1740 Journal o f Food Protection, Vol. 77, No. 10, 2014, Pages 1740-1746 doi: 10.4315/0362-028X. JFP-14-014 Copyright © , International Association for Food Protection

Chemical Composition, Antibacterial Activity, and Mechanism of Action of the Essential Oil from Am om um kravanh WEN-RUI DIAO,1 LIANG-LIANG ZHANG,1 SAI-SAI FENG,2 A N D JIAN-GUO XU1-2* 'College o f Life Sciences and 2College o f Engineering, Shanxi Normal University, Linfen City 041004, People's Republic o f China MS 14-014: Received 12 January 2014/Accepted 27 May 2014

ABSTRACT Amomum kravanh is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of A. kravanh fruits was analyzed by gas chromatography-mass spectrometry, and 34 components were identified. 1,8-Cineole (68.42%) was found to be the major component, followed by a-pinene (5.71%), a-terpinene (2.63%), and P-pinene (2.41%). The results of antibacterial tests showed that the sensitivities to the essential oil of different foodbome pathogens tested were different based on the Oxford cup method, MIC, and MBC assays, and the essential oil exhibited the best antibacterial activity against Bacillus suhtilis, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. Growth in the presence of Amomum kravanh at the MIC, as measured by monitoring optical density over time, demonstrated that the essential oil was bacteriostatic after 12 h to both B. subtilis and E. coli. Observations of cell membrane permeability, cell constituent release assay, and transmission electron microscopy indicated that this essential oil may disrupt the cell wall and cell membrane permeability, leading to leakage of intracellular constituents in both B. subtilis and E. coli.

In recent years, the number of reported cases of infectious diseases, especially those caused by microbial contamination of foods, has increased dramatically through­ out the world (10, 24). The contamination of raw and/or processed foods with microflora can take place at various stages from production to sale, which is becoming one of the most important concerns of the food industry (26, 29). Some synthetic antimicrobial chemicals have been made to control microbial growth and to reduce the incidence of food poisoning and spoilage. However, consumers have become increasingly concerned about the side effects of synthetic antimicrobial chemicals and want safer materials for preventing and controlling pathogenic microorganisms in foods, which has led to research and use of “ naturally derived” antimicrobials (11, 27). Some natural substances of plant origin, especially of aromatic and medicinal plants, have good antimicrobial properties and have been used as preservatives for centuries (9, 22). One such possibility is the use of essential oils as antibacterial additives. Many studies have reported that the essential oils extracted from many species of plants have potent activity against microorganisms (8, 21, 25). Considering their excellent antimicrobial function, natural essential oils possess great potential as antimicrobial agents in food systems to increase the safety and shelf life of foods. Amomum kravanh Pierre ex Gagnep. (Zingiberaceae) is a tropical plant, widely distributed in Cambodia, Thailand, Vietnam, and South China. The fruits are used as spices * Author for correspondence. Tel: + 8 6 357 2051714; Fax: + 8 6 357 2051000; E-mail: [email protected].

throughout the world and are commonly used as a traditional Chinese medicine to treat stomach diseases and digestive disorders (31). Some studies have reported the chemical composition (28, 31-33) and have also indicated that plants of the genus Amomum showed antioxidant, antimicrobial (15,18), and anti-inflammatory activities (19). Although the chemical compositions and antimicrobial properties of the essential oil of A. kravanh have been studied, this information is still limited. Most importantly, to the best of our knowledge, no work has been reported on the mechanism of action of the essential oil of A. kravanh fruits on the growth of microorganisms. Therefore, the aim of the present study was to investigate the chemical composition and antibacterial activity on several foodbome pathogens of the essential oil from A. kravanh fruits and, further, to evaluate the possible mechanism of action responsible for the antibacterial activity against sensitive strains by assays of growth as monitored by optical density (OD) over time, cell membrane permeability, and cell constituent release, as well as make transmission electron microscopy (TEM) observations. MATERIALS AND METHODS Plant materials and chemicals. Fruits from A. kravanh, which were grown in Wanning County of Hainan Province, were obtained as commercial products from the local market in 2012. The sample was identified as the dry and the ripe fruits of A. kravanh by Professor Xuefeng Wu. Nutrient agar and nutrient broth were from Beijing Aoboxing Bio-tech Co. Ltd. (Beijing, People's Republic of China). Other chemicals used were all of analytical grade.

