ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 22 (2005) 37–45
www.elsevier.nl/locate/jnlabr/yfmic

Volatile components and antibacterial effects of pine needle

(Pinus densiflora S. and Z.) extracts
Yong-Suk Kima, Dong-Hwa Shinb,*
a
Research Center for Industrial Development of BioFood Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea
b
Faculty of Biotechnology (Food Science & Technology Major), Chonbuk National University, Dukjin-Dong, Jeonju, Chonbuk 561-756,
Republic of Korea
Received 10 February 2004; accepted 11 May 2004

Abstract

The antibacterial effects of the volatile components extracted from Pinus densiflora S. and Z., by simultaneous steam distillation
and solvent extraction (SDE), were examined on six foodborne bacteria using Bioscreen C (a computer-controlled shake–incubator–
reader). The SDE extracts of P. densiflora obtained after 1.5 or 2.0 h at pH 3.6 exhibited a strong growth inhibitory effect on
Escherichia coli O157:H7 and their overall antibacterial activities against the various bacteria tested tended to increase with
increased extraction time and with a lower extraction pH. The major volatile components of the SDE extracts obtained at pH 3.6
and 1.5 h, as determined by gas chromatography, were a-ocimene (29.3%), sabinene (10.9%), b-myrcene (9.6%), b-caryophyllene
(8.0%), b-cadinene (7.3%), a-terpinolene (4.9%), 2-hexanal (4.5%), and b-pinene (4.3%). The addition of 8% or 10% (v/v) of the
SDE extracts to culture broth completely inhibited the growths of Bacillus cereus, Salmonella Typhimurium, and Staphylococcus
aureus. The intracellular adenosine triphosphate (ATP) concentration of S. Typhimurium treated with P. densiflora extract reduced
to 0.165 mm from 0.595 mm, whereas the ATP concentration in culture supernatants was increased to 0.469 mm form 0.065 mm.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Pinus densiflora; Antibacterial activity; Volatile components; SDE; Foodborne bacteria

1. Introduction medical affects of the needles are strongest in the winter,

Since materials added to prolong shelf-life should not
remain in the food, the use of volatile antibacterial
materials as food preservatives and as a means of the
preventing micro-organism development has become the
subject of study (Kim and Shin, 2003).
Pinus densiflora S. and Z. belongs to the Pinaceae
family. It is an evergreen needle-leafed tree indigenous
to East Asia, and has bitter tasting leaves, which are
gathered between spring and autumn (Korea Food &
Drug Administration, 1997). Its leaves contain an
essential oil (0.3–1.3%), which contains a-pinene, b-
pinene, camphene, phellandrene, limonene, borneol
(6.8%), and bornyl acetate (3.8%) (Im, 1998). The fresh
needles untreated as a folk medicine. Apparently, the
*Corresponding author. Tel.: +82-63-270-2570; fax: +82-63-270-
2572.
E-mail address: dhshin@moak.chonbuk.ac.kr (D.-H. Shin).
and they said to expel pathogenic wind, remove
dampness, and to stop bleeding (Korea Food & Drug
Administration, 1997). Various parts of this tree, i.e.,
needles, cones, cortices, and pollen, have been widely
used for health promoting purposes as a folk medicine
or as a food (Dongeuhak Institute, 1994).
Recently, the composition and the various biological
functions of the volatile fraction of P. densiflora were
described: growth-inhibiting effects of the constituents
of leaves on human intestinal bacteria (Jeon et al., 2001),
components of the root or needles (Watanabe et al.,
1991; Jung et al., 2001), volatile constituents and the
extraction solvent used (Cho et al., 1999a), essential oils
from needles and twigs (Koukos et al., 2000), and the
antimicrobial effects of ethanol extracts on lactic acid
bacteria (Lim et al., 2001).
Given the lack of research information in this field, we
examined the antibacterial effects of the essential oil of
the leaves of P. densiflora extracted under different

