CADERNO DE CIÊNCIAS AGRÁRIAS
Agrarian Sciences Journal
Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential
oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses
Larissa de Oliveira
1
*, Sabrina de Souza Sales
2
Abstract
Syzygium aromaticum L. and Ocimum gratissimum L. essential oils were tested for their effectiveness in reduction the
counts of inoculated Staphylococcus aureus and Escherichia coli in sheepmeat. The inhibitory effect was analysed by
disk diffusion and broth macrodilution method with four strains and seven concentrations. Disinfectant activity of the
oils was assessed using the suspension test with two strains and two concentrations. The inhibition was observed at
concentrations 400, 200 and 100 µL/mL. Syzygium aromaticum L. oil was better than Ocimum gratissimum L. against
bacteria isolated from sheep carcasses. The minimum bactericidal concentration of the essential oils aginst inocula
ranged from 50 to 200 µL/mL. The suspension test showed that after 5 minutes of contact, Syzygium aromaticum L.
disinfectant at 400 µL/mL deactivated the inoculants completely. The results demonstrated that essential oils exerted
a significant bactericidal and bacteriostatic action against pathogens.
Keywords: “Alfavacão”. Antiseptic. Clove. Food safety. Microorganisms.
Efeito inibitório e atividade antisséptica de óleos essenciais de Syzygium aromaticum L. e Ocimum
gratissimum L contra Escherichia coli e Staphylococcus aureus isolados de carcaça de ovinos
Resumo
Verificou-se a efetividade dos óleos essenciais de Syzygium aromaticum L. e Ocimum gratissimum L. em reduzir a
contagem de Staphylococcus aureus e Escherichia coli inoculadas em carne de ovino. Foram utilizadas quatro cepas e
sete concentrações dos óleos nas técnicas de disco-difusão e macrodiluição em caldo empregadas na análise do efeito
inibitório. No teste de suspensão, trabalhou-se com duas cepas e duas concentrações na determinação da atividade
desinfetante. A inibição foi observada nas concentrações de 400, 200 e 100 µL/mL. O óleo de Syzygium aromaticum L.
foi melhor em relação ao alfavacão para inativar as bactérias isoladas das carcaças de ovinos. A concentração bactericida
mínima dos óleos contra os inóculos variou de 50 a 200 µL/mL. O teste de suspensão mostrou que após 5 minutos de
contato com o desinfetante de cravo na concentração de 400 µL/mL os inóculos foram completamente inativados. Os
resultados mostraram significativa atividade bactericida e bacteriostática dos óleos essenciais frente aos patógenos.
Palavras-chave: Alfavacão. Antisséptico. Cravo da índia. Segurança alimentar. Microrganismos.
1
Faculdade Senac Minas, unidade Belo Horizonte. Belo Horizonte, MG. Brasil.
https://orcid.org/0000-0001-6426-1945
2
Instituto Português do Mar e da Atmosfera (IPMA). Departamento do Mar e Recursos Marinhos, Instituto Português do Mar e da Atmosfera. Lisboa.
Portugal.
https://orcid.org/0000-0003-4822-1518
*Autora para correspondência: larisoliveira@hotmail.com
Recebido para publicação em: 01 de novembro de 2019. Aceito para publicação em 28 de novembro de 2019.
e-ISSN: 2447-6218 / ISSN: 2447-6218 / © 2009, Universidade Federal de Minas Gerais, Todos os direitos reservados.
Oliveira, L.; Sales, S. S.
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Cad. Ciênc. Agrá., v. 11, p. 01–12, 2019. e-ISSN: 2447-6218 / ISSN: 1984-6738
Introduction
Foods are excellent vehicles for the dissemina-
tion of pathogens such as bacteria, viruses and parasites
(Newell et al., 2010). Many of them, such as E. coli and
S. aureus cause diseases.
In the last twenty years, different cases of diar-
rhea have been attributed to the consumption of food
containing toxin producing E. coli (Hussein and Bollinger,
2005; Newell et al., 2010), Shigella sp. or Salmonella sp.
(Borowsky et al., 2006; Brasil, 2005). Staphylococci are
often related to outbreaks of foodborne illness due to the
ability of some strains to produce enterotoxins (Viçosa
et al., 2010).
Many pathogenic organisms are resistant to tra-
ditionally used antibiotics due to the indiscriminate and
inappropriate use of these drugs (Sartoratto et al., 2004).