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ANTIBACTERIAL ACTIVITY OF THE ESSENTIAL OIL FROM A. KRAVANH

Microbial strains and culture. The in vitro antimicrobial activity of the essential oil was tested against six different microorganisms. The three gram-positive strains were Staphylo­ coccus aureus ATCC 25923, Staphylococcus alhus ATCC 8799, and Bacillus subtilis ATCC 6051. The three gram-negative bacteria were Salmonella enterica serovar Typhimurium ATCC 19430, Shigella dysenteriae CMCC (B) 51252, and Escherichia coli ATCC 25922. The strains were provided by the College of Life Sciences, Shanxi Normal University, and cultured at 37°C on nutrient agar or nutrient broth medium. Extraction of essential oil. The dried A. kravanh fruits were ground with a micro-plant grinding machine (Taisite Instruments, Tianjin, People's Republic of China) and hydrodistilled for 4 h using a Clevenger-type apparatus. The oil was separated from the water, dried over anhydrous sodium sulfate, and stored in tightly closed dark vials at 4°C until use. The essential oil was obtained as a light yellow transparent liquid and had the characteristic A. kravanh aroma. GC-flame ionization detection analysis. The essential oil was analyzed using a Hewlett-Packard 5890II gas chromatography (GC) system equipped with a flame ionization detector and HP-5 mass spectrometry (MS) capillary column (30 m by 0.25 mm; film thickness, 0.25 pm), with the injector and detector temperatures at 280 and 250°C, respectively. The oven temperature was pro­ grammed to rise from 60°C for 2 min to 200°C at a rate of 2°C/ min. Helium was the carrier gas, at a flow rate of 1 ml/min. A sample of 0.1 pi of the essential oil was injected manually, and the GC split ratio used was 1:50. The percentages of the constituents were calculated by electronic integration of flame ionization detection peak areas without the use of response factor correction. GC-MS analysis. The analysis of the essential oil was performed using a Hewlett-Packard 5890 II GC equipped with an HP-5 MS capillary column (30 m by 0.25 mm; film thickness, 0.25 pm) and an HP 5972 mass selective detector for the separation. The mass selective detector was operated in electron impact ionization mode with a mass scan range from m/z 50 to 550 at 70 eV. The GC conditions were the same as described above. The components were identified by using the National Institute of Standards and Technology mass spectral search program (version 2.0, National Institute of Standards and Technology) and mass spectra with published data. Antibacterial activity, (i) Oxford cup method. The Oxford cup assay was carried out according to the method described by Diao et al. (6) with minor modifications. The essential oil was dissolved in ethanol to prepare a 50% concentration, which was then sterilized by filtration through 0.22-pm Millipore filters. The bacterial strains were incubated at 37°C for 10 h on nutrient broth medium (the bacteria were incubated under the same conditions for the following tests). Two hundred microliters of suspension containing 1 x 10° CFU/ml of bacteria was spread on nutrient agar medium. Oxford cups (6 mm in diameter) were placed on the inoculated agar, and then 200 pi of essential oil was added with a micropipette. The diameter of the inhibition zone (DIZ) was measured after 24 h of incubation at 37°C. (ii) MIC and MBC assays. MIC and MBC were determined according to the method described by Diao et al. (6) with minor modifications. Briefly, a stock solution of the essential oil was prepared in ethanol. Two-fold serial dilutions of essential oil were filtered through 0.22-pm Millipore filters and prepared in sterile

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nutrient broth medium. To each tube, 50 pi of the exponentially growing bacterial cells was added into the medium, and the cell concentration in each medium was approximately 1 x 10 6 CFU/ ml. A control test was also performed containing inoculated broth supplemented with ethanol only. The maximum final concentration of ethanol in each medium was 2%, which did not affect the growth of the tested strains. The tubes were then incubated at 37°C for 24 h and examined for evidence of growth. The MIC was determined as the lowest concentration of the essential oil that demonstrated no visible growth. The MBC was determined as follows. After the determination of the MIC, 100-fold dilutions with antimicrobial-free nutrient broth of culture from each tube showing no turbidity were incubated at 37°C for 48 h. The MBC was the lowest concentration of the essential oil for which the culture showed no visible growth in the antimicrobial-free cultivation. All experiments were performed in triplicate. (iii) Growth as monitored by OD over time. The assay was performed according to the method described by Muroi and Kubo (20) with some modifications. The effects of different concentra­ tions (0.5 x MIC and 1 x MIC) of the essential oil on the growth of tested bacteria were studied. Fifty microliters of the essential oil filtered through 0.22-pm Millipore filters was added to 4.9 ml of the sterile nutrient broth medium and then mixed with 50 pi of a 10-h culture of test bacteria (1 x 10 RCFU/ml). A control test was also performed, using inoculated broth supplemented with ethanol only. The cultures were incubated at 37°C and shaken with a rotary shaker at 120 rpm. During the culture, seven samplings were carried out at 1,2, 3, 5, 7, 9, and 12 h, and the absorbance of each sample was measured at an OD of 600 nm (OD60o). Cell membrane permeability. The permeability of the cell membrane is expressed as the relative electrical conductivity, which was determined according to the method described by Kong et al. (14). After incubation at 37°C for 10 h, B. subtilis and E. coli cells were centrifuged at 3,214 x g for 10 min to form pellets. Then, the bacteria were washed with 5% glucose until their electrical conductivities were near to that of 5% glucose. The essential oil at three different concentrations (control, 1 x MIC, and 2 x MIC) was added to 5% glucose, and the electrical conductivities of the mixture were marked as L\. Then, different concentrations of the essential oils were added into the isotonic bacterial solution. After complete mixing, the samples were incubated at 37°C for 8 h, and then the conductivities were measured and marked as L2. The conductivity of bacteria in 5% glucose treated in boiling water for 5 min served as the control and was marked as L0. The conductivity of the culture medium was measured at selected time intervals. The permeability of the bacterial membrane was calculated according to the following formula: % relative electrical conductivity = 100 x (L2 —L\)/L0. Cell constituent release. The release of cell constituents into the supernatant was examined according to the method described by Rhayour et al. (23) with some modifications. Cells from the 50-ml working culture of tested microorganisms were collected by centrifugation for 15 min at 5,000 x g, washed three times, and resuspended in 0.1 M phosphate buffer solution (phosphatebuffered saline [PBS], pH 7.2). One hundred milliliters of cell suspension was incubated at 37°C under agitation for 4 h in the presence of the essential oil at three different concentrations (control, 1 x MIC, and 2 x MIC). Then, 25-ml amounts of samples were collected and centrifuged at 11,000 x g for 5 min. After that, the concentrations of proteins and reducing sugars in the supernatant were detennined according to the method described by

