ARTICLE IN PRESS

FOOD MICROBIOLOGY
Food Microbiology 22 (2005) 3745

www.elsevier.nl/locate/jnlabr/yfmic

Volatile components and antibacterial effects of pine needle (Pinus densiora S. and Z.) extracts
Yong-Suk Kima, Dong-Hwa Shinb,*
b a Research Center for Industrial Development of BioFood Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea 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 densiora S. and Z., by simultaneous steam distillation and solvent extraction (SDE), were examined on six foodborne bacteria using Bioscreen C (a computer-controlled shakeincubator reader). The SDE extracts of P. densiora 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. densiora 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 densiora; Antibacterial activity; Volatile components; SDE; Foodborne bacteria

1. Introduction 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 densiora 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 (0.31.3%), which contains a-pinene, bpinene, 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: +82-63-2702572. E-mail address: dhshin@moak.chonbuk.ac.kr (D.-H. Shin). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.05.002

medical affects of the needles are strongest in the winter, 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. densiora 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 eld, we examined the antibacterial effects of the essential oil of the leaves of P. densiora extracted under different

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38 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 3745

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. densiora were determined and related to observed bactericidal effects of SDE on several bacteria. Specically, 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 limitedstorage instant foods.

2.3. Analysis and identication 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 lm thickness) were employed for the analysis. Helium was used as the carrier gas at a ow 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 identied 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 shakeincubatorreader (Flower, 2001) The extracts of P. densiora 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 lter (0.2 mm) (Naigre et al., 1996; Kim and Shin, 2004). To determine antibacterial activity, 0.06 ml of bacteria (105106 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, antibacterial 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) Luciferaseluciferin (Sigma Chemical Co., Missouri, USA) stock was prepared by dissolving the luciferaseluciferin 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 luciferaseluciferin stock and used as a standard.

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 parahaemolyticus (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. densiora 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 LikensNickerson 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 ask. 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 ow. 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.

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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 discarded. 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. densiora 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 suspension, 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.

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. densiora 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. densiora 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 identied at the SDE times of 0.5, 1.0, 1.5, and 2.0 h, respectively. The main volatile components identied in P. densiora leaf extract were aocimene (24.529.3%, peak area), sabinene (9.910.9%), b-myrcene (9.611.0%), b-caryophyllene (8.010.4%), bcadinene (7.310.2%), a-terpinolene (4.96.3%), b-pinene (4.15.1%), and 2-hexanal (3.14.5%). These components accounted for 78.081.7% 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. densiora hexane extract were a-pinene (12.4%), bthugene (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-caryophyllene (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 uid extraction or by the SDE

3. Results and discussion 3.1. Antibacterial activity of the extracts obtained using different SDE conditions The antibacterial activities of the extracts of P. densiora 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. densiora 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 densiora versus SDE extraction time Micro-organisms Extraction time (pH 3.6) 0.5 h Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028 Vibrio parahaemolyticus ATCC 33844 Listeria monocytogenes ATCC 19111 Staphylococcus aureus ATCC 25923 Escherichia coli O157:H7 ATCC 43894 21.971.4 8.770.7 16.671.1 14.270.8 22.771.2 32.472.1
a

1.0 h 30.072.1 16.470.7 20.371.3 17.671.2 26.771.7 43.572.4

1.5 h 45.372.5 29.171.3 28.571.2 19.170.5 29.670.6 46.471.4

2.0 h 47.571.5 35.171.0 30.970.7 21.371.5 33.671.2 47.271.1

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 3). a Growth inhibition rate (%)=100(B/A 100).