Moreover, there are products on the market with moderate
or even poor antiseptic effect (Pitten et al., 2003), the
need of finding new substances with antimicrobial action
becoming of utmost importance (Duarte et al., 2007).
Foodstuffs are subject to microbial spoilage during
their useful life. In the Brazilian semiarid, meat sheep
of the small producers are slaughtered and sold, often
without proper hygienic conditions, affecting the sensory
and microbiological quality of the meat (Sá et al., 2007).
Sheep meat, lamb or mutton, is tender and juicy, with a
characteristic aroma has considerable economic impor-
tance (Costa et al., 2008) and the consumer market for
this product is constantly growing (Barros et al., 2003).
Lately, there has been increasing demand for safe
products (Rajkovic et al., 2010; Todd, 2004), natural and
free from chemical preservatives, leading the industry to
investigate elements capable of maintaining and impro-
ving the sensory characteristics while maintaining the
nutritional properties of the food (Goñi et al., 2009).
Studies have confirmed the bactericidal and/or
bacteriostatic action of spices, seasonings and extracts or
essential oils. Essential oils, plant secondary metabolites
isolates, have won over the market and consumer tastes,
owing to their health benefits and lower environmen-
tal impact (Pereira et al., 2008). Among the plants and
condiments currently researched and used are clove (S.
aromaticum), an oriental spice used in cooking (Brune-
ton, 1995) and the Alfavacão” (O. gratissimum), or wild
basil, an aromatic plant known for its medicinal aspects
(Di Stasi et al., 2002) and use in cooking (Pereira and
Maia, 2007).
The objective here in was to evaluate the inhibi-
tory effect, the minimum bactericidal concentration and
disinfectant activity of essential oils of S. aromaticum
and O. gratissimum on the bacteria S. aureus and E. coli,
isolated from sheep carcasses.
Material and methods
Sample collection
Samples were collected from 36 carcasses, ga-
thered during three visits to a butcher in Montes Claros
- MG. The slaughter happened informally, at a different
location from the butcher shop, according to the owner.
We used a 5-point surface smear technique (neck,
shoulder, ribs, loin and leg), and the swabs were trans-
ported in of 50 mL bottles with 0.1% peptone water
(Himedia
®
) (Midura and Bryant, 2001). The volumes of
bottles with the swabs (solution 10
-1
) were homogenized
in 0.1% peptone water and then underwent dilution to
obtain the other two solutions (10
-2
and 10
-3
) (Swanson
et al., 2001).
Upon collection, the samples were taken in a
cooled container to the laboratory in the same city, and
cultivation being conducted on the same day.
Research on thermotolerant fecal coliforms, E. coli
and S. aureus
These strains were identified by microbiological
analyses conducted previously (QUINN et al., 2005) and
were stored in glycerol at -18°C.
Plant material and essential oil extraction
The extraction of essential oils was conducted by
hydrodistillation in a Clevenger apparatus. There were
five extractions with 188 g each of fresh of O. gratissimum,
leaves, collected in the of Medicinal Plant Garden of the
Institute of Agricultural Sciences, Federal University of
Minas Gerais, and two extractions, each using 118 g of
S. aromaticum, inflorescences, acquired in the commerce
of Montes Claros. The samples were placed separately
into 1 liter flasks and kept boiling for 3 hours, at constant
temperature. The content was expressed in volume of
oil per dry matter of the material used in the extraction
(Souza et al., 2010).
The Ocimum gratissimum specimen collected was
identified by Professor Alexandre Salino and a voucher
specimen deposited in the BHCB Herbarium at the Fe-
deral University of Minas Gerais, under voucher number
150123.
Chromatographic analysis of essential oils
The qualitative analyses to identify the essential
oil constituents were carried out at the Natural Products
Laboratory - Centro de P&D Recursos Genéticos Vegetais,
at the Agronomic Institute of Campinas in Campinas - SP.
Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses
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Separation and quantification (area normaliza-
tion method) of the substances was performed by gas
chromatography with a flame ionization detector (GC-
-FID, Shimadzu GC-2010) under the following analytical
conditions: DB-5 capillary column (J & W Scientific; 0.25
mm x 30.0 x 0.25 mM); Injector and detector temperatu-
res maintained respectively at 220°C and 230°C; helium
carrier gas at a flow rate of 1.0 mL/min); split: 1/20;
Injection volume: 1 µL of essential oil solution (1 µL of
essential oil/1 mL of ethyl acetate); oven temperature
program: Initial temperature 60°C up to 240°C at 3°C/
min.