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TABLE 1. Chemical composition of essential oil from Amomum kravanh fruits RT (min)0

Compound

5.14 6.25 6.78 7.61 8.14 8.60 9.52 10.16 10.54 11.24 11.50 11.75 12.82 14.39 15.21 16.74 19.11 20.25 21.51 22.08 25.51 28.55 30.26 32.12 32.89 34.41 35.46 38.14 39.84 41.25 42.13 42.62 43.05 44.66

a-Thujene a-Pinene Camphene Sabinene P-Pinene P-Myrcene a-Phellandrene a-Terpinene 5-3-Carene p-Cymene 1,8-Cineole 3,7-Dimethyl-1,3,6-octatriene y-Terpincne a-Terpinolene Fenchone Linalool Camphor 4-Terpineol a-Terpineol Phellandrene epoxide 2-Carene Thymol l-p-Menthen-8-ol acetate a-Copaene P-Elemene P-Phellandrene Caryphyllene a-Humulene Eremophilene P-Bisabolene y-Cadinene 5-Cadinene D-nerolidol a-Cedrol

Peak area (%)b 0.06 5.71 0.18 0.08 2.41 0.76 0.38 2.63 0.04 1.24 68.42 0.32 0.63 2.25 0.19 0.25 0.11 0.83 1.36 0.12 0.07 0.04 0.94 0.55 0.34 0.08 0.25 0.25 0.32 0.26 0.29 0.54 0.23 0.10

± + ± ± + + ± ± + ± ± ± ± ± ± ± + ± ± ± ± ± ± + ± ± + + ± ± +

0.01 D 0.25 b 0.03 d 0.01 d 0.36 cd 0.05 cd 0.01 cd 0.22 c 0.01 d 0.14 cd 4.36 a 0.03 cd 0.08 cd 0.31 cd 0.02 d 0.05 d 0.02 d 0.07 cd 0.14 cd 0.05 d 0.02 d 0.01 d 0.06 cd 0.03 cd 0.04 cd 0.01 d 0.05 d 0.02 d 0.02 cd 0.05 cd 0.02 cd ± 0.04 cd ± 0.01 d ± 0.02 d

a RT, retention time. h Peak area obtained by GC-flame ionization detection. Different letters within a column indicate statistically significant differ­ ences between the means (P < 0.05). Xu et al. (30). In addition, to determine the concentration o f the constituents released, 3 ml o f supernatant was used to measure UV absorption at 260 nm. Correction was made for the absorption of the suspension with the same PBS containing the same concentration of the essential oil after 2 min of contact with tested strains. The untreated cells were corrected with pH 7.2 PBS.

TEM observation. TEM observation was performed accord­ ing to the method described by Diao et al. (6) with minor modifications. The bacterial cells were incubated at 37°C in nutrient broth for 10 h. To the suspension was added 1 x MIC of the essential oil. All suspensions were incubated at 37°C for 4 h and then centrifuged. The cells were washed twice with 0.1 M PBS (pH 7.2) and fixed with 2.5% (vol/vol) glutaraldehyde in 0.1 M PBS overnight at 4°C. Next, the cells were postfixed with 1% (wt/ vol) O s04 in 0.1 M PBS for 2 h at room temperature and washed three times with the same buffer and then dehydrated by a graded series of ethanol solutions (30, 50, 70, 90, and 100%). Stained bacteria were viewed and photographed with a TEM (H-7650, Hitachi Ltd., Tokyo, Japan) operated at 80 kV and were analyzed

TABLE 2. DIZs, MICs, and MBCs of the essential oil from Amomum kravanh fruits against foodborne pathogens Bacterium

DIZ (mm)‘

MIC (mg/ml)

MBC (mg/ml)

12.2 ± 0.5 c 10.8 ± 0.4 d 15.2 ± 0.3 b

> 5 .0 5.0 2.5

NT* 5.0 5.0

Gram positive

S. aureus S. alhus B. suhtilis Gram negative

S. enterica S. dysenteriae E. coli

18.2 + 0.7 15.3 + 0.6 15.2 ± 0.3

a b b

2.5 1.25 2.5

5.0 2.5 2.5

a Values represent means o f three independent replicates + standard deviations. Different letters within a column indicate statistically significant differences between the