0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2004.05.002
 ARTICLE IN PRESS
38 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45
conditions. In addition, the concentration of ATP in
cells and in culture media treated with the simultaneous
steam distillation and solvent extraction (SDE) extract
of P. densiflora were determined and related to observed
bactericidal effects of SDE on several bacteria. Specifi-
cally, efforts were made to identify and quantity the
volatiles present, and to assess their antibacterial effects
with a view to increasing the shelf lives of limited-
storage instant foods.
2. Materials and methods
2.1. Micro-organisms and cultures
Six different foodborne bacteria were used. Bacillus
cereus (ATCC 11778) and Salmonella Typhimurium
(ATCC 14028) strains were grown at 30
C in nutrient
broth or nutrient agar (Oxoid Ltd., Basingstoke,
Hampshire, England). E. coli O157:H7 (ATCC 43894)
and Staphylococcus aureus (ATCC 25923) strains were
grown at 37
C, and Listeria monocytogenes (ATCC
19111) at 30
C, in tryptic soy broth or tryptic soy agar
(Difco, Detroit, Michigan, USA). Vibrio parahaemoly-
ticus (ATCC 33844) strain was grown at 37
C in tryptic
soy broth or tryptic soy agar supplemented with 3% (w/
v) NaCl. All bacteria were grown for 24 h in sterilized
broth medium. An aliquot of each culture (0.1 ml) was
then transferred to a 9.9 ml new broth medium and
culture for 18 h.
2.2. Extraction of volatile components
The leaves of P. densiflora were collected from the
Jeonju Arboretum (Jeonju, Korea) in September 2001,
washed and stored at 20
C. Volatile leaf extracts were
obtained by SDE using the ‘improved’ Likens–Nick-
erson apparatus (Parliament, 1997). After circulating
50 ml of the extracting solvent (redistilled diethyl ether)
through the apparatus at 36
C, 100 g of the leaves were
ground in a Waring blender (Waring, New Hartford,
Connecticut, USA) and mixed with 1000 ml of distilled
water in a round-bottomed flask. The SDE times used
were 0.5, 1.0, 1.5, and 2.0 h at pH 3.6 (the control pH),
then the pH was increased to 4.6, 5.6, or 6.6 and these
mixtures and the control pH mix were extracted for
1.5 h. Anhydrous sodium sulfate (B15 g) was then
added to remove water. The ether mixture was then
cooled to 20
C for 12 h, and evaporated to 1 ml using
a nitrogen flow. Ten microliters of 1-pentanol (n-amyl
alcohol) was then added to the extracts as the internal
gas chromatography (GC) standard. The extracts
obtained were tested for antibacterial activity and their
volatile components were analysed.
2.3. Analysis and identification of volatile constituents
GC (GC-17A V3) and GC-MS (QP5050, Shimadzu
Co., Kyoto, Japan) using Supelcowax 10 fused silica
capillary column (60 m  0.25 mm; 0.25 mm film thick-
ness) were employed for the analysis. Helium was used
as the carrier gas at a flow rate of 1 ml/min. The GC
oven temperature was maintained at 50
C for 5 min,
then increased to 230
C at the rate of 2
C/min and held
for 10 min.The temperature of the injector was 250
C
and that of the FID detector was 260
C. The GC split
ratio was 1:60 and 0.5 ml of the extract was injected per
GC run. The mass spectra ranged from m/e 28 to 400
and the ionizing voltage used was 70 eV. Extracts
components were identified by comparing the spectra
obtained with a mass spectrum library (Wiley NBS 139),
and by comparing the GC retention indices against
known standards.
2.4. Antibacterial activities of SDE extracts
The antibacterial activities of the SDE extracts were
determined using a Bioscreen C Microbiology Reader
(Labsystem, Helsinki, Finland). Bioscreen C is a
computer-controlled shake–incubator–reader (Flower,
2001)
The extracts of P. densiflora leaves (4.38 ml from 100 g
of fresh leaves) were obtained by completely evaporating
off the diethyl ether with nitrogen. They were then
resuspended in 0.5 ml water containing 10% Tween 80
(v/v) (Showa Chemical Co. Ltd., Tokyo, Japan).
Extracts were sterilized by passing them through a
membrane filter (0.2 mm) (Naigre et al., 1996; Kim and
Shin, 2004). To determine antibacterial activity, 0.06 ml
of bacteria (105–106 cfu/ml) were incubated in 5.82 ml
media and 0.12 ml of either sterilized extract or the
control containing 10% Tween 80. In addition, anti-
bacterial experiments were conducted using different
concentrations (2, 4, 8, or 10% (v/v) in 10% Tween 80
(v/v)) of the SDE extracts. Aliquots of these cultures
(0.3 ml) were dispensed into Bioscreen C wells and
incubated as described in the Material and methods
section. The optical densities (600 nm) of the media were
measured every 12 h for 3 days using Tween 80 as
control.
2.5. Measurement of adenosine triphosphate (ATP)
Luciferase–luciferin (Sigma Chemical Co., Missouri,
USA) stock was prepared by dissolving the luciferase-
luciferin reagent in 5.0 ml of 25 mm 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) (pH 7.4) and
stored at –20
C. Just before use, 500 ml of stock solution
