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Inhibitory Effects of Spice Essential Oils on the

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Article in Journal of Medicinal Food · February 2006

DOI: 10.1089/jmf.2006.9.418 · Source: PubMed

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JOURNAL OF MEDICINAL FOOD
J Med Food 9 (3) 2006, 418–421
© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Short Communication

Inhibitory Effects of Spice Essential Oils on the Growth of Bacillus Species

Mehmet Musa Özcan,1 Osman Sağdıç,2 and Gülcan Özkan3
1Department of Food Engineering, Agricultural Faculty, Selçuk University, Konya; 2Department of Food
Engineering, Engineering Faculty, Erciyes University, Kayseri; and 3Agricultural Faculty, Suleyman
Demirel University, Isparta, Turkey

ABSTRACT A series of essential oils of 11 Turkish plant spices [black thyme, cumin, fennel (sweet), laurel, marjoram,
mint, oregano, pickling herb, sage, savory, and thyme], used in foods mainly for their flavor, aromas, and preservation, in
herbal tea, in alternative medicines, and in natural therapies, were screened for antibacterial effects at 1:50, 1:100, 1:250, and
1:500 dilutions by the paper disc diffusion method against six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacil-
lus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger
ATCC 10). All of the tested essential oils (except for cumin) showed antibacterial activity against one or more of the Bacil-
lus species used in this study. Generally, the essential oils at 1:50 and 1:100 levels were more effective. Only one essential
oil (laurel) was not found effective against the tested bacteria. The bacterium most sensitive to all of the spice essential oils
was B. amyloliquefaciens ATCC 3842. Based on the results of this study, it is likely that essential oils of some spices may
be used as antimicrobial agents to prevent the spoilage of food products.

KEY WORDS: • Bacillus species • essential oils • inhibitory effect

INTRODUCTION the food industries as troublesome spoilage organisms. They

F OR YEARS, INVESTIGATIONS have been conducted into the
antimicrobial effects of various spices and derivatives.1–11
Spices are rich in extracts and essential oils with known an-
timicrobial activity; for this reason, these materials can be
used to delay or inhibit the growth of microorganisms. Dif-
ferent additives are used to increase the storage period of
foods and to prevent the growth of microorganisms. How-
ever, there is a great interest in the use of alternatives, espe-
cially natural products such as spices and plants.12,13 The leaf
parts of plants such as sage, thyme, oregano, and savory be-
longing to the Labiatae family have been added to meat, fish,
and food products for years. In addition to improving flavor,
certain spices and essential oils prolong the storage life of
foods because of their antimicrobial activity.3
Bacillus species are aerobic, sporulating, rod-shaped bac-
teria that are ubiquitous in nature. Because the spores of
many Bacillus species are resistant to heat, radiation, disin-
fectants, and desiccation, they are difficult to eliminate from
medical and pharmaceutical materials and are a frequent
cause of contamination. Bacillus species are well known in
are associated with a variety of foods, including meat and
vegetable dishes, sauces, pastas, desserts, and dairy prod-
ucts. The symptoms of Bacillus cereus food poisoning are
caused by a toxin or toxins produced in the food during bac-
terial multiplication. Toxins have not yet been identified for
other Bacillus species that cause food poisoning.14
The aim of this study was to test a large number of es-
sential oils against six Bacillus species. The purpose of this
was to measure directly comparable, quantitative, antimi-
crobial data and to generate data for oils for which few data
presently exist.
MATERIALS AND METHODS
Spice samples
The plant spices used in this study were collected from
plants grown in Turkey and dried at room temperature. Plants
were identified botanically at the Biology Department of
Selçuk University, Konya, Turkey. The plant and botanical
names of the spices tested in this study are given in Table 1.

Bacterial cultures
Manuscript received 11 February 2005. Revision accepted 17 May 2005.

