Lebensm.-Wiss. u.-Technol.

36 (2003) 467473
Sensitivity of four pathogenic bacteria to Turkish thyme and
oregano hydrosols
Osman Sa$ gdi@*
Department of Food Engineering, Faculty of Agriculture, Suleyman Demirel University, 32260 Isparta, Turkey
Received 31 May 2002; accepted 21 January 2003
Abstract
The two thyme (Thymus vulgaris L. and Thymus serpyllum L.) and three oregano (Origanum vulgare L., Origanum onites L. and
Origanum majorana L.) hydrosols, widely used in food products and as drinks, were tested for their inhibitory effects against four
pathogenic bacteria (Escherichia coli ATCC 25922, E. coli O157:H7 ATCC 33150, Staphylococcus aureus ATCC 2392 and Yersinia
enterocolitica ATCC 1501). Two principles were used in determining the antibacterial effects of spice hydrosols: inhibition zones
were determined by paper disc diffusion method, and the bacteriostatic effects, bactericidal effects and the growth inhibition levels
were studied as colony forming units (cfu) in broth cultures. All tested bacteria were inhibited by all of the spice hydrosols using
paper disc diffusion method. According to serial dilution method, while all spice hydrosols at 10 and 25 mL/100 mL concentrations
had generally bacteriostatic activities, the same hydrosols appeared to have bactericidal effects at concentrations above
50 mL/100 mL. The most sensitive of the bacteria against the spice hydrosols was S. aureus. In addition, the most inhibitive of the
spice hydrosols on the four pathogenic bacteria were O. onites L. and O. majorana L.
r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Spice hydrosols; Thyme and oregano; Antibacterial effect
1. Introduction
Recently, there has been an increasing interest in the
discovery of new natural antimicrobials, because of an
increase in risk in the rate of infections with antibiotic-
resistant microorganisms. The leafy part of plants, such
as thyme and oregano belonging to the Labiatae family,
has been added to meat, sh and food products for
years. In addition to improving the avour, certain
spices and essential oils prolong the storage life of foods
by an antimicrobial activity. Being natural foodstuffs,
they appeal to consumers who tend to question the
safety of synthetic additives (Farag, Daw, Hewedi, &
El-Baroty, 1989).
Experiments on the antimicrobial properties of spices,
especially thyme and oregano and their derivatives, have
been documented in recent years and interest continues
to the present (Akg. ul & Kivan@, 1988a;
.
Ozcan, 1998;
Cosentino et al., 1999; Dorman & Deans, 2000;
Aligiannis, Kalpoutzakis, Mitaki, & Chinou, 2001;
Marino, Bersani, & Comi, 2001;
.
Ozcan & Erkmen,
2001). Generally, these studies were concentrated
exclusively on spices, essential oils and extracts of thyme
and oregano (Akg. ul & Kivan@, 1988b, 1989; Panizzi,
Flamini, Cioni, & Morelli, 1993; Hammer, Carson, &
Riley, 1999; Karahan, Sa& gdi@,
.
Ozcan, &
.
Ozkan, 2000;
.
Ozcan & Boyraz, 2000; Sa& gdi@, Ku- s@u.
.
Ozcan, &
.
Oz@elik, 2002). Research on thyme and oregano
hydrosols (also called thyme and oregano waters) has
been limited (Sa& gdi@, &
.
Ozcan, 2003).
Hydrosols, also known as oral water, distillate water
or aromatic water, are the co-products or the bypro-
ducts of hydro- and steam distillation of plant material.
Hydrosols are quite complex mixtures containing traces
of the essential oil and, of course, several water-soluble
components. They have practically been used as
beverages for a long time in Turkey. Thyme and
oregano have commonly been used in foods mainly for
their avour, aromas and preservation, herbal tea,
alternative medicines and natural therapies (Huntanen,
1980; Aureli, Costantini, & Zolea, 1992).
Thyme and oregano species are rich in essential oils
characterised by a notable antimicrobial activity; for this
*Tel.: +90-246-2111542; fax: +90-246-2370437.
E-mail address: osagdic@ziraat.sdu.edu.tr (O. Sa$ gdi@).
0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0023-6438(03)00037-9
reason, these substances can be used to delay or inhibit
the growth of pathogenic or spoilage microorganisms
(Marino et al., 2001). Their activity depends on the type,
composition and concentration of the spice or the
derivatives, the type and concentration of the target
microorganism, the composition of the substrate, the
processing and the storage conditions (Pandit & Shelef,
1994; Marino et al., 2001).
In this research, thyme and oregano hydrosols, typical
of Turkey, were examined for their potential to inhibit
common bacteria encountered in foods as pathogens,
namely Escherichia coli ATCC 25922, E. coli O157:H7
ATCC 33150, Staphylococcus aureus ATCC 2392 and
Yersinia enterocolitica ATCC 1501.
2. Material and methods
2.1. Plant materials
Two thyme species (Thymus vulgaris L. and Thymus
serpyllum L.) and three oregano species (Origanum
vulgare L., Origanum onites L. and Origanum majorana
L.) were collected from different regions in Turkey, and
dried spices were nely ground. Plant materials were
identied by the scientists of botany at Suleyman
Demirel University in Isparta, Turkey.
2.2. Preparation of test bacteria
Four bacterial species including E. coli ATCC 25922,
E. coli O157:H7 ATCC 33150, S. aureus ATCC 2392
and Y. enterocolitica ATCC 1501 were used to
determine the antibacterial activity of each sample of
the hydrosols.
Bacterial species obtained from stock cultures were
grown in nutrient broth. E. coli, E. coli O157:H7 and
S. aureus were incubated at 37