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40 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 3745 Table 2 Volatile components of Pinus densiora versus SDE extraction time at pH 3.6 Peak No. Components RIa Peak area (%)b 0.5 h 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 ethyl alcohol a-pinene trans-a-ocimene camphene n-hexanal b-pinene b-phellandrene 1-pentene-3-ol b-myrcene limonene sabinene 2-hexanal a terpinolene cis-3-hexen-1-ol a-copaene b-bourbonene junipene bornyl acetate b-caryophyllene b-elemene a-humulene unknown (M.W. 204) a-terpineol borneol b-cadinene b-selinene a-muurolene germacrene B d-cadinene unknown (M.W. 204) patchulane d-cadinene spathulenol torreyol juniper camphor unknown (M.W. 226) d-cadinol biformene sandaracopimar-15-ene-8, b-acetate unknown (M.W. 256) aromadendrene total 4.197 5.523 6.000 7.004 7.515 8.317 8.736 10.190 10.629 12.028 12.609 13.110 16.369 22.224 28.432 30.072 32.965 34.168 35.051 35.595 39.412 40.686 40.712 41.545 42.166 42.463 42.936 43.395 44.898 55.198 57.182 61.162 64.889 67.310 68.110 68.703 70.362 70.906 76.850 77.289 86.195 0.970.1 0.970.1 29.171.3 3.070.1 0.470.0 4.870.1 0.370.2 0.570.0 11.070.1 2.770.1 10.770.2 3.670.1 6.170.1 0 0.270.0 0 0 0.570.1 8.570.2 0.270.0 1.270.1 0.570.1 0.270.1 0 7.970.1 0.470.1 0.370.0 1.470.1 2.570.1 0 0 0.370.0 0 0 tr 0 0.370.1 0 0.270.0 0 0.370.0 98.9 1.0 h 0 0.870.1 24.570.2 2.670.1 0.270.0 4.170.1 0.270.1 0.370.0 10.070.1 2.670.2 9.970.3 3.370.1 6.070.1 0 0.270.0 0.270.1 0.270.1 0.770.1 10.470.1 0.370.0 1.670.1 0.770.1 0 0.670.1 10.270.2 0.570.1 0.470.0 1.870.1 3.770.1 0 0 0.270.0 0.270.0 0.270.1 0.370.1 0 0.770.1 0 0.470.0 0 0.470.1 98.4 1.5 h 1.470.2 0.670.1 29.370.2 2.870.1 0.770.1 4.370.3 0.270.1 0.470.1 9.670.2 2.570.1 10.970.2 4.570.1 4.970.1 0.170.0 0.170.0 0 0.270.0 1.570.1 8.070.2 0.470.1 1.370.0 0.470.1 0.170.0 1.470.1 7.370.1 0.370.0 0.270.1 1.170.1 1.370.1 0.270.0 0 0.170.0 0 0.270.0 0.370.0 0.170.0 0.870.1 0 0.770.1 0.170.0 0 98.3 2.0 h 0.470.1 0.870.1 25.870.2 2.770.1 0.370.1 5.170.2 0.270.0 0.370.0 10.370.1 2.77o.1 10.870.2 3.170.1 6.370.2 0 0.270.0 0.170.0 0.270.0 0.770.1 8.770.3 1.970.1 1.370.1 0.670.1 0 1.270.1 7.970.3 0.470.1 0.370.1 1.470.2 2.470.3 0 0.170.0 0.170.0 0.270.0 0.370.0 0.470.0 0.170.0 1.070.1 0.170.0 0.470.0 0.170.0 0.570.1 99.4 A, A, A A A, A, A A, A A A A A A, A, A A A A A A, A A, A A A A A A, A A A A A A A A A A A A B B IMc

B B B

B B

B B

Mean7standard deviation (n 3). a RI: retention index. b Peak area (%) on the gas chromatogram. c IM: identication mode. Components identied by GC-Mass are designated as A, and by retention index of authentic compounds are designated as B.

method were limonene (32.643.4%), b-pinene (10.8 19.1%), b-myrcene (11.517.3%), 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-phellandrene (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%), germacrene D (19.4%), a-terpinene (6.8%), and bcaryophyllene (4.6%), and by dynamic headspace analysis the components were bornyl acetate (33.0%), a-cubebene (16.8%), terpinolene (8.7%), and a-phellandrene (5.5%). Thus, we believe that observed differences between the main components and peak areas of P. densiora extracts were based upon variations in collection time, collection place, and extraction conditions.