The identification of compounds was performed
on a gas chromatograph coupled to mass spectrometer
(GC-MS, Shimadzu QP-5000) operating at an electron
impact (70 eV) under the same chromatographic condi-
tions described above, using capillary column: OV -5 (Ohio
Valley Specialty Chemical, Inc.; 30.0 x 0.25 mm x 0.25
um.). The identification of the substances was performed
by comparing their mass spectra with the database of
the GC-MS system (Nist. 62 lib.) and the retention ratio
(Adams, 2007). To obtain the retention index (RI), we
used a standard hydrocarbon mixture (C9-C24) injected
under the same operating conditions as the samples, by
applying the Van den Dool; Kratz equation (1963).
Evaluation of inhibitory activity and bactericidal oils
For in vitro evaluation of the antimicrobial activity
of S. aromaticum and O. gratissimum oils against strains
of S. aureus and E. coli isolated from sheep carcasses and
S. aureus ATCC 25923 and E. coli ATCC 8733, we used
the broth macrodilution and diffusion methodologies in
Clinical and Laboratory Standards Institute plates - CLSI
(2018a).
In plate diffusion, one aliquot of each strain was
placed in BHI broth (Himedia®) and taken for activation
to an oven at 37°C for 24 hours. Activated cultures were
transplanted on TSA agar (Triptic Soy Agar Himedia®)
and incubated under the same conditions. The inoculum
was prepared by placing five isolated colonies from the
plates in 5 mL of saline solution. to the Turbidity was
adjusted to the 0.5 McFarland standard solution equiva-
lent (CLSI, 2018b).
With the aid of a sterile swab 200 µL of the
inoculum was seeded on the surface of Petri dishes con-
taining Mueller-Hinton agar (Himedia®). Whatman
filter paper discs number one, 6 mm in diameter and
impregnated with 10 µL of each test concentration were
uniformly distributed across the plate with the aid of a
sterile tweezer.
For the first concentration we used, 400 µL of
essential oil, 27 µL of Tween 80 and the volume was
completed to 1 mL with sterile distilled water according
to the methodology proposed by Oliveira et al. (2006).
The final volume was vortexed for 5 minutes. The con-
centrations evaluated were 400; 200; 100; 50; 25; 12.5
and 6.25 µL/mL, obtained by the serial dilution method
at a ratio of 2 (CLSI, 2018a). The paper disc impregnated
with Tween 80 represents the negative control, while
commercial antibiotic disks served as positive control,
using 10 µg Gentamycin and 1 µg Oxacillin on the plates
with Staphylococcus sp. and 5 µg Ciprofloxacin and 1 µg
Oxacillin the plates with E. coli, all DME® brand. The
plates were placed in an oven for 24 hours at 37°C. After
the incubation period, the microbial growth inhibition
zone diameter was measured with a ruler and recorded
in millimeters.
The experiment was conducted according to a
randomized block design. The factorial design was 4 x
2 x 7, four bacteria (S. aureus and E. coli isolated from
sheep carcass and S. aureus ATCC 25923 and E. coli
ATCC 8733), two oil-seven concentrations, totaling 56
treatments with two replications. Data were subjected
to analysis of variance, the SAEG 9.1 program, and the
averages compared by Scott-Knott test at 5% significance
level.
In the minimum bactericidal concentration test,
was employed the broth macrodilution technique descri-
bed by Chanwitheesuk et al. (2007) with modifications.
The seven concentrations used in the diffusion test were
tested, using the same principles as in serial dilution, but
replacing the distilled water by BHI broth (Himedia®),
preserving the volumes. At the end, we added 2.5 µL of
each inoculum obtained from isolates of the sheep meat
adjusted in a manner equivalent to the McFarland 0.5
standard solution and maintained the tubes incubated
for 24 hours at 37°C. After incubation, an aliquot of each
tube was plated on agar plates (TSA Triptic Soy Agar
Himedia®) which were taken to an oven under the same
conditions. The absence of bacterial growth was defined
as the MBC. The test was conducted in duplicate.