C for 18 h. In addition,
Y. enterocolitica was grown in nutrient broth and
incubated at 22

C for 18 h. Broth cultures were

prepared overnight in nutrient broth and later adjusted
so that the nal concentration of each sample following
inoculation was approximately 10
6
10
7
cfu/mL (Il@im,
Di & grak, & Ba& gci, 1998).
2.3. Preparation of spice hydrosols
Hydrosols of thyme and oregano were produced by
the Clevenger hydrodistillation method (Deans &
Svoboda, 1990). Plant materials (about 50 g), cut into
small pieces, were placed in a ask (1 L) with 500 mL of
double distilled water and hydrodistilled for 1 h. After
hydrodistillation, the oil was collected in cooling vapour
to separate the essential oil of the plant. After
hydrodistillation, the mixture without essential oil in
the ask was identied as hydrosol. The amounts of
obtained hydrosols were approximately 100 mL. The
hydrosol was then ltered and preserved in sterile dark
bottles (500 mL) in a cool environment (4

C) until
further use (Sa& gdi@ &
.
Ozcan, 2003).
2.4. Paper disc diffusion method
The spice hydrosols were tested for antibacterial
activity using the paper disc diffusion method (Kelman-
son, J. ager, & Van Staden, 2000), using four bacteria.
Nutrient agar (15 mL) was inoculated with fresh broth
culture (0.15 mL, containing 10
6
10
7
cfu/mL) and
poured in sterile 9 cm Petri dishes. Fifty microlitres of
spice hydrosols or sterilised water (control) were applied
to a sterile lter paper disc (Whatman No. 1, 5 mm in
diameter). After evaporation of some water in a
biological safety cabinet with laminar ow (Nuaire
Laminar Flow Products, USA), the discs were placed on
the agar surface inoculated with test bacteria. Each plate
contained four paper discs soaked with spices hydrosols
(50 mL) and a disc with sterile water (50 mL) as control.
The plates inoculated with E. coli, E. coli O157:H7 and
S. aureus were incubated at 37

C for 18 h. In addition,
the plate inoculated with Y. enterocolitica was incubated
at 22