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Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 3745 Table 3 Antibacterial activity of the volatile essential oil from Pinus densiora versus the pH of SDE dispersion medium Micro-organisms Extraction pH (1.5 h) pH 3.6 Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028 Vibrio parahaemolyticus ATCC 33844 Listeria monocytogenes ATCC 19111 Staphylococcus aureus ATCC 25923 Escherichia coli O157:H7 ATCC 43894 Mean7standard deviation (n 3). a See footnote in Table 1. 43.571.5 29.172.4 28.571.5 19.171.0 29.671.9 46.472.4
a

41

pH 4.6 38.271.8 26.171.7 29.270.7 19.371.0 29.771.6 49.772.9

pH 5.6 24.370.3 19.371.7 23.771.6 18.971.8 22.371.0 47.371.3

pH 6.6 21.372.4 13.670.9 16.271.1 16.571.4 14.271.8 43.871.4

Table 4 Volatile components of Pinus densiora versus the pH of the dispersion medium in 1.5 h SDEs Peak no. Components RIa Peak area (%)b pH 3.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Ethyl alcohol a-pinene trans-a-ocimene Camphene n-Hexanal b-Pinene b-Phellandrene 1-Pentene-3-ol b-myrcene limonene sabinene 2-hexanal a-terpinolene cis-3-hexen-1-ol a-copaene b-bourbonene junipene bornyl acetate b-caryophyllene b-elemene a-humulene unknown (M.W. 204) a-terpineol borneol b-cadinene b-selinene a-muurolene germacrene B d-cadinene unknown (M.W. 204) patchulane d-cadinene spathulenol torreyol juniper camphor unknown (M.W. 222) d-cadinol biformene sandaracopimar-15-ene-8, b-acetate unknown (M.W. 256) aromadendrene total 4.197 5.523 6.000 7.004 7.515 8.317 8.736 10.190 10.629 12.028 12.609 13.110 16.369 22.224 28.432 30.072 32.965 34.168 35.051 35.595 39.412 40.686 40.712 41.545 42.166 42.463 42.936 43.395 44.898 55.198 57.182 61.162 64.889 67.310 68.110 68.703 70.362 70.906 76.850 77.289 86.195 1.470.2 0.670.1 29.370.2 2.870.1 0.770.1 4.370.3 0.270.1 0.470.1 9.670.2 2.570.1 10.970.2 4.570.1 4.970.1 0.170.0 0.170.0 0 0.270.0 1.570.1 8.070.2 0.470.1 1.370.0 0.470.1 0.170.0 1.470.1 7.370.1 0.370.0 0.270.1 1.170.1 1.370.1 0.270.0 0 0.170.0 0 0.270.0 0.370.0 0.170.0 0.870.1 0 0.770.1 0.170.0 0 98.3 pH 4.6 0.370.0 0.870.1 26.170.3 2.870.1 0.470.1 4.870.1 0.370.0 0.470.1 10.370.2 2.770.3 11.170.2 4.270.1 6.270.2 0.270.0 0.270.0 0.170.0 0.270.0 0.870.1 7.270.2 0.270.0 1.270.1 0.570.1 0 0.370.0 8.070.1 0.470.1 0.370.0 1.470.1 2.770.1 0 0.270.0 0.270.0 0.370.0 0.370.0 0.470.0 0.170.0 0.970.0 0.170.0 0.470.0 0.170.0 0.670.0 97.7 pH 5.6 0.670.1 0.570.0 24.770.3 2.570.1 0.370.1 4.670.2 0.470.1 0.370.1 9.270.2 2.470.1 10.370.1 3.670.1 5.470.2 0.270.0 0.270.0 0.170.0 0.270.0 0.770.1 7.670.1 1.070.1 1.370.1 0.370.0 0.270.0 0.270.0 9.170.2 0.170.0 0.370.0 1.870.2 2.970.1 0.270.0 0.270.0 0.970.0 0.370.0 0.470.1 0.470.0 0.170.0 1.070,1 0.270.0 0.770.1 0.170.0 1.170.1 96.6 pH 6.6 0.570.1 0.870.1 25.270.2 2.670.2 0.370.1 4.470.1 0.470.1 0.370.0 9.470.1 2.470.1 10.670.3 3.170.1 5.570.2 0.270.0 0.270.0 0.170.0 0.270.0 0.670.1 8.570.3 0.270.1 1.370.1 0.370.1 0.270.0 0.170.0 9.170.2 0.470.0 0.370.0 1.970.1 2.870.2 0.270.0 0.270.0 1.170.2 0.370.0 0.470.0 0.470.1 0.170.0 1.070.1 0.270.0 0.770.1 0.170.0 1.570.2 98.1 A, A, A A A, A, A A, A A A A A A, A, A A A A A A, A A, A A A A A A, A A A A A A A A A A A A B B IMc