Verification of oil efficiency disinfectant
In determining the disinfectant activity, the metho-
dology described by Medeiros et al. (2009) was employed
with suspension test techniques defined by the Minis-
try of Agriculture, Livestock and Supply (Brasil, 1993).
Concentrations of 400 and 200 µL/mL were tested, the
most effective in diffusion test plates and the minimum
bactericidal concentration of each essential oil against
S. aureus ATCC 25923 and E. coli ATCC 8733. Bacterial
suspensions were prepared in 0.85% saline, equivalent
to a 0.5 McFarland standard solution with approximately
1.5x10
8
CFU/mL.
A 900 µL volume of disinfectants - used in item 2.5,
prepared with the essential oil, Tween 80 and distilled
water - was distributed aseptically into tubes and added
to 100 µL of sterile organic matter, being 10 g of sheep
meat, homogenized with 100 ml of distilled water and tyn-
dallized once for 30 minutes. Subsequently, 10 µL of the
bacterial suspension was added and exposure durations
Oliveira, L.; Sales, S. S.
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timed. After 5, 15, 30 and 60 minutes, via bacteriologi-
cal loop, an aliquot was transferred to tubes containing
5 mL Brain Heart Infusion broth (Himedia®), shaken
and incubated at 37°C for 96 hours. Also at 5 minutes,
aliquots were placed on Baird-Parker agar (Himedia®)
and CP (Oxoid®) for counting the number of colonies
after this of contact period between disinfectant solution,
the organic substance and inoculum. After inoculation,
the plates went into an oven at 37°C for 24 hours.
At 24, 48, 72 and 96 hours, presence of turbidity of
tube contents, surface film formation and/or precipitate
on the bottom of the glassware was examined. Turbidity,
or not, was considered, respectively as active bacteria
(negative) or inactive (positive). The tubes classified as
positive were transferred to solid media for specific for
S. aureus, (Baird-Parker Himedia®) and E. coli (EMB,
Oxoid®), incubated at 37°C for 24 hours, to confirm
the effectiveness of the disinfectant by the absence of
bacterial growth on the plates.
The test with each concentration was performed
in duplicate and we used descriptive statistical analysis.
Result and discussion
Yield and chemical characterization of essential oils
The essential oil yields, 2.77% for Ocimum gra-
tissimum and 5.04% for S. aromaticum, was higher than
that found in the literature. Pereira (2006) obtained a
yield of 2.32% from S. aromaticum inflorescences, and
the literature reports a range from 0.29% to 1.12% in
the yield of O. gratissimum (Craveiro et al., 1981; Faria
et al., 2006; Ngassoum et al., 2003). According to Ci-
manga et al. (2002), these variations can be attributed
to factors such as climate, plant age, harvest time and the
extraction method. Silva et al. (1999) demonstrated the
sunlight influence on eugenol production. The content
of this aromatic substance ranged from 98.0% in the oil
extracted from the leaves of O. gratissimum harvested at
12:00 p.m., to 11.4% in the collection at 5 p.m.
According to the literature, the essential oil of O.
gratissimum has two chemical types: thymol and eugenol
(Silva et al., 1999; Silva et al., 2010). Freire et al. (2006)
added geraniol to the list. According to the chromato-
graphic analysis, the essential oil of this present study is
the eugenol type, with 61.28% of the substance (Figure
1).
Figure 1 – Chromatogram of total ions of O. gratissimum essential oil
1- tricyclene (1.07%), 2- α-pinene (0.30%), 3- sabinene (0.21%), 4- β-pinene (0.17%), 5- myrcene (1 21%), 6- α-phellandrene (0.18%), 7- α-ter-
pinene (1.02%), 8- p-cymene (5.23%), 9- limonene (0.33%) 10- cis-β-ocimene (1.14%), 11- trans-β-ocimene (0.17%), 12- γ-terpinene (8.88%),
13- unidentified compound (0.19% ), 14- m-cymene (0.45%), 15- linalool (0.58%), 16- unidentified substance (0.30%), 17- thymol (8.07%), 18-
o-methoxyacetophenone (0.22%), 19- carvacrol (0.27%), 20- eugenol (61.28%), 21- trans-caryophyllene (1.60%), 22- α-humulene (0.20% ), 23-
β-farnesene (0.15%), 24- germacrene D (0.25%), 25- β-selinene (0.38%), 26- α-selinene (0.19%), 27- β-bisabolene (4.98%), 28- eugenyl acetate
(0.60%), 29- unidentified substance (0.40%).