was mixed with 1.0 ml of HEPES. ATP was mixed with
luciferase–luciferin stock and used as a standard.
 ARTICLE IN PRESS
Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45
B. cereus grown in nutrient broth medium for 12 h at
30
C was centrifuged for 5 min at 2000 rpm. Cell pellets
were retained, and residual culture supernatants dis-
carded. The microbial cell suspension was neutralized to
pH 7.0 with 100 mm glycine buffer (pH 7.0) or 1 m
glycine buffer (pH 12.6) and then centrifuged. The cell
suspension was divided into two treatment groups. For
the control group, 500 ml of the cell suspension was
placed in a centrifugal tube and adjusted the pH with
100 mm glycine buffer. For P. densiflora treatment,
100 mm glycine buffer and 10 ml SDE extracts were
added to 500 ml cell suspension. Both treatments were
reacted for 30 min at 37
C, centrifuged for 30 s at
7000 rpm, and placed on ice to terminate the reaction.
The ATP concentration of the supernatant, which
represents the exterior of the cells, was determined using
a luminometer (Lumac LB 9507, EG&G Berthold,
Germany) after adding 80 ml of 100 mm glycine buffer
to 20 ml of supernatant and 100 ml of the prepared
luciferase. The ATP concentration of the cell suspen-
sion, representing residual cells, was determined by
adding 100 ml of the cell suspension to 2% Triton-X100
of 25 mm HEPES in a versatile homogenizer (HD-S,
Hanil Ind. Co., Korea) for 1 min at 50/60 Hz until the
microorganisms had been ruptured. Subsequently,
100 ml of luciferase was added to the mixture, and the
ATP concentration was measured using a luminometer.
3. Results and discussion
3.1. Antibacterial activity of the extracts obtained using
different SDE conditions
The antibacterial activities of the extracts of P.
densiflora needles obtained by SDE over 0.5, 1.0, 1.5,
and 2.0 h at pH 3.6 are shown in Table 1.
The extracts of P. densiflora leaves showed strong
growth inhibitory effects on E. coli O157:H7, and the
overall antibacterial activity against the microorganisms
Table 1
Antibacterial activity of the volatile essential oil from Pinus densiflora versus SDE extraction time
Micro-organisms Extraction time (pH 3.6)
0.5 h
a
Bacillus cereus ATCC 11778 21.971.4
Salmonella Typhimurium ATCC 14028 8.770.7
Vibrio parahaemolyticus ATCC 33844 16.671.1
Listeria monocytogenes ATCC 19111 14.270.8
Staphylococcus aureus ATCC 25923 22.771.2
Escherichia coli O157:H7 ATCC 43894 32.472.1
A: Total area of growth curve of sample with only 10% Tween 80 by Bioscreen C for a 72 h incubation.
B: Total area of growth curve of treated sample by Bioscreen C for a 72 h incubation.
Mean7standard deviation (n ¼ 3).
a
Growth inhibition rate (%)=100–(B/A  100).
39
tested tended to increase with increased extraction time.
Seo et al. (1996) reported that the antibacterial activity
of the extract of mustard leaves was weak initially, and
increased considerably after 12 h of extraction, reached a
maximum at 24 h of extraction, and thereafter remained
constant. Our data support this result. The antibacterial
activity of P. densiflora extracts increased up to 1.5 h of
extraction and remained constant thereafter. In the later
experiments, 1.5 h was selected as the extraction time.
Moreover, prolonged heat treatment can break down
the effective volatile components (Kim et al., 1982).
3.2. The compositions of the volatile components changed
with extraction time
It has been reported that the volatile components of
extracts obtained by SDE may vary with extraction time
(Au-Yeung and MacLeod, 1981).
The volatile components of P. densiflora leaf extracts
are shown in Table 2. As the extraction time increased,
the number of volatile components also increased, 29, 33,
36, and 38 components were identified at the SDE times
of 0.5, 1.0, 1.5, and 2.0 h, respectively. The main volatile
components identified in P. densiflora leaf extract were a-
ocimene (24.5–29.3%, peak area), sabinene (9.9–10.9%),
b-myrcene (9.6–11.0%), b-caryophyllene (8.0–10.4%), b-
cadinene (7.3–10.2%), a-terpinolene (4.9–6.3%), b-pinene
(4.1–5.1%), and 2-hexanal (3.1–4.5%). These compo-
nents accounted for 78.0–81.7% of the total peak area
observed, but the peak area of these components did not
show constant tendency with extraction time.
Kang et al. (1996) reported that the main components
of P. densiflora hexane extract were a-pinene (12.4%), b-
thugene (5.4%), trans-caryophyllene (4.8%), b-myrcene
(3.4%) and b-cubebene (3.1%), and that those of the
SDE extract were b-cubebene (11.4%), trans-caryophyl-
lene (11.0%), 2-hexenal (9.3%), t-muurolol (7.8%), and
d- cadinene (7.09%). However, Woo et al. (1999)
reported that the main components of pine twig extracts
obtained by supercritical fluid extraction or by the SDE
1.0 h 1.5 h 2.0 h
30.072.1 45.372.5 47.571.5
16.470.7 29.171.3 35.171.0
20.371.3 28.571.2 30.970.7
17.671.2 19.170.5 21.371.5
26.771.7 29.670.6 33.671.2
43.572.4 46.471.4 47.271.1
 ARTICLE IN PRESS
40 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45