B B B

B B

B B

Mean7standard deviation (n 3). a,b,c See footnote in Table 2.

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3.3. Effect of extraction pH on antibacterial activity Table 3 shows the antibacterial activity of the extracts prepared for 1.5 h at pH 3.6 (control), 4.6, 5.6, or 6.6. The overall antibacterial activity against these strains tended to increase as the extraction pH decreased. The antibacterial activities of P. densiora extracts against B. cereus, S. Typhimurium, V. parahaemolyticus, and S. aureus at pH 3.6 (unadjusted) were approximately twice those at pH 6.6. However, the activity on E. coli O157:H7 was unaffected by extraction pH. 3.4. Effect of extraction pH on the volatile compositions of the extracts It has been reported that for the SDE method, the composition of volatile components is affected by salt concentration (Ebeler et al., 1988) and the pH (Schultz et al., 1977; Bredie et al., 2002) of the dispersion medium.

Similarly, we observed that the number of volatile compounds varied with pH and we identied 36, 39, 41, and 41 compounds at pH 3.6 (control), 4.6, 5.6, and 6.6, respectively (Table 4). Choi and Lee (1996) reported that the number of volatile compounds of Capsella bursapastoris was affected by the medium pH, and they identied 10, 23, 51, and 21 compounds at pH values of 3, 5, 7, and 9, respectively, which is in line with the results of the present study. The peak area of the main volatile compound, aocimene, was 29.3% of the total peak area when extracted at pH 3.6, but this decreased to 24.726.1% at pH 4.66.6. In addition, the peak area of 2-hexanal was 4.5% at pH 3.6, but this reduced to 3.1% at pH 6.6. However, the peak area of b-cadinene tended to increase with increased extraction pH. The levels of b-pinene, bmyrcene, sabinene, a-terpinolene, and b-caryophyllene were unaffected by the extraction pH. Thus the extraction efcacies of some compounds appears to be affected by pH whereas others are unaffected. Studies on volatile avors in the extrusion cooking of wheat

Bacillus cereus ATCC 11778 Optical density (600 nm). 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 Incubation time (hr) 60 72 Optical density (600 nm). 1.2 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

Salmonella Typhimurium ATCC 14028

12

24 36 48 Incubation time (hr)

60

72

Vibrio parahaemolyticus ATCC 33844 Optical density (600 nm). Optical density (600 nm). 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 Incubation time (hr) 60 72 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

Listeria monocytogenes ATCC 19111

12

24 36 48 Incubation time (hr)

60

72

Staphylococcus aureus ATCC 25923 Optical density (600 nm). Optical density (600 nm). 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 Incubation time (hr) 60 72

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

Escherichia coli O157:H7 ATCC 43894

12

24 36 48 Incubation time (hr)

60

72

Fig. 1. Antibacterial activities of volatile oil obtained by 1.5 h SDE at pH 3.6 from Pinus densiora against several foodborne micro-organisms.

