Essential Oil From Five Zingiberaceae For Anti Food-Borne Bacteria
Essential Oil From Five Zingiberaceae For Anti Food-Borne Bacteria
Essential Oil From Five Zingiberaceae For Anti Food-Borne Bacteria
Introduction
Food-borne diseases are still a major problem of
the world, even in well-developed countries (Mead
et al., 1999). A variety of microorganisms also lead
food spoilage that is encountered as one of the most
important matter concerning the food industry.
So far, many pathogenic microorganisms, such
as Escherichia coli, Staphylococcus aureus, Klebsiella
pneumoniae, Listeria monocytogenes and Campylobacter
jejuni have been reported as the causal agents of
food-borne diseases and/ or food spoilage (Deak
and Beuchat, 1996; Betts et al., 1999). Thus,
at present, it is a necessity to use the chemical
preservatives to prevent the growth of food spoiling
microbes in the food industry (Sagdc and Ozcan,
2003). Due to the consumers concerned about the
safety of food containing preservative as synthetic
chemicals, therefore, there is a growing interest to
use natural antibacterial compounds. Extracts of
herbs and spices can be the preservation of foods, as
these possess a characteristic flavor and sometimes
show antioxidant activity as well as antimicrobial
activity (Smid and Gorris, 1999). For centuries,
indigenous plants have been used in herbal
medicine for curing various diseases (Cowan, 1999).
Recently, the acceptance of traditional medicine
as an alternative form for health care and the
development of microbial resistance to the available
*Corresponding author
Email: [email protected]
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Extraction procedure
Hydrodistillation
Essential oils of five Zingiberaceae species were
extracted by hydrodistillation, and all operations
were carried out at room temperature. The fresh
rhizomes of Zingiberaceae were washed to remove
soil, peeled and sliced. Sliced rhizomes of fresh
Zingiberaceae (2 Kg) were mixed with distilled
water (5 L). The essential oils were extracted by
hydrodistillation using a vertical hydrodistillation
unit. A flask containing the homogenate was
heated during 24 h and the vapor condensed and
separated throughout an auto-oil/water separator.
Each essential oil extraction was running in
triplicate. Yield percentages were recorded as dry
basis material.
Solvent extraction
Plant material was oven dried at 50C for 24 h to
reduce water content. Extracts were prepared by
blending preserved plant material (approximately
200 g dry weight) in 99% ethanol and petroleum
ether (1:3 w/v ratio). After 24 h, the mixture was
filtered through Whatman filter paper (No.1) using
a Buchner funnel. The solvent was removed with a
rotary vacuum evaporator at 40C (30 mmHg). The
oil was stored in dark vials at 4C before analyzing.
The waste or residue after extracted by petroleum
ether of plant materials was repeated once with
ethanol, called secondary extraction, similar to the
above condition.
The objectives of this study were to compare
the antimicrobial activity of the essential oils and
extracts from Zingiberaceae against common
food-borne pathogen and/or spoilage bacteria,
including Escherichia coli, Staphylococcus aureus,
Bacillus cereus and Listeria monocytogenes. Evaluating
minimal inhibitory concentrations and the main
components of the extracts by GC/MS, in an
attempt to contribute to the use of these as
alternative products for microbial control and food
preservation were determined.
Materials and methods
Plant material
Fresh rhizomes of five Zingiberaceae (ginger,
galanga, turmeric, kaempferia, bastard cardamom)
were purchased from a local vegetable and
fruit market of the Tungkru District, Bangkok,
Thailand.
Antibacterial screening
The antibacterial activity of the plant extracts was
carried out by disc diffusion assay that was used to
screen (Kumar et al., 2001; Gulluce et al., 2003).
Muller Hinton agar (MHA) plates were swabbed
with the respective broth culture of the organisms
(diluted to 0.5 McFarland Standard with saline)
and kept for absorption to take place. Sterile 6 mm
diameter filter paper discs were impregnated with
100 mg/ml of plants extract that dissolved in sterile
dimethylsulfoxide (DMSO). Negative controls
were prepared using the same solvents employed
to dissolve the plant extracts. Streptomycin (5
g/ml) was used as positive reference standards
to determine the sensitivity of one strain in each
bacterial species tested. The plates were incubated
overnight at 37C. The antimicrobial activity was
evaluated by measuring the zone expressed as mm
of inhibition against test organism. Five discs per
plate and three plates were used, and each test was
run in triplicate.
Determination of minimum inhibitory concentration
(MIC)
The minimum inhibition concentration (MIC)
values were also studied for the bacteria which
were determined as sensitive to the extracts in disc
diffusion assay. The inoculated bacteria as prepared
from 24 h nutrient broth cultures and suspensions
were adjusted to 0.5 McFarland turbidity standard.