Eugenol is the main bacterial agent present in the
oil extracted from Ocimum gratissimum leaves, but the
presence of thymol (8.07%), a recognized antimicrobial
(Burt, 2004) was also found as one of the components.
Cimanga et al. (2002) speculate on the involvement of
less abundant constituents, such as thymol, carvacrol,
Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses
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Cad. Ciênc. Agrá., v. 11, p. 01–12, 2019. e-ISSN: 2447-6218 / ISSN: 1984-6738
linalool and γ-terpinene, in the biological activity of the
essential oils. For Maciel et al. (2002), numerous plant
constituents or classes of compounds with different struc-
tures, contribute to the same activity. This responsibility
does not lie only among the substances present in higher
proportions, the synergistic effect among the components
also being suggested. Bakkali et al. (2008) corroborate
this statement. For them, it is more significant to study
the oils as a whole than just some of their components,
because it is the complex mixtures whose biological effects
can be the result of key molecules present or the synergy
among all the molecules identified by chromatographic
analysis.
The phenolic compound, eugenol, is also the
principal component of the essence of Syzygium aroma-
ticum (Figure 2). Studied as a food preservative (Pereira,
2006), it is found in Syzygium aromaticum and other
plants. Attributed to this substance are antifungal (Faria et
al., 2006), anti-mycotoxigenic (Dambolena et al., 2010),
antibacterial (Santurio et al., 2007) and antioxidant ac-
tivity (Pereira and Maia, 2007). Wenqiang et al. (2007)
and Oliveira et al. (2009) also found eugenol as the most
abundant principle component in S. aromaticum oil.
Figure 2 – Chromatogram of total ions of S. aromaticum essential oil
1- unidentified substance (0.29%), 2- eugenol (88.93%), 3- trans-caryophyllene (3.23%), 4- α-humulene (0.49%), ethyl 5- eugenyl (6.80%), 6-
caryophyllene oxide (0.26%).
In vitro antimicrobial activity of essential oils on E.
coli and S. aureus
In the diffusion plate tests, strains react differently
to oils, independent of both having eugenol as the prin-
ciple component. Halo diameter averages are shown in
Table 1. Inhibition zones were observed in only three of
the seven concentrations (400, 200, and 100 µL/mL),
which lowered the average of the results, but did not
interfere with their interpretation. The inhibition zone
is the area with no detectable growth of microorganisms
with the naked eye (CLSI, 2018b) and is directly related
to the sensitivity of the bacterial sample (Brasil, 2008).
The means 4.00 mm and 5.14 mm show the best
S. aromaticum oil action, compared to the Ocimum gratis-
simum (2.79 and 3.43 mm), against the bacteria isolated
from sheep meat. The same was not observed with the
standard ATCC microorganisms that statistically res-
ponded analogously to both oil activities. The results
reported by Oussalah et al. (2007) and Pereira (2006)
confirm these data, because the former tested 28 plants
against E. coli O157: H7, S. Typhimurium, S. aureus and L.
monocytogenes and seven of them, S. aromaticum among
them, showed strong antimicrobial activity against the
four bacteria, and the latter, also assigned the best E. coli
and S. aureus inhibition results S. aromaticum.
Oliveira, L.; Sales, S. S.
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Table 1 – Inhibition halo diameters (mm) of the essential oils of S. aromaticum and O. gratissimum against S. aureus
and E. coli
Oil
S. aureus
from sheep
carcass
S. aureus
ATCC 25923
E. coli
from sheep
carcass
E. coli
ATCC 8733
Halo diameter (mm)
O. gratissimum
2.57 Bb 2.36 Ab 3.71 Ba 4.79 Aa
S. aromaticum
4.00 Ab 2.79 Ab 5.14 Aa 3.43 Ab
Means followed by the same letter do not differ by the Scott-Knott test at 5% significance level. Uppercase letters compare the means in columns
and lowercase, on the lines.
The essential oil of Ocimum gratissimum was more
effective against Gram-negative bacteria, with average
diameters of 3.71 and 4.79 mm. In the work of Kotzekidou
et al. (2008), among the tested species, Gram-negative
were the most sensitive to oils and plant extracts and
Gram-positive, more resistant, confirming the results.
