indicadores higiênicos-sanitários em queijos produzidos
no Estado do Rio Grande do Norte. Ciência e
Tecnologia de Alimentos, v. 23, p. 162-165, 2003.
FERNANDEZ-LOPEZ, J., ZHI, N., ALESONCARBONELL, L., PEREZ-ALVAREZ, J.A., KURI, V.
Antioxidant and antibacterial activities of natural
extracts: application in beef meatballs. Meat Science,
v. 69, n. 3, p. 371-380, 2004.
FLORENTINO, E.R., MARTINS, R.S. Características
microbiológicas do “queijo de coalho” produzido no
Estado da Paraíba. Revista Higiene Alimentar, v. 13,
n. 59, p. 43-48, 1999.
GACHKAR, L., YADEGARI, D., REZAEI, M.B.,
TAGHIZADEH, M., ASTANEH, S.A., RASOOLI, I.
Chemical and biological characteristics of Cuminum
cyminum and Rosmarinus officinalis essential oils.
Food Chemistry, v. 102, n. 3, p. 898-904, 2007.
HAMMER, K.A., CARSON, C.F., RILEY, T.V.
Antimicrobial activity of essential oils and other plant
extracts. Journal of Applied Microbiology, v. 86, n. 6,
p. 985–990, 1999.
HARRIS, R. Sinergism in the essential oil world. The
International Journal of Aromatherapy, v. 12, n. 4,
p. 179-186, 2003.
HENTZ, S. M., SANTIN, N.C. Avaliação da atividade
antimicrobiana do óleo essencial de alecrim
(Rosmarinus officinalis l.) contra Salmonella sp.
Evidência, Joaçaba, v. 7, n. 2, p. 93-100, 2007.
JAY, J.M. Introdução aos microrganismos
causadores de doenças de origem alimentar. In:
LAMBERT P.A. Cellular impermeability and uptake of
biocides and antibiotics in Gram positive bactéria and
mycobacteria. Journal of Applied Microbiology, v.
92, n. 1, p. 46S-54, 2002.
LAMBERT, R.J.W. SKANDAMIS, P.N., COOTE, P.,
NYCHAS, G.-J.E. A study of the minimum inhibitory
concentration and mode of action of oregano essential
oil, thymol and carvacrol. Journal of Applied
Microbiology, v. 91, n. 3, p. 453–462, 2001.
LEITE JÚNIOR, A.F.S., FLORENTINO, E.R.,
OLIVEIRA, E.B., TORRANO, A.D.M. Qualidade
microbiológica do queijo de coalho comercializado à
temperatura ambiente ou sob refrigeração, em Campina
Grande-PB. Revista Higiene Alimentar, v. 14, n. 73,
p. 53-59, 2000.
LEITE, C.C., GUIMARÃES, A.G., RIBEIRO, N.S.,
SILVA, M.D., ASSIS, P.N. Pesquisa de Listeria
monocytogenes e Escherichia coli em queijo do tipo
“coalho” comercializado em Salvador (BA).
Importância para saúde pública. Revista Analytica, v.
2, p. 8-41, 2000.
LEUSCHNER, R. G. K., ZAMPARINI, J. Effects of
spices on growth and survival of Escherichia coli 0157
and Salmonella enterica serovar enteridis in broth
model systems and mayonnaise. Food Control, Oxford,
v. 13, n. 6-7, p. 399-404, 2002.
LOPEZ, P., SANCHEZ, C., BATLE, R., NERIN, C.
Solid and vapor phase antimicrobial activities of six
essential oils: susceptibility of selected foodborne
bacterial and fungal strains. Journal of Agricultural
and Food Chemistry, v. 53, n.7, p. 6939–6946, 2005.
LUQMAN, S., DWIVEDI, G.R., DAROKAR, M.P.,
KALRA, A., KHANUJA, S.P. Potential of rosemary oil
to be used in drug-resistant infections. Alternative
Therapies in Health and Medicine, v. 13, n. 5, p. 5459, 2007.
MEJLHOLM, O., DALGAARD, P. Antimicrobial
effect of essential oils on the seafood spoilage microorganism Photobacterium phosphoreum in liquid media
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
8
and fish products. Letters in Applied Microbiology, v.
34, n. 1, p. 27– 31, 2002.
ethanol extracts of Lonicera japonica.
Chemistry. v. 116, n. 3, p. 670–675, 2009.
MENDES, E.S., LIMA, E.C., NUMERIANI, A.K.M.,
COELLHO, M.I.S. Staphylococcus aureus, Salmonella
sp. e coliformes em queijo de “coalho” comercializado
em Recife. Revista Higiene Alimentar, v. 13, n. 66/67,
p. 122-126, 1999.
RÍOS, J.L., RECIO, M.C. Medicinal plants and
antimicrobial activity. Journal Ethnopharmacology.
v. 100, n. 1/2, p. 80-84, 2005.
MENDOZA-YEPES, M.J., SANCHEZ-HIDALGO,
L.E., MAERTENS, G. MARIN-INIESTA, F. Inhibition
of Listeria monocytogenes and other bacteria by a plant
essential oil (DMC) en Spanish soft cheese. Journal of
Food Safety, v. 17, n. 1, p. 47–55, 1997.
MORENO, S., SCHEYER, T., ROMANO, C.S.,
VOJNOV, A.A. Antioxidant and antimicrobial
activities of rosemary extracts linked to their
polyphenol composition. Free Radical Research, v.