Table 2
Volatile components of Pinus densiflora versus SDE extraction time at pH 3.6

Peak No. Components RIa Peak area (%)b IMc

0.5 h 1.0 h 1.5 h 2.0 h

1 ethyl alcohol 4.197 0.970.1 0 1.470.2 0.470.1 A, B

2 a-pinene 5.523 0.970.1 0.870.1 0.670.1 0.870.1 A, B
3 trans-a-ocimene 6.000 29.171.3 24.570.2 29.370.2 25.870.2 A
4 camphene 7.004 3.070.1 2.670.1 2.870.1 2.770.1 A
5 n-hexanal 7.515 0.470.0 0.270.0 0.770.1 0.370.1 A, B
6 b-pinene 8.317 4.870.1 4.170.1 4.370.3 5.170.2 A, B
7 b-phellandrene 8.736 0.370.2 0.270.1 0.270.1 0.270.0 A
8 1-pentene-3-ol 10.190 0.570.0 0.370.0 0.470.1 0.370.0 A, B
9 b-myrcene 10.629 11.070.1 10.070.1 9.670.2 10.370.1 A
10 limonene 12.028 2.770.1 2.670.2 2.570.1 2.77o.1 A
11 sabinene 12.609 10.770.2 9.970.3 10.970.2 10.870.2 A
12 2-hexanal 13.110 3.670.1 3.370.1 4.570.1 3.170.1 A
13 a –terpinolene 16.369 6.170.1 6.070.1 4.970.1 6.370.2 A
14 cis-3-hexen-1-ol 22.224 0 0 0.170.0 0 A, B
15 a-copaene 28.432 0.270.0 0.270.0 0.170.0 0.270.0 A, B
16 b-bourbonene 30.072 0 0.270.1 0 0.170.0 A
17 junipene 32.965 0 0.270.1 0.270.0 0.270.0 A
18 bornyl acetate 34.168 0.570.1 0.770.1 1.570.1 0.770.1 A
19 b-caryophyllene 35.051 8.570.2 10.470.1 8.070.2 8.770.3 A
20 b-elemene 35.595 0.270.0 0.370.0 0.470.1 1.970.1 A
21 a-humulene 39.412 1.270.1 1.670.1 1.370.0 1.370.1 A, B
22 unknown (M.W. 204) 40.686 0.570.1 0.770.1 0.470.1 0.670.1 A
23 a-terpineol 40.712 0.270.1 0 0.170.0 0 A, B
24 borneol 41.545 0 0.670.1 1.470.1 1.270.1 A
25 b-cadinene 42.166 7.970.1 10.270.2 7.370.1 7.970.3 A
26 b-selinene 42.463 0.470.1 0.570.1 0.370.0 0.470.1 A
27 a-muurolene 42.936 0.370.0 0.470.0 0.270.1 0.370.1 A
28 germacrene B 43.395 1.470.1 1.870.1 1.170.1 1.470.2 A
29 d-cadinene 44.898 2.570.1 3.770.1 1.370.1 2.470.3 A, B
30 unknown (M.W. 204) 55.198 0 0 0.270.0 0 A
31 patchulane 57.182 0 0 0 0.170.0 A
32 d-cadinene 61.162 0.370.0 0.270.0 0.170.0 0.170.0 A
33 spathulenol 64.889 0 0.270.0 0 0.270.0 A
34 torreyol 67.310 0 0.270.1 0.270.0 0.370.0 A
35 juniper camphor 68.110 tr 0.370.1 0.370.0 0.470.0 A
36 unknown (M.W. 226) 68.703 0 0 0.170.0 0.170.0 A
37 d-cadinol 70.362 0.370.1 0.770.1 0.870.1 1.070.1 A
38 biformene 70.906 0 0 0 0.170.0 A
39 sandaracopimar-15-ene-8, b-acetate 76.850 0.270.0 0.470.0 0.770.1 0.470.0 A
40 unknown (M.W. 256) 77.289 0 0 0.170.0 0.170.0 A
41 aromadendrene 86.195 0.370.0 0.470.1 0 0.570.1 A
total 98.9 98.4 98.3 99.4