In general, gram-negative bacteria such as E. coli and
Salmonella sp., are less sensitive to the action of essential
oils. This is probably due to the difficulty of the compou-
nds to act on the complex cell wall structure of these
microorganisms (Cimanga et al., 2002; Holley and Patel,
2005). Burt (2004) cites other studies that strengthen
this proposition. The cell wall of Gram-positive bacteria
is typically permeable and does not restrict the passa-
ge of antimicrobial agents. The outer membrane of the
Gram-negative favors resistance and therefore does not
allow, or at least complicates the introduction of toxic
molecules into the cytoplasm or other destination (Denyer
and Maillard 2002; Lambert, 2002).
The diffusion plate method (disk diffusion and
agar diffusion) is simple, reliable (Brasil, 2008) and
one of the most used in verification testing of essential
oil antimicrobial activity (Tajkarimi et al., 2010), as it
ascertains, quickly, the susceptibility of microorganisms
to many substances (Ponce et al., 2003). In addition to
the structural diversity of the cell, the metabolic diffe-
rences between microorganisms should be considered
(Holley and Patel, 2005), since the antimicrobial action
of the constituents vary considerably according to species
lineage (Silva et al., 2009).
The biological effect of phenolic compounds such
as eugenol and essences in general, is easily observed in
the experiments, but the action mechanism has not been
fully elucidated. Normally, it is considered as the start of
the cell wall degradation mechanism, causing damage
to protein and cytoplasmic membrane, interrupting the
proton motive force, the flow of electrons and active
transport, favoring coagulation of the cytoplasm (Kotze-
kidou et al., 2008; Silva et al., 2010). According to the
results, 400µL/mL was the most effective concentration
for the strains two, three and four, with halos of 10.25,
12.25 and 13.75 mm (Table 2), respectively, being more
efficient for S. aromaticum oil, with a 13.38 mm inhibition
zone (Table 3). Strain one reacted similarly to 400 and
200 µL/mL. The research of Duarte et al. (2007) diffe-
red from this present work. When testing the activity of
essential oils extracted from Brazilian medicinal plants
against different E. coli serotypes they observed Ocimum
gratissimum inhibitory activity at concentrations from
600 to > 1000 µL/mL, or at least 50% higher. Pereira
(2006), with the essence of S. aromaticum tested on S.
aureus and E. coli, observed inhibition zone formation
at virtually all concentrations, but the most effective
for S. aureus was 50%, higher than that of the present
work, and 10% for E. coli, a more satisfactory result. In
analogy, it appears that 400 µL/mL represents 40% oil
in solution.
The lower the inhibitory concentration, the
better for use in foods because these compounds have
outstanding flavor and aroma and can alter the organo-
leptic characteristics of foodstuffs (Nedorostova et al.,
2009; Tajkarimi et al., 2010). The use of essential oils
to promote food safety is a viable technique (Goñi et al.,
2009), but having determined the concentration capable
of preventing microbial growth, any change can modify
the nature of the inhibitory effect (Holley and Patel,
2005), compromising the microbiological quality of the
product.
The concentration used is also important in the
verification of the inhibitory activity of essential oils
(Ponce et al., 2003), because together there can be a
cytotoxic effect on the living cell (Bakkali et al., 2008).
Hardly only one natural antimicrobial, however potent,
could be used effectively in a single concentration in all
kinds of food and against all microorganisms (Holley and
Patel, 2005; Yossa et al., 2010).
The biological activity of essential oils of E. coli
and S. aureus is represented by regression equations re-
presented in Figure 3. The results were discussed in the
text and confirm the action and the power of the tested
antimicrobials. The straight lines show the halo diameter
behavior in relation to the interaction of the bacteria
with the oils and concentrations tested. The inhibitory
Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses
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effect, that is, the antibiotic sensitivity represented by the
inhibition zone increased as the concentration increased,
but varied according to the oil applied and test-microor-
ganisms used. Concentrations below 100 µL/mL showed
no biological activity.
Table 2 – Relationship between O. gratissimum and S. aromaticum essential oil concentrations and Gram-positive and
negative bacteria
Bacteria
Concentration (µL/mL)
400 200 100 50 25 12,5 6,5
Halo diameter (mm)
1 11.50 Aa 9.25 Aa 2.25 Bb 0 Ab 0 Ab 0 Ab 0 Ab
2 10.25 Aa 6.00 Bb 1.75 Bc 0 Ac 0 Ac 0 Ac 0 Ac
3 12.25 Aa 10.25 Ab 8.50 Ab 0 Ac 0 Ac 0 Ac 0 Ac
4 13.75 Aa 11.00 Ab 4.00 Bc 0 Ad 0 Ad 0 Ad 0 Ad
Means followed by the same letter do not differ by the Scott-Knott test at 5% significance level. Uppercase letters compare the means in columns
and lowercase on the lines.