C for 18 h. Then, inhibition zones were recorded in

millimetre (mm) (Aureli et al., 1992; Kelmanson et al.,
2000). All experiments were conducted in duplicate and
the results are expressed as average values of inhibition.
2.5. Effect of thyme and oregano hydrosols on the growth
of pathogens in broth media by serial dilution method
The inhibitory effects of spice hydrosols against
bacterial species were measured using a nutrient broth-
dilution method. Nutrient broth (10 mL) was autoclaved
at 121

C for 15 min and maintained at 5560

C in liquid
state. The spice hydrosols were sterilised by 0.45 mm
millipore lters (Schleicher & Schuell, Germany). The
sterilised spice hydrosols at concentrations of 10, 25, 50
and 75 mL/100 mL were added to the sterilised nutrient
broths. Tubes containing nutrient broth and various
concentrations of hydrosols (total 10 mL) were inocu-
lated with activated bacterial cultures (0.1 mL). Tubes
inoculated with E. coli, E. coli O157:H7 and S. aureus
were incubated at 37

C for 4 days. Another tube

inoculated with Y. enterocolitica was incubated at
22

C for 4 days. Each day, the number of colonies in

the tubes was counted using serial dilution method in
nutrient agar. The growth inhibition level caused by
each concentration of the spice hydrosols on test
bacteria was determined according to the following
equation:
growth inhibition level