40, n. 2, p. 223-231, 2006.
NASCIMENTO P.F.C., NASCIMENTO, A.C.,
RODRIGUES, C.S., ANTONIOLLI A.A., SANTOS,
P.O., BARBOSA JUNIOR, A.M., TRINDADE R.C.
Atividade antimicrobiana dos óleos essenciais: uma
abordagem multifatorial dos métodos. Revista
Brasileira de Farmacognosia, v. 17, n. 1, p. 108-113,
2007.
NASCIMENTO, M.G.F., NASCIMENTO, R.E.,
CUNHA, C.P., CORBIA, G.C.A. Estudo transversal
sobre alguns fatores de risco na contaminação natural
de coliformes fecais em queijo minas frescal. Revista
Higiene Alimentar, v. 15, n. 86, p. 55-59, 2001.
OLIVEIRA, K.A., EVÊNCIO NETO, J., PAIVA, J.E.,
MELO, L.E.H. Qualidade microbiológica do queijo de
coalho comercializado no município do Cabo de Santo
Agostinho, Pernambuco, Brasil. Arquivos do Instituto
Biológico, v. 77, n. 3, p. 435-440, 2010.
OLUWATUYI, M., KAATZ, G. W., GIBBONS, S.
Antibacterial and resistance modifying activity of
Rosmarinus officinalis. Phytochemistry, v. 65, n. 24,
p. 3249-3254, 2004.
PANDIT, V.A., SHELEF, L.A. Sensitivity of Listeria
monocytogenes to rosemary (Rosmarinus officinalis L.).
Food Microbiology. v. 11, n. 1, p. 57– 63, 1994.
PORTE, A., GODOY, R. L. O. Alecrim (Rosmarinus
officinalis L.): Propriedades antimicrobianas e químicas
do óleo essencial. Boletim Centro de Pesquisa de
Processamento de Alimentos, v. 19, n. 2, p. 193-210,
2001.
Food
SANTOYO, S.; CAVERO, S.; JAIME, L.; IBANEZ,
E.; SENORANS, F. J.; REGLERO, G. Chemical
composition and antimicrobial activity of Rosmarinus
officinalis L. essential oil obtained via supercritical
fluid extraction. Journal of Food Protection, v. 68, n.
4, p. 790–795, 2005.
SILVA, N., JUNQUEIRA, V. C. A., SILVEIRA, N. F.
A., TANIWAKI, M.H., SANTOS, R.F.S., GOMES,
R.A.R.
Manual
de
Métodos
de Análise
Microbiológica de Alimentos. 3ª edição. São Paulo:
Livraria Varela, p. 552, 2007a.
SILVA, J.P.L. Avaliação da ação de antimicrobianos
naturais no controle de Salmonella Enteretides em
salada de legumes com maionese. (Doctoral
Dissertation).
Retrieved
from
http://www.teses.usp.br/teses/disponiveis/9/9131/tde15022009-210130/, 2007b.
SOUZA, S. M. C., PEREIRA, M.C., ANGÉLICO,
C.L., PIMENTA, C.J. Avaliação de óleos essenciais de
condimentos sobre o desenvolvimento micelial de
fungos associados a produtos de panificação. Ciência e
Agrotecnologia, v. 28, n. 3, p. 685-690, 2004.
SOUZA, E. L., STAMFORD, T. L.M., LIMA, E.O.,
BARBOSA FILHO, J.M., MARQUES, M.O.M.
Interference of heating on the antimicrobial activity and
chemical composition of Origanum vulgare L.
(Lamiaceae) essential oil. Ciência e Tecnologia de
Alimentos, v. 28, n. 2, p. 418-422, 2008.
SOUZA, E.L., LIMA, E.O., FREIRE, K.R.L., SOUSA,
C.P. Inhibitory action of some essential oils and
phytochemicals on the growth of moulds isolated from
foods. Brazilian Archivies of Biology and
Technology, v. 48, n. 2, p. 245-250, 2005.
SMITH-PALMER, A., STEWART, J., FYFE, L. The
potential application of plant essential oils as natural
food preservatives in soft cheese. Food Microbiology.
v. 18, n. 4, p. 463– 470, 2001.
TASSOU, C., DROSINOS, E.H., NYCHAS, G.-J.E.
Effects of essential oil from mint (Mentha piperita) on
Salmonella enteritidis and Listeria monocytogenes in
model food systems at 4ºC and 10ºC. Journal of
Applied Bacteriology. v. 78, n. 6, p. 593– 600, 1995.
RAHMAN, T. A., KANG, S.C. In vitro control of foodborne and food spoilage bacteria by essential oil and
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013
Ribeiro, D. S. et al.
9
ZAGO, J.A.A., USHIMARU, P.I., BARBOSA, L.N.,
FERNANDES JUNIOR, A. Synergism between
essential oils and antimicrobial drugs against
Staphylooccus aureus and Escherichia coli strains from
human
infections.
Brazilian
Journal
of
Pharmacognosy, v. 19, n. 4, p. 828-33, 2009.
Recebido: 08/11/2012
Received: 11/08/2012
Aprovado: 31/01/2013
Approved: 01/31/2013
J. Biotec. Biodivers. v. 4, N.1: pp. 1-9, Feb. 2013