Address reprint requests to: Mehmet Musa Özcan, Department of Food Engineering, Agri-
The six Bacillus species used as test organisms were as
cultural Faculty, Selçuk University, 42031 Konya, Turkey, E-mail: mozcan@selcuk.edu.tr follows: Bacillus amyloliquefaciens ATCC 3842, Bacillus

418
 SPICE ESSENTIAL OILS AND BACILLUS GROWTH
TABLE 1. PLANT SPICE MATERIALS
Plant name Botanical name
Black thyme Thymbra spicata L.
Cumin Cuminum cyminum L.
Fennel (sweet) Foeniculum vulgare
Laurel Laurus nobilis L.
Mint Mentha spicata L.
Marjoram Origanum majorana
Oregano Origanum vulgare L.
Pickling herb Echinophora tenuifolia L.
Sage Salvia aucheri L.
Savory Satureja cuneifolia
Thyme Thymbra sintenesi
brevis FMC 3, B. cereus FMC 19, Bacillus megaterium
DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger
ATCC 10.
Preparation of spice essential oils
The plant materials (about 100 g), cut into small pieces,
were placed in a flask (2 L) together with doubly distilled
water (1.5 L) and subjected to hydrodistillation for 3 hours
using a Clevenger-type apparatus. The oils obtained were
dried over anhydrous sodium sulfate. All essential oils were
held at refrigerator temperature (4°C) and stored in the dark
when not in use.
Determination of antibacterial effects by paper disc
diffusion method
Stock cultures of B. amyloliquefaciens and B. brevis were
grown in nutrient broth (Acumedia Manufacturers, Inc., Bal-
timore, MD) at 25°C for 22 hours. The other Bacillus species
were grown in the same medium at 35°C for 22 hours.15 Fi-
nal cell concentrations were 106–107 colony-forming units/
mL. Each bacterium grown in tubes was inoculated as 1%
of tested bacteria into a flask containing 20 mL of sterile
nutrient agar (Acumedia Manufacturers) at 43–45°C. The
prepared bacterial cultures were poured into Petri plates (9
cm in diameter), and then the agar was allowed to solidify
at 4°C for 1 hour. The paper disc diffusion method was used
to detect the antibacterial activity of spice essential oils. Ster-
ilized filter paper discs (5 mm in diameter) were soaked with
40
L of a solution of 1%, 2%, 0.4%, or 0.2% of each spice
essential oil in absolute ethanol (Merck, Darmstadt, Ger-
many); absolute ethanol was used as the control. The soaked
discs were put in the middle of the plates. B. amyloliquefa-
ciens and B. brevis were incubated at 25°C for 18–24 hours;
the other Bacillus species were grown in the same medium
at 35°C for 18–24 h.10,16–19 Then, inhibition zones were
recorded in mm. All experiments were conducted in dupli-
cate, and the results are expressed as average values of in-
hibition.
419
FROM WHICH ESSENTIAL OILS WERE OBTAINED
Family Part used
Labiatae Leaves  flowers
Umbelliferae Fruits
Umbelliferae Fruits
Lauraceae Leaves
Labiatae Leaves  flowers
Labiatae Leaves
Labiatae Leaves  flowers
Umbelliferae Leaves  stem
Labiatae Leaves
Labiatae Leaves
Labiatae Leaves  flowers
Statistical analysis
Results were tested for statistical significance by analy-
sis of variance using SPSS (Chicago, IL) version 10.0.
RESULTS AND DISCUSSION
The antibacterial effects of black thyme, cumin, fennel
(sweet), marjoram, mint, oregano, pickling herb, sage, sa-
vory, and thyme volatile oils against tested Bacillus spp. are
shown in Table 2. The different essential oils caused dif-
ferent inhibition zones against the tested bacteria. Control
treatment (absolute alcohol) did not show an inhibitory ef-
fect against any of the Bacillus species.
The most active of essential oils was marjoram,
with 1:50, 1:100, and 1:250 concentrations inhibiting
completely the growth of all the bacteria. Also, black
thyme, cumin, fennel, mint, oregano, pickling herb, sage,
savory, and thyme oils were found to have an inhibitory
effect; the lowest effect was obtained with laurel oil,
which did not inhibit the growth of any Bacillus species.
While the essential oils of all spices (except laurel) at
low concentration (1:250) showed antibacterial activity
against one or more bacteria, the oil of marjoram at
the same concentration showed antibacterial activity
against all the Bacillus species. However, 1:250 and 1:500
concentrations of black thyme, cumin, fennel, mint,
marjoram, oregano, pickling herb, sage, savory, and
thyme oils completely inhibited the growth of B. amy-
loliquefaciens. Therefore, B. amyloliquefaciens was the
most sensitive of Bacillus species. Results showed
that 1:50 and 1:100 dilutions of these oils could be
used in foods in which Bacillus species are considered
a health hazard. Similar findings have been reported by
others.1,3,6,8,11,20,21
Differences between concentrations for each essential
oil were statistically significant at P
.001. Also, differ-
ences between each bacterium were statistically signifi-
cant at P
.001. The effectiveness of the inhibitors fol-
lowed the sequence marjoram  oregano  black thyme
 thyme  savory  mint  cumin  sage  fennel 
420 ÖZCAN ET AL.