control population treated population

control population
100
O. Sa$ gdic / Lebensm.-Wiss. u.-Technol. 36 (2003) 467473 468
The control tube was prepared as described above
except that the tube contained no spice hydrosols (Ismaiel
& Pierson, 1990). Microbial counts (as log cfu/mL) were
carried out in a biological safety cabinet with laminar
ow (Nuaire Laminar Flow Products, USA).
2.6. Statistical analysis
Analysis of variance was performed on data obtained
at different stages of experiment. SPSS 7.5 for
Windows was used for the statistical analysis. The
experimental data for the spice hydrosols and patho-
genic bacteria were subjected to analyses of variance
using ANOVA.
3. Results and discussion
3.1. Paper disc diffusion method
The antibacterial activity of thyme and oregano
hydrosols against E. coli, E. coli O157:H7, S. aureus
and Y. enterocolitica are shown in Table 1.
All spice hydrosols showed inhibitory effect (inhibi-
tion X12 mm) against all bacteria. As expected, the
control treatment had no inhibitory effect on any
of the test bacteria. O. majorana L. (OM) showed
the highest activity against E. coli (19 mm inhibition
zone) and Y. enterocolitica (18 mm zone). O. vulgare L.
(OV) exhibited the lowest activity against E. coli
(13 mm zone) and Y. enterocolitica (12 mm zone).
E. coli O157:H7 was extremely prevented (19 mm zone)
by O. onites L. (OO), while slightly inhibited (12 mm
zone) by T. vulgaris L. (TV) and T. serpyllum L. (TS).
OO was the most active against S. aureus (30 mm zone),
while TV was the least active on this bacteria (17 mm
zone).
The most effective and the least effective of the spice
hydrosols against the four pathogenic bacteria are given
in Table 2. Statistically, the differences between each of
the spice hydrosols and pathogenic bacteria were
signicant at Po0:01: The effectiveness of the spice
hydrosols on pathogens followed the sequence: OOX
OM>OV>TS>TV. OO and OM had the highest
activity against all pathogens. S. aureus was the most
sensitive bacteria against all of the spice hydrosols.
OO was the most active of the hydrosols and showed a
30 mm inhibition zone on S. aureus. Generally, the
antibacterial effects of the oregano hydrosols were
stronger than thyme hydrosols.
Sa& gdi@ and
.
Ozcan (2003) indicated that O. vulgare
had inhibitory effect against E. coli, E. coli O157:H7,
S. aureus and Y. enterocolitica. However, the researches
on the thyme and oregano hydrosols were limited.
3.2. Inhibitory effects and growth inhibition levels of spice
hydrosols against pathogenic bacteria by serial dilution
method
The growth inhibition levels at different concentra-
tions of two thyme and three oregano hydrosols against
the four bacteria are shown in Table 3. The increasing of
the hydrosol concentrations generally showed an in-
crease in the growth inhibition levels and a reduction in
the rate of growth of the different microorganisms. In
other words, all spice hydrosols had an inhibitory effect
against all test bacteria during the 4 days. Also, the spice
hydrosols at 10 and 25 mL/100 mL concentrations had a
bacteriostatic effect, while the hydrosols at 50 and
75 mL/100 mL concentrations had a bactericidal effect.
Generally, the 50 and 75 mL/100 mL concentrations of
the spice hydrosols completely inhibited the test
bacteria. The hydrosol of OO was the most active on
all bacteria in serial dilution method. In addition,
S. aureus was the most sensitive to all of the spice
hydrosols.
Generally, TV and TS hydrosols at 10 and 25 mL/
100 mL concentrations had bacteriostatic effects against
E. coli, E. coli O157:H7, S. aureus and Y. enterocolitica
during the 4 days with respect to the control treatment.
However, TV hydrosol at the same concentrations had a
bactericidal effect on the fourth day and completely
Table 1
Antibacterial activity of spice hydrosols against pathogenic bacteria
(inhibition zone diameter in mm; diameter of paper disc, 5 mm,
included)
Spice hydrosols
nn
Pathogenic bacteria
nn
E. coli E. coli
O157:H7
S. aureus Y. entero-
colitica
Control (sterile water) 0 0 0 0
T. vulgaris L. 14 12 17 13
T. serpyllum L. 14 12 18 14
O. vulgare L. 13 15 21 12
O. onites L. 17 19 30 16
O. majorana L. 19 18 22 18
nn
Statistically, the differences were signicant at Po0:01:
Table 2
The most and least effective of spice hydrosols against four pathogens
Pathogens Spice
hydrosols
showed
the highest
activity
Inhibition
zones
(mm)
Spice
hydrosols
showed
the least
activity
Inhibition
zones (mm)
E. coli OM 19 OV 13
E. coli O157:H7 OO 19 TV, TS 12
S. aureus OO 30 TV 17
Y. enterocolitica OM 18 OV 12
O. Sa$ gdic / Lebensm.-Wiss. u.-Technol. 36 (2003) 467473 469
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1
6
4
.
5
8
4
.
1
1
0
0
5
6
.
4
6
7
.
9
7
3
.
9
1
0
0
5
8
.
7
6
3
.
5
1
0
0
1
0
0
4
7
.
9
7
0
.
4
8
5
.
7
1
0
0
7
5
6
6
.
7
7
4
.
2
1
0
0
1
0
0
7
0
.
5
1
0
0
1
0
0
1
0
0
7
7
.
3
1
0
0
1
0
0
1
0
0
6
5
.
8
1
0
0
1
0
0
1
0
0
O. Sa$ gdic / Lebensm.-Wiss. u.-Technol. 36 (2003) 467473 470
inhibited the growth of S. aureus on the fourth day. TS
hydrosol at a concentration of 25 mL/100 mL had a
complete inhibitory effect on S. aureus. TV and TS
hydrosols at 50 and 75 mL/100 mL concentrations had
bactericidal effects against all test bacteria (Table 3).
S. aureus was the most sensitive against TV and TS
hydrosols at all concentrations. The researches on the
antibacterial activity of the TV and TS hydrosols were
lacking. However, Sa& gdi@ and
.
Ozcan (2003) reported
that hydrosol of Thymbra spicata had inhibitory effect
against E. coil, S. aureus and Y. enterocolitica.
At concentrations of 10 and 25 mL/100 mL, OV, OO
and OM hydrosols showed bacteriostatic effects on
E. coli, E. coli O157:H7, S. aureus and Y. enterocolitica.
These spice hydrosols at the same concentrations had a
bactericidal effect on S. aureus on the fourth day. With
respect to the control treatment, concentrations of 50
and 75 mL/100 mL of OV, OO and OM hydrosols
showed bactericidal effects against all bacteria (Table 3).
Some researchers indicated that OV, OO and OM had
antibacterial activity (Huntanen, 1980; Akg. ul & Kivan@,
1988a, b; Deans & Svoboda, 1990; Hammer et al., 1999;
Dorman & Deans, 2000; Marino et al., 2001;
.
Ozcan &
Erkmen, 2001) and antifungal activity (Karapinar, 1985;
.
Ozcan, 1998;
.
Ozcan & Boyraz, 2000). Sa& gdi@ and
.
Ozcan
(2003) indicated that O. vulgare had inhibitory effect
against E. coli, E. coli O157:H7, S. aureus and
Y. enterocolitica. But the studies on the antibacterial
activity of the OO and OM hydrosols were lacking.
The bactericidal effects of spice hydrosols at a
concentration of 50 mL/100 mL on E. coli, E. coli
O157:H7, S. aureus and Y. enterocolitica are shown in
Figs. 1, 2, 3 and 4, respectively. In addition, the
bactericidal effects of spice hydrosols at 75 mL/100 mL
concentration on pathogenic bacteria are given in
Figs. 5, 6, 7 and 8, respectively. Based on all of these
results, OO hydrosol on the four pathogens showed the
strongest inhibitory effect compared to the other spice
0
1
2
3
4
5
6
7
8
0 1 2 3 4
time (days)
b
a
c
t
e
r
i
a
l