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Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 3745 43

our (Bredie et al., 2002) and on volatile avor components of Capsella bursa-pastoris (Choi and Lee, 1996) concur with the results of the present study. Lis-Balchin et al. (1998) reported that a direct or inverse relationship between the antimicrobial activity and the chemical composition of 105 commercial essential oils was showed with the type of main components. However, in the present study no consistent relationship was observed between antibacterial activity and the chemical composition of P. densiora extract with respect to extraction conditions. 3.5. Effect of SDE extract concentration on antibacterial activity Fig. 1 shows the antibacterial activities of the SDE extracts of P. densiora, which were measured by varying the concentrations of SDE extracts (i.e., 2, 4, 8, 10%, v/v). The growths of B. cereus, S. Typhimurium, and V. parahaemolyticus were slightly inhibited by 2% or 4% of P. densiora extract, and the addition of 8% or 10% of the extract inhibited its growth for up to 48 or 72 h, respectively. The growth of L. monocytogenes was signicantly inhibited by 8% or 10% of the extract. 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. Cho et al. (1999b) reported that the methanol extracts of P. densiora needles have weak growth inhibitory effects on S. aureus and E. coli. However, Kim et al. (2000) reported that the butanol and water layer of the methanol extracts of P. densiora needles strongly inhibited S. Typhimurium, L. monocytogenes, E. coli O157:H7, and S. aureus. In addition, Shin et al. (1997) reported that the growth of S. aureus, but not of E. coli, was inhibited by the SDE extract of P. densiora leaves. These results indicate that the antibacterial activity of P. densiora is depend on the micro-organisms type, which agrees with our ndings. 3.6. Measurement of ATP Figs. 2 and 3 show the intracellular- and extracellular ATP concentration in cell pellets and in the culture medium of B. cereus and S. Typhimurium treated with the SDE extracts of P. densiora. In the case of treating B. cereus (Fig. 2) with the SDE extracts of P. densiora, the concentration of intracellular ATP reduced to 0.235 mm from the control level of 0.618 mm (Fig. 2A). The ATP concentration in culture supernatants of P. densiora treatment was 0.383 mm (Fig. 2B), which was 6.2 times the control level of 0.062 mm. A similar tendency was showed in S. Typhimurium. The concentration of intracellular ATP reduced to

ATP concentration (M)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control P. densiflora Treatments

(A) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control (B)

ATP concentration (M)

P. densiflora Treatments

Fig. 2. Adenosine triphosphate (ATP) concentration in cell pellets (A) and culture supernatant (B) of Bacillus cereus ATCC 11778 treated with the SDE extracts of Pinus densiora.

0.7 ATP concentration (M) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control (A) Treatments P. densiflora

0.7 ATP concentration (M) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control (B) Treatments P. densiflora

Fig. 3. Adenosine triphosphate (ATP) concentration in cell pellets (A) and culture supernatant (B) of Salmonella Typhimurium ATCC 14028 treated with the SDE extracts of Pinus densiora.