TABLE 2. INHIBITORY EFFECT OF ESSENTIAL OILS OF TURKISH SPICES AT DIFFERENT CONCENTRATIONS

AGAINST BACILLUS SPECIES IN THE PAPER DISC DIFFUSION METHOD

Bacillus strainsa

B. B.
B. B. cereus B. B. subtilis
amyloliquefaciens brevis FMC megaterium subtilis var. niger
Concentrationa ATCC 3842 FMC 3 19 DSM 32 IMG 22 ATCC 10

Ethanol (control) Absolute — — — — — —

Black thyme 1
500 28 32 27 31 28 29
1
100 31 21 31 30 31 29
1
250 27 — — 30 — 27
1
500 26 — — — — —
Cumin 1
500 33 23 31 — — 27
1
100 33 14 — — — —
1
250 33 — — — — —
1
500 30 — — — — —
Fennel (sweet) 1
500 35 — — — — —
1
100 33 — — — — —
1
250 30 — — — — —
1
500 30 — — — — —
Marjoram 1
500 35 36 38 33 37 36
1
100 32 32 33 32 35 31
1
250 32 16 30 32 30 19
1
500 30 — — — — —
Mint 1
500 31 — 29 32 33 30
1
100 33 — — 32 — 30
1
250 31 — — — — —
1
500 33 — — — — —
Oregano 1
500 33 35 33 27 33 32
1
100 33 29 31 28 32 36
1
250 33 — — 30 30 —
1
500 30 — — — — —
Pickling herb 1
500 34 — — — — —
1
100 32 — — — — —
1
250 30 — — — — —
1
500 28 — — — — —
Sage 1
500 33 — — 26 — 29
1
100 30 — — — — 26
1
250 30 — — — — —
1
500 28 — — — — —
Savory 1
500 34 30 32 25 26 31
1
100 34 21 32 — 27 32
1
250 34 — 32 — — —
1
500 32 — — — — —
Thyme 1
500 35 12 30 32 33 32
1
100 33 — 26 31 32 28
1
250 33 — — — — —
1
500 33 — — — — —

A dash indicates inhibition not detectable.

significant at P
.001.
aStatistically

pickling herb  laurel, and the effectiveness of the spices
concentrations followed the sequence 1:50  1:100 
1:250  1:500.
Of the oils, marjoram was found to be the most in-
hibitory and was considered to be among the most active of
the essential oils tested. The variation of antibacterial ac-
tivities of the essential oils tested may be due to their con-
stituents and the probable nonvolatile compounds of spices.
Generally, the extent of the inhibitory effect of the oils could
be attributed to the presence of an aromatic nucleus con-
taining a polar functional group.8,21
The extent of bacterial inhibition by essential oils in food
systems remains to be demonstrated, however, and further
investigations are needed.
 SPICE ESSENTIAL OILS AND BACILLUS GROWTH
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