1- S. aureus from sheep carcasses; 2- S. aureus ATCC 25923; 3- E. coli from sheep carcasses; 4- E. coli ATCC 8733.
Table 3 – Relationship between O. gratissimum and S. aromaticum essential oil concentrations
Oil
Concentration (µL/mL)
400 200 100 50 25 12,5 6,5
Halo diameter (mm)
O. gratissimum
10.50 Ba 8.25 Ab 4.75 Ac 0 Ad 0 Ad 0 Ad 0 Ad
S. aromaticum
13.38 Aa 10.00 Ab 3.50 Ac 0 Ad 0 Ad 0 Ad 0 Ad
Means followed by the same letter do not differ by the Scott-Knott test at 5% significance level. Uppercase letters compare the averages in columns
and lowercase on the lines.
The results obtained by Nakamura et al. (1999) are
consistent with those of this study, since the bactericidal
concentration of the Ocimum gratissimum essence for S.
aureus was lower, 1.5 µg/mL, compared to that obtained
for E. coli, 12 µg/mL. In contrast, Silva et al. (2010) found
antagonistic results, i.e., lower MBC for Gram-negative
bacteria and higher for Gram-positive; however, the
researchers used the oil from the inflorescences and not
the leaves.
As with the Ocimum gratissimum, the essence of
Syzygium aromaticum presented the lowest minimum
bactericidal concentration against S. aureus, with 50 µL/
mL. In research of Hoffmann et al. (1999), S. aromaticum
oil showed to be a good antimicrobial as it completely
inhibited bacteria such as S. aureus and S. enteritidis, in
addition to yeast, at 10%. Moreira et al. (2005) in their
work, observed the effectiveness of S. aromaticum against
strains of E. coli O157: H7 only at concentrations 30 ti-
mes higher than that found in this present study. Other
favorable results for S. aromaticum, but contrary to the
results obtained, i.e., with lower concentrations identified
for Gram-negative bacteria, were Fu et al. (2007) and
Silva et al. (2009), whose units used in dilution were
different from those in the present work.
To increase the chances of complete inactivation
of the colonies, Berrington and Gould (2001) suggest
the application of the highest concentration used in the
tests. Ponce et al. (2003), however, suggest small changes
in low concentrations to achieve significant changes in
cell behavior. Upon altering the highest values, not even
the most relevant changes promoted an inactivation that
justifies the use.
Inequalities in the effects of susceptibility testing
can be explained by the lack of uniformity in the criteria
established by the researchers (Ríos and Recio, 2005).
The contradictions observed and the difficulty in com-
paring the results obtained in researche that verifies the
antimicrobial activity of medicinal plants may result from
changes in environmental conditions at collection time,
the plant part studied, the procedures and protocols fol-
lowed (Auricchio and Bacchi, 2003), the physicochemical
characteristics of the essential oils (Nakamura et al, 1999),
reciprocity of its constituents, and in the extraction form
(Moreira et al., 2005; Ponce et al., 2003), among others.
The data shows the sanitizing potential of essential
oils of Ocimum gratissimum and clove. Confirmation of
the antiseptic character was observed in in vitro testing
of the efficiency as a disinfectant.
Oliveira, L.; Sales, S. S.
8
Cad. Ciênc. Agrá., v. 11, p. 01–12, 2019. e-ISSN: 2447-6218 / ISSN: 1984-6738
Figure 3 - Inhibition halo (mm) determined by the antimicrobial effect of essential oil of O. gratissimum. (A) and S.
aromaticum (B) on E. coli and S. aureus in function of the concentrations tested (µL/mL)
BAC1- S. aureus of sheep carcasses, BAC2- S. aureus ATCC 25923, BAC3- E. coli of sheep carcasses, BAC4- E. coli ATCC 8733
The minimum bactericidal concentration (MBC) is
defined as the lowest concentration of essential oil that
can completely destroy the inoculum (Fu et al., 2007;
Pozzo et al., 2011). The MBC of essential oils of Ocimum
gratissimum and S. aromaticum against S. aureus was 50
µL/mL. As for E. coli, it was 200 and 100 µL/mL, respec-
tively.