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44 Y.-S. Kim, D.-H. Shin / Food Microbiology 22 (2005) 3745 Cho, J.E., Lee, M.J., Lee, Y.B., Yoon, J.R., 1999a. Comparisons of volatile compounds of Pinus densiora on kinds of extraction solvent and parts of Pinus. J. Korean Soc. Food Sci. Nutr. 28, 973979. Cho, S.H., Jeon, H.J., Han, Y.K., Yeon, S.H., Ahn, Y.J., 1999b. In vitro growth-inhibiting effects of leaf extracts from pinus species on human intestinal bacteria. Agric. Chem. Biotechnol. 42, 202204. Choi, H.S., Lee, M.S., 1996. The effect of dispersion medium on intensity of volatile avor components and recovery of essential oil from Capsella bursa-pastoris by steam distillation. Korean J. Food Sci. Technol. 28, 827833. Do, U., LA, S., 1996. Bioluminescence measurements of the antilisterial activity of nisin: comparison with ampicillin and streptomycin. J. Biolumin. Chemilumin. 11, 169173. Dongeuhak Institute, 1994. Dongeubogam (Original author; Hur, J.), p1329, p2216, p2794. Ryo-gang Pub. Co., Seoul, Korea. Ebeler, S.E., Pangborn, R.M., Jennings, W.G., 1988. Inuence of dispersion medium on aroma intensity and headspace concentration of menthone and isoamyl acetate. J. Agric. Food Chem. 36, 791796. Flower, A.M., 2001. Secg function and phospholipid metabolism in Escherichia coli. J. Bacteriol. 183, 20062012. Im, R.J., 1998. Flora Medica Coreana, Vol. 1. Part Modern Medicine. Agricultural Pub. House, Pyongyang, North Korea, pp. 6768. Jeon, H.J., Lee, K.S., Ahn, Y.J., 2001. Growth-inhibiting effects of constituents of Pinus densiora leaves on human intestinal bacteria. Food Sci. Biotechnol. 10, 403407. Jung, M.J., Choi, J.H., Chung, H.Y., Jung, J.H., Choi, J.S., 2001. A new C-methylated avonoid glycoside from Pinus densiora. Fitoterapia 72, 943945. Kang, S.K., Kang, K.H., Choi, O.J., Kim, Y.W., Kim, Y.D., 1996. Volatile avor compounds of Pinus densiora Sieb and Zucc according to extracting solvents and steam distillation method. Korean J. Diet. Cult. 11, 403408. Kim, K.R., Zlatkis, A., Park, J.W., Lee, U.C., 1982. Isolation of essential oil from tobacco by gas co-distillation/solvent extraction. Chromatographia 15, 559. Kim, K.Y., Davidson, P.M., Chung, H.J., 2000. Antimicrobial effectiveness of pine needle extract on food borne illness bacteria. J. Microbiol. Biotechnol. 10, 227232. Kim, Y.S., Shin, D.H., 2003. A reviewresearches on the volatile antimicrobial compounds from edible plants and their food application. Korean J. Food Sci. Technol. 35, 159165. Kim, Y.S., Shin, D.H., 2004. Volatile constituents from the leaves of Callicarpa japonica Thunb and their antibacterial activities. J. Agric. Food Chem. 52, 781787. Korea Food & Drug Administration, 1997. An Illustrated Guide to Medicinal Plants. Woojin Pub. Co, Seoul, Korea, p. 95. Koukos, P.K., Papadopoulou, K.I., Patiaka, D.Th., Papagiannopoulos, A.D., 2000. Chemical composition of essential oils from needles and twigs of balkan pine (Pinus peuce Grisebach) grown in northern Greece. J. Agric. Food Chem. 48, 12661268. Lee, M.J., Jung, E.J., Lee, S.J., Cho, J.E., Lee, Y.B., Cho, H.J., Yoon, J.R., 2002. Comparisons of volatile compounds extracted from Pinus densiora by headspace analysis. J. Korean Soc. Food Sci. Nutr. 31, 2631. Lim, Y.S., Park, K.N., Bae, M.J., Lee, S.H., 2001. Antimicrobial effects of ethanol extracts of Pinus densiora Sieb and Zucc on lactic acid bacteria. J. Korean Soc. Food Sci. Nutr. 30, 11581163. Lis-Balchin, M., Deans, S.G., Eaglesham, E., 1998. Relationship between bioactivity and chemical composition of commercial essential oils. Flavour Fragr. J. 13, 98104. Naigre, R., Kalck, P., Roques, C., Roux, I., Michel, G., 1996. Comparison of antimicrobial properties of monoterpenes and their carbonylated products. Planta Med. 62, 275277.

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. densiora was 0.469 mm (Fig. 3B), which was 7.2fold that of the control level of 0.065 mm. These results indicate that intracellular ATP reduced while supernatant ATP increased following treatment with the SDE extracts of P. densiora. 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. densiora on S. Typhimurium was relatively higher than that on B cereus (Fig. 1). Do and La (1996) reported that in the presence of nisin (312 mg/ml), intracellular ATP and the number of L. monocytogenes Scott A reduced rapidly during the rst 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 nding 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 bioluminescence 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 Chonbuk National University, Chonju, Korea. The center is designated as a Regional Research Center appointed by the Korea Science and Engineering Foundation (KOSEF), Jeollabuk-do Provincial Government and Chonbuk National University.

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