c
o
u
n
t

(
l
o
g

c
f
u
/
m
L
)
0
1
2
3
4
5
6
7
8
TV OV OO
control TS OM
Fig. 1. The inhibitory effect of spice hydrosols at 50 mL/100 mL
concentration against E. coli.
0
1
2
3
4
5
6
7
8
0 1 2 3 4
time (days)
b
a
c
t
e
r
i
a
l

c
o
u
n
t

(
l
o
g

c
f
u
/
m
L
)
0
1
2
3
4
5
6
7
8
TV OV OO
control TS OM
Fig. 2. The inhibitory effect of spice hydrosols at 50 mL/100 mL
concentration against E. coli O157:H7.
0
1
2
3
4
5
6
7
8
0 1 2 3 4
time (days)
b
a
c
t
e
r
i
a
l

c
o
u
n
t

(
l
o
g

c
f
u
/
m
L
)
0
1
2
3
4
5
6
7
8
TV OV OO
control TS OM
Fig. 3. The inhibitory effect of spice hydrosols at 50 mL/100 mL
concentration against S. aureus.
0
1
2
3
4
5
6
7
8
0 1 2 3 4
time (days)
b
a
c
t
e
r
i
a
l

c
o
u
n
t

(
l
o
g

c
f
u
/
m
L
)
0
1
2
3
4
5
6
7
8
TV OV OO
control TS OM
Fig. 4. The inhibitory effect of spice hydrosols at 50 mL/100 mL
concentration against Y. enterocolitica.
O. Sa$ gdic / Lebensm.-Wiss. u.-Technol. 36 (2003) 467473 471
hydrosols. Generally, the effects of all of the spice
hydrosols on the pathogens were found to be similar,
depending on concentrations and bacterial species.
Some researchers reported that there is a relationship
between the chemical structures of the most abundant
compounds in the tested spices and the antimicrobial
activity (Farag et al., 1989; Deans & Svoboda, 1990).
4. Conclusion
Based on the results of this study, the two thyme and
three oregano hydrosols appeared to inhibit the growth
of the four pathogens tested. Thyme and oregano
hydrosols at 50 and 75 mL/100 mL concentrations were
completely inhibitive on bacterial growth in broth
culture. The results of this study conrmed the
possibility of using thyme and oregano hydrosols in
food preservation and drinks. These hydrosols can be
used in different concentrations to protect some stored
food products from pathogens, since these hydrosols
have GRAS status.
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0
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