Mean7standard deviation (n ¼ 3).

a
RI: retention index.
b
Peak area (%) on the gas chromatogram.
c
IM: identification mode. Components identified by GC-Mass are designated as A, and by retention index of authentic compounds are designated
as B.

method were limonene (32.6–43.4%), b-pinene (10.8–
19.1%), b-myrcene (11.5–17.3%), and a-pinene (5.3–
12.0%). In addition, Koukos et al. (2000) reported that
the main components of Pinus peuce needles similarly
extracted were a-pinene (23.1%), b-pinene (22.0%),
citronellol (13.4%), bornyl acetate (9.8%), b-phellan-
drene (6.8%), camphene (5.5%), and b-caryophyllene
(3.1%). Lee et al. (2002) reported that the main
components of pine needles by solid phase microextrac-
tion were b-pinene (30.7%), camphene (23.8%), germa-
crene D (19.4%), a-terpinene (6.8%), and b-
caryophyllene (4.6%), and by dynamic headspace
analysis the components were bornyl acetate (33.0%),
a-cubebene (16.8%), terpinolene (8.7%), and a-phellan-
drene (5.5%). Thus, we believe that observed differences
between the main components and peak areas of P.
densiflora extracts were based upon variations in collec-
tion time, collection place, and extraction conditions.
 ARTICLE IN PRESS
Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45 41

Table 3
Antibacterial activity of the volatile essential oil from Pinus densiflora versus the pH of SDE dispersion medium

Micro-organisms Extraction pH (1.5 h)

pH 3.6 pH 4.6 pH 5.6 pH 6.6

a
Bacillus cereus ATCC 11778 43.571.5 38.271.8 24.370.3 21.372.4
Salmonella Typhimurium ATCC 14028 29.172.4 26.171.7 19.371.7 13.670.9
Vibrio parahaemolyticus ATCC 33844 28.571.5 29.270.7 23.771.6 16.271.1
Listeria monocytogenes ATCC 19111 19.171.0 19.371.0 18.971.8 16.571.4
Staphylococcus aureus ATCC 25923 29.671.9 29.771.6 22.371.0 14.271.8
Escherichia coli O157:H7 ATCC 43894 46.472.4 49.772.9 47.371.3 43.871.4

Mean7standard deviation (n ¼ 3).

a
See footnote in Table 1.