Plant extracts dissolved in DMSO were first diluted
to the highest concentration (50 g/ml) to be
339
Table 1: % Relative peak areas and RIs of volatile compounds in ginger extracts
Compound
RIs
calminol
1233
1.4
3.3
neral
1247
10.2
4.8
1.3
1.1
chavicol
1249
1.2
geranial
1261
15.1
1.9
0.6
linalool acetate
1277
0.5
curcumene
1471
6.3
3.3
4.2
2.9
zingiberene
1489
30.7
51.4
46.0
41.5
-farnesene
1491
15.2
16.0
17.6
22.8
-bisabolene
1518
6.9
7.3
9.0
6.9
-sesquiphellandrene
1560
11.3
12.2
15.1
17.6
guaiol
1589
0.7
0.6
1.3
0.9
gingerone
1718
0.5
1.1
3.6
5.7
A, B. C and D refer to the different extraction methods used (A: hydrodistillation; B: extraction with petroleum ether; C: secondary
extraction with ethanol of plant residue after extraction by method B and D: extraction with ethanol).
340
The result obtained by GC-MS analysis of
turmeric is presented in Table 3. Fifteen compounds
were identified. The oil profile shows turmerone as
the main compound (A; 50.0%, B; 58.5%, C; 66.7%,
D; 64.7%); other major compounds were curlone,
-farnesene and -zingiberene, respectively.
Table 2: % Relative peak areas and RIs of volatile compounds in galanga extracts
Compound
RIs
1,8-cineole
1029
33.6
4.1
2.4
3.3
methyl chavicol
1033
37.9
Camphor
1139
4.5
16.0
9.8
5.0
-thujene
1158
0.7
2.4
-caryophyllene
1432
4.2
5.9
7.4
1.5
-farnesene
1491
4.2
4.6
4.8
1.8
-elemene
1494
1.2
3.5
0.3
-selinene
1497
3.0
2.4
2.2
1.6
-farnesene
1500
5.9
6.7
2.4
5.3
-selinene
1503
4.2
4.3
2.0
2.4
-cadinene
1507
0.8
0.5
ethyl p-methoxy-cinnamate
1711
49.8
68.2
74.6
eugenol acetate
1935
0.8
4.2
A, B. C and D refer to the different extraction methods used (A: hydrodistillation; B: extraction with petroleum ether; C: secondary
extraction with ethanol of plant residue after extraction by method B and D: extraction with ethanol).
Table 3: % Relative peak areas and RIs of volatile compounds in tumeric extracts
Compound
RIs
1,8-cineole
1025
1.1
1.4
-terpinene
1055
5.5
1.7
isocaryophyllene
1409
1.8
0.7
0.5
-caryophyllene
1449
1.1
0.4
0.4
ar-curcumene
1471
2.9
1.2
1.3
1.3
-zingiberene
1489
7.8
3.6
1.8
3.5
-bisabolene
1494
1.7
0.5
0.5
-farnesene
1500
10.8
4.1
2.5
4.1
cubenol
1602
3.1
0.9
1.4
1.1
ar-turmerol
1610
0.9
1.9
2.5
2.1
turmerone
1643
50.0
58.5
66.7
64.7
curlone
1645
12.9
20.9
21.4
20.1
(6S,7R)-bisabolene
1666
0.9
-atlantone
1669
0.5
1.3
0.9
0.7
(E)--atlantone
1689
1.8
1.1
1.0
A, B. C and D refer to the different extraction methods used (A: hydrodistillation; B: extraction with petroleum ether; C: secondary
extraction with ethanol of plant residue after extraction by method B and D: extraction with ethanol).
The profile of kaempferia in Table 4 shows
-terpinene (44.0%), which was only found most
in this essential oil by hydrodistillation. The other
major compounds of kaempferia extracts were
geraniol (A; 20.6%, B; 55.3%, C; 37.2%, D; 40.7%)
and 6-camphenone (A; 18.7%, B; 29.6%, C; 25.2%,
D; 31.3%).
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The most abundant compound in rhizome
of bastard cardamom obtained by hydrodistillation
and petroleum ether was methyl chavicol (93.1%
and 48.7%, respectively), whereas in the ethanol
extract and secondary extraction with ethanol was
anethole (Table 5).
Table 4: % Relative peak areas and RIs of volatile compounds in kaemferia extracts extracts
Compound
RIs
limonene
1025
0.7
1,8-cineole
1029
12.8
1.8
3.6
5.2
-terpinene
1055
44.0
3.5
1.9
4.7
linalool
1090
0.8
1.2
0.8
1.2
terpineol
1135
0.3
6-camphenone
1141
18.7
29.6
25.2
31.3
borneol
1154
0.3
0.4
nerol
1234
0.6
0.6
citral
1254
0.3
geraniol
1276
20.6
55.3
37.2
40.7
methyl cinnamate
1320
2.1
2.6
3.7
4.2
-farnesene
1448
0.8
0.2
0.3
nerolidol
1543
0.3
0.4
11-dodecen-1-ol
1692
2.2
1.1
1.1
pinostrobin chalcone
2502
2.7
25.1
10.1
A, B. C and D refer to the different extraction methods used (A: hydrodistillation; B: extraction with petroleum ether; C: secondary
extraction with ethanol of plant residue after extraction by method B and D: extraction with ethanol).