The results show a lower bactericidal concentration,
50 mL, for Ocimum gratissimum oil against S. aureus,
whereas for E. coli the same effect was only observed at
200 µL/mL, contrary to the response obtained with pre-
viously run diffusion plate tests (Table 1). The dilution
method, microdilution, macrodilution or agar dilution, is
applied to define the lowest bactericidal or bacteriostatic
concentration of an antimicrobial agent (Alves et al.,
2008). It is the most efficient to establish the potency of
a substance, since in the diffusion method, for example,
the possibility of failure is increased due to possible errors
in the preparation of the culture medium, the suspension
Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses
9
Cad. Ciênc. Agrá., v. 11, p. 01–12, 2019. e-ISSN: 2447-6218 / ISSN: 1984-6738
density, the halo measurement, unsuitable incubation
time and/or temperature etc. (Ríos e Recio, 2005).
In vitro efficacy of essential oils as disinfectants
According to the Ministry of Agriculture, Livestock
and Supply (Brasil, 1993), disinfectant is a substance, or
a group of them, “capable of destroying non-sporulated
pathogenic microorganisms in a short period of time
when applied to inanimate objects.” The results herein
are consistent with the aforementioned definition (Brasil,
1993). There was a reduction in the number of cells within
the first 5 minutes. The antiseptic composed of Ocimum
gratissimum at the highest concentration reduced E. coli
and S. aureus by 87 and 88%, respectively, and at a con-
centration of 200 µL/mL 58 and 78% less colonies were
observed. The product based on Ocimum gratissimum at
400 µL/ml completely eliminated the two species early in
the process. When the test was conducted with the lowest
concentration, there was only a cell count decrease in S.
aureus and E. coli of 67 and 27%, respectively.
Avancini and Wiest (2008) tested the effective-
ness of disinfectant composed of a medicinal plant in the
presence of organic matter in this present work whereas
Oliveira et al. (2010) and Tresoldi (2008) developed the
tests in the absence of this matter. These research works
strengthen the results in that in all of them, likewise, found
a direct relationship between infectious dose inactivation
and time, i.e., the longer the disinfectant contact time,
the higher the microorganism inactivation. However,
Chorianopoulos et al. (2008) found that, in general,
significant reductions in the number of colonies occur
within the first 60 minutes of contact with the natural
product. For the authors, extending this period does not
improve the disinfection efficiency
The growing interest in antibacterial compounds,
such as antibiotics and disinfectants, derived from plants
arises from the need to create new strategies (Oliveira et
al., 2010) to control, among other things, the pathogens
present in the air (Bouaziz et al., 2009), zoonotics (Pa-
laniappan and Holley, 2010), the formation of biofilms
and also to meet increasing consumer demand averse to
synthetic compounds. Among the alternatives to the use
of disinfectants and commercial chemicals, some resear-
chers propose the use of medicinal plants (Avancini et
al., 2000).
The number of research work seeking to verify the
effectiveness of natural compounds, like disinfectants, is
lower compared to work with commercial chemicals. The
presence of compounds with proven inhibitory activity
stimulates further study to obtain safer and more natural
ways to preserve food. In vitro study results show oils and
plant extracts as potentially rich sources for medicine and
the food industry, due to the broad spectrum of activity
against pathogens (Fu et al., 2007; Kotzekidou et al.,
2008).
In addition to the lower cost, these substances
can circumvent the negative effects caused by the indis-
criminate use of conventional antibiotics, favoring veteri-
nary medicine, farmers, consumers and the environment
(Avancini and Wiest, 2008; Bouaziz et al., 2009; Tresoldi,
2008).
Conclusion
The essential oils of O. gratissimum and S. aroma-
ticum showed satisfactory in vitro bactericidal inhibitory
and antiseptic activity on the bacteria S. aureus and E.
coli isolated from sheep carcasses and being promising
in the use as an alternative to antibiotics and commercial
antiseptics. However, more extensive studies are necessary
to verify adverse effects.
Financing
Banco do Nordeste do Brasil, Fundação de Amparo à
Pesquisa do Estado de Minas Gerais FAPEMIG, e Coor-
denação de Aperfeiçoamento de Pessoal de Nível Supe-
rior – CAPES
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