Table 4
Volatile components of Pinus densiflora versus the pH of the dispersion medium in 1.5 h SDEs

Peak no. Components RIa Peak area (%)b IMc

pH 3.6 pH 4.6 pH 5.6 pH 6.6

1 Ethyl alcohol 4.197 1.470.2 0.370.0 0.670.1 0.570.1 A, B

2 a-pinene 5.523 0.670.1 0.870.1 0.570.0 0.870.1 A, B
3 trans-a-ocimene 6.000 29.370.2 26.170.3 24.770.3 25.270.2 A
4 Camphene 7.004 2.870.1 2.870.1 2.570.1 2.670.2 A
5 n-Hexanal 7.515 0.770.1 0.470.1 0.370.1 0.370.1 A, B
6 b-Pinene 8.317 4.370.3 4.870.1 4.670.2 4.470.1 A, B
7 b-Phellandrene 8.736 0.270.1 0.370.0 0.470.1 0.470.1 A
8 1-Pentene-3-ol 10.190 0.470.1 0.470.1 0.370.1 0.370.0 A, B
9 b-myrcene 10.629 9.670.2 10.370.2 9.270.2 9.470.1 A
10 limonene 12.028 2.570.1 2.770.3 2.470.1 2.470.1 A
11 sabinene 12.609 10.970.2 11.170.2 10.370.1 10.670.3 A
12 2-hexanal 13.110 4.570.1 4.270.1 3.670.1 3.170.1 A
13 a-terpinolene 16.369 4.970.1 6.270.2 5.470.2 5.570.2 A
14 cis-3-hexen-1-ol 22.224 0.170.0 0.270.0 0.270.0 0.270.0 A, B
15 a-copaene 28.432 0.170.0 0.270.0 0.270.0 0.270.0 A, B
16 b-bourbonene 30.072 0 0.170.0 0.170.0 0.170.0 A
17 junipene 32.965 0.270.0 0.270.0 0.270.0 0.270.0 A
18 bornyl acetate 34.168 1.570.1 0.870.1 0.770.1 0.670.1 A
19 b-caryophyllene 35.051 8.070.2 7.270.2 7.670.1 8.570.3 A
20 b-elemene 35.595 0.470.1 0.270.0 1.070.1 0.270.1 A
21 a-humulene 39.412 1.370.0 1.270.1 1.370.1 1.370.1 A, B
22 unknown (M.W. 204) 40.686 0.470.1 0.570.1 0.370.0 0.370.1 A
23 a-terpineol 40.712 0.170.0 0 0.270.0 0.270.0 A, B
24 borneol 41.545 1.470.1 0.370.0 0.270.0 0.170.0 A
25 b-cadinene 42.166 7.370.1 8.070.1 9.170.2 9.170.2 A
26 b-selinene 42.463 0.370.0 0.470.1 0.170.0 0.470.0 A
27 a-muurolene 42.936 0.270.1 0.370.0 0.370.0 0.370.0 A
28 germacrene B 43.395 1.170.1 1.470.1 1.870.2 1.970.1 A
29 d-cadinene 44.898 1.370.1 2.770.1 2.970.1 2.870.2 A, B
30 unknown (M.W. 204) 55.198 0.270.0 0 0.270.0 0.270.0 A
31 patchulane 57.182 0 0.270.0 0.270.0 0.270.0 A
32 d-cadinene 61.162 0.170.0 0.270.0 0.970.0 1.170.2 A
33 spathulenol 64.889 0 0.370.0 0.370.0 0.370.0 A
34 torreyol 67.310 0.270.0 0.370.0 0.470.1 0.470.0 A
35 juniper camphor 68.110 0.370.0 0.470.0 0.470.0 0.470.1 A
36 unknown (M.W. 222) 68.703 0.170.0 0.170.0 0.170.0 0.170.0 A
37 d-cadinol 70.362 0.870.1 0.970.0 1.070,1 1.070.1 A
38 biformene 70.906 0 0.170.0 0.270.0 0.270.0 A
39 sandaracopimar-15-ene-8, b-acetate 76.850 0.770.1 0.470.0 0.770.1 0.770.1 A
40 unknown (M.W. 256) 77.289 0.170.0 0.170.0 0.170.0 0.170.0 A
41 aromadendrene 86.195 0 0.670.0 1.170.1 1.570.2 A
total 98.3 97.7 96.6 98.1

Mean7standard deviation (n ¼ 3).

a,b,c
See footnote in Table 2.
 ARTICLE IN PRESS
42 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45

3.3. Effect of extraction pH on antibacterial activity Similarly, we observed that the number of volatile
compounds varied with pH and we identified 36, 39, 41,
Table 3 shows the antibacterial activity of the and 41 compounds at pH 3.6 (control), 4.6, 5.6, and 6.6,
extracts prepared for 1.5 h at pH 3.6 (control), 4.6, respectively (Table 4). Choi and Lee (1996) reported that
5.6, or 6.6. The overall antibacterial activity against the number of volatile compounds of Capsella bursa-
these strains tended to increase as the extraction pH pastoris was affected by the medium pH, and they
decreased. The antibacterial activities of P. densiflora identified 10, 23, 51, and 21 compounds at pH values of
extracts against B. cereus, S. Typhimurium, V. para- 3, 5, 7, and 9, respectively, which is in line with the
haemolyticus, and S. aureus at pH 3.6 (unadjusted) results of the present study.
were approximately twice those at pH 6.6. However, The peak area of the main volatile compound, a-
the activity on E. coli O157:H7 was unaffected by ocimene, was 29.3% of the total peak area when
extraction pH. extracted at pH 3.6, but this decreased to 24.7–26.1%
at pH 4.6–6.6. In addition, the peak area of 2-hexanal
3.4. Effect of extraction pH on the volatile compositions was 4.5% at pH 3.6, but this reduced to 3.1% at pH 6.6.
of the extracts However, the peak area of b-cadinene tended to increase
with increased extraction pH. The levels of b-pinene, b-
It has been reported that for the SDE method, the myrcene, sabinene, a-terpinolene, and b-caryophyllene
composition of volatile components is affected by salt were unaffected by the extraction pH. Thus the
concentration (Ebeler et al., 1988) and the pH (Schultz extraction efficacies of some compounds appears to be
et al., 1977; Bredie et al., 2002) of the dispersion affected by pH whereas others are unaffected. Studies
medium. on volatile flavors in the extrusion cooking of wheat

Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028

1.2 1.2
Optical density (600 nm).
Optical density (600 nm).

1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Incubation time (hr) Incubation time (hr)

Vibrio parahaemolyticus ATCC 33844 Listeria monocytogenes ATCC 19111

1.2 1.2
Optical density (600 nm).

Optical density (600 nm).

1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Incubation time (hr) Incubation time (hr)

Staphylococcus aureus ATCC 25923 Escherichia coli O157:H7 ATCC 43894

1.2 1.4
Optical density (600 nm).

Optical density (600 nm).

1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2 0.2
0.0 0.0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Incubation time (hr) Incubation time (hr)

Fig. 1. Antibacterial activities of volatile oil obtained by 1.5 h SDE at pH 3.6 from Pinus densiflora against several foodborne micro-organisms.
 ARTICLE IN PRESS
Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45 43

flour (Bredie et al., 2002) and on volatile flavor 0.7

ATP concentration (µM)

components of Capsella bursa-pastoris (Choi and Lee, 0.6
1996) concur with the results of the present study.
0.5
Lis-Balchin et al. (1998) reported that a direct or
0.4
inverse relationship between the antimicrobial activity
and the chemical composition of 105 commercial 0.3
essential oils was showed with the type of main 0.2
components. However, in the present study no consis- 0.1
tent relationship was observed between antibacterial 0.0
activity and the chemical composition of P. densiflora Control P. densiflora
(A) Treatments
extract with respect to extraction conditions.

3.5. Effect of SDE extract concentration on antibacterial 0.7

ATP concentration (µM)

activity 0.6
0.5
Fig. 1 shows the antibacterial activities of the SDE 0.4
extracts of P. densiflora, which were measured by 0.3
varying the concentrations of SDE extracts (i.e., 2, 4, 0.2
8, 10%, v/v).
0.1
The growths of B. cereus, S. Typhimurium, and V.
0.0
parahaemolyticus were slightly inhibited by 2% or 4% of Control P. densiflora
P. densiflora extract, and the addition of 8% or 10% of (B) Treatments
the extract inhibited its growth for up to 48 or 72 h,
Fig. 2. Adenosine triphosphate (ATP) concentration in cell pellets (A)
respectively. The growth of L. monocytogenes was and culture supernatant (B) of Bacillus cereus ATCC 11778 treated
significantly inhibited by 8% or 10% of the extract. with the SDE extracts of Pinus densiflora.
The antibacterial effect of 2% or 4% of the extract on E.
coli O157:H7 was weak. The antibacterial activity of 8%
or 10% extracts on E. coli O157:H7 lasted for 24 h or
60 h, respectively. 0.7
Cho et al. (1999b) reported that the methanol extracts
ATP concentration (µM)