Table 5: % Relative peak areas and RIs of volatile compounds in bastard cardamom extracts
Compound
RIs
methyl chavicol
1175
93.1
48.7
5.2
19.2
anethole
1282
0.3
44.2
79.4
63.4
-copaene
1378
1.0
1.1
0.5
aromadendrene
1443
0.2
0.3
0.8
0.2
-himachalene
1450
0.2
caryophellene
1432
0.4
-farnesene
1448
0.3
0.3
0.6
-selinene
1494
0.6
0.5
1.0
0.8
-cadinene
1508
3.3
4.2
4.8
13.5
-chamigrene
1547
0.3
0.3
1.0
0.5
-farnesol
1697
0.2
0.5
bornyl benzoate
1766
1.5
0.3
eugenolacetate
1935
6.4
1.1
A, B. C and D refer to the different extraction methods used (A: hydrodistillation; B: extraction with petroleum ether; C: secondary
extraction with ethanol of plant residue after extraction by method B and D: extraction with ethanol).
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Antibacterial activity
The antibacterial activity of essential oils and
extracts from five Zingiberaceae species against
the microorganisms considered in the present
study were qualitatively and quantitatively assessed
evaluating the presence of inhibition zone and
zone diameter (Table 6). Among Gram-positive
bacteria, B. cereus was the most sensitive organism
to plant extracts that this finding is in agreement
with a previous report (Alzoreky et al., 2003). Gramnegative bacteria showed resistance (no inhibition
zone) to the 18 extracts of plant. Water-distilled
essential oils of kaempferia (%yield = 0.26 dry
basis (d.b.)) and bastard cardamom (%yield = 0.27
d.b.) were only inhibitory for E. coli. Kaempferia
Table 6: Results of the antibacterial tests of the investigated plants in agar diffusion assay
S. aureus
B. cereus
E.coli
L. monocytogenes
Hydrodistillation
16a
20a
0c
22a
Ethanol
9hi
10h
0c
10i
Petroleum ether
10g
12f
0c
11h
8j
11g
0c
8l
Hydrodistillation
8k
9hi
0c
11h
Ethanol
12e
13d
0c
9k
Petroleum ether
12e
12f
0c
9k
13
14
10i
10h
0c
16c
Plants species
Boesenbergia pandurata
(kaempferia)
Amomum xanthioides
(bastard cardamom)
Extract
Hydrodistillation
9i
cd
Ethanol
11fg
12f
0c
13f
Petroleum ether
10
10
10i
11fg
11e
0c
11g
Hydrodistillation
15ab
16b
9b
19b
Ethanol
11f
14c
0c
15d
Petroleum ether
14b
12cf
0c
13f
14b
16b
0c
14e
Hydrodistillation
12c
13d
10a
13f
Ethanol
0l
8i
0c
0m
Petroleum ether
8j
9hi
0c
0m
0m
24
26
22
24
Streptomycin, 5 mg/ml
* Inhibition zone including the diameter of the paper disc (6 mm).
a,b,c,the letters in the same column are significant difference at p 0.05.
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In this study, the major compounds in five
Zingiberaceae essential oils were terpenes which
effect on membrane of bacteria (Gram positive
and negative), as shown in Figure 1. Terpenes in
ginger, galanga, turmeric, kaempferia, bastard
cardamom were zingiberene and farnescene;
methyl chavicol and ethyl-p-methoxycinnamate;
tumerone, farnescene, curlone and zingiberene;
terpinene, geraniol, and 6-camphenone; methyl
chavicol, respectively.
Minimum inhibitory concentration (MIC)
The MICs of each plant extracts are presented
in Table 7. Of the 7 plant extracts tested, ginger,
turmeric, and bastard cardamom extracted
by hydrostillation, kaempferia extracted by
Figure 1: Structures of major components in essential oils from five Zingiberaceace sp.
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Hydrodistillation
Ethanol
ginger
turmeric
kaempferia
bastard
cardamom
galanga
kaempferia
kaempferia
S. aureus
12.5
none
12.5
none
100
50
12.5
B. cereus
6.25
none
12.5
none
25
12.5
12.5
E. coli
none
none
50.0
25
none
none
none
L. monocytogenes
6.25
25
6.25
none
none
6.25
6.25
Conclusion
References
Acknowledgements
The authors would like to thank Department of
Applied Microbiology, King Mongkuts University
of Technology Thonburi in Thailand for supplying
the bacterial strains.
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