0.6
of P. densiflora needles have weak growth inhibitory
0.5
effects on S. aureus and E. coli. However, Kim et al.
(2000) reported that the butanol and water layer of the 0.4
methanol extracts of P. densiflora needles strongly 0.3
inhibited S. Typhimurium, L. monocytogenes, E. coli
0.2
O157:H7, and S. aureus. In addition, Shin et al. (1997)
reported that the growth of S. aureus, but not of E. coli, 0.1
was inhibited by the SDE extract of P. densiflora leaves. 0.0
These results indicate that the antibacterial activity of P. Control P. densiflora
densiflora is depend on the micro-organisms type, which (A) Treatments
agrees with our findings.
0.7
3.6. Measurement of ATP
ATP concentration (µM)

0.6

Figs. 2 and 3 show the intracellular- and extracellular 0.5

ATP concentration in cell pellets and in the culture 0.4
medium of B. cereus and S. Typhimurium treated with 0.3
the SDE extracts of P. densiflora. In the case of treating
0.2
B. cereus (Fig. 2) with the SDE extracts of P. densiflora,
the concentration of intracellular ATP reduced to 0.1
0.235 mm from the control level of 0.618 mm (Fig. 2A). 0.0
The ATP concentration in culture supernatants of P. Control P. densiflora
densiflora treatment was 0.383 mm (Fig. 2B), which was (B) Treatments
6.2 times the control level of 0.062 mm. Fig. 3. Adenosine triphosphate (ATP) concentration in cell pellets (A)
A similar tendency was showed in S. Typhimurium. and culture supernatant (B) of Salmonella Typhimurium ATCC 14028
The concentration of intracellular ATP reduced to treated with the SDE extracts of Pinus densiflora.
 ARTICLE IN PRESS
44 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 37–45
0.165 mm from a control level of 0.595 mm, which was
relatively low compared with that in B. cereus (Fig. 3A).
The extracellular ATP concentration of cells treated
with P. densiflora was 0.469 mm (Fig. 3B), which was 7.2-
fold that of the control level of 0.065 mm. These results
indicate that intracellular ATP reduced while super-
natant ATP increased following treatment with the SDE
extracts of P. densiflora. Therefore, we believe that the
since the cell contents effused out of the cells due to an
unknown mechanism, more extracellular ATP was
detected than in normal cells. These results are
consistent with those of previous experiments, which
found that the growth inhibitory effect of a 2% extract
of P. densiflora on S. Typhimurium was relatively higher
than that on B cereus (Fig. 1).
Do and La (1996) reported that in the presence of
nisin (3–12 mg/ml), intracellular ATP and the number of
L. monocytogenes Scott A reduced rapidly during the
first hour of treatment at 35
C, whereas extracellular
ATP increased. Furthermore, Ahn et al. (2001) reported
that a shape change in the intracellular shape or the
disruption of the cell wall leads to the death of L.
monocytogenes treated with allyl isothiocyanate. This
finding was similar to that observed in our study, which
showed a tendency for the ATP of B. cereus and S.
Typhimurium to reduce after treatment with volatile
antimicrobial components.
In summary, by using the described ATP biolumines-
cence assay, we found that intracellular ATP effuses out
of cells by an unknown mechanism, and consequently,
that the bacteria died. However, further research is
needed to elucidate the mechanism of this biological
inactivation.
Acknowledgements
This research was supported by Research Center for
Industrial Development of Biofood Materials in Chon-
buk National University, Chonju, Korea. The center is
designated as a Regional Research Center appointed by
the Korea Science and Engineering Foundation (KO-
SEF), Jeollabuk-do Provincial Government and Chon-
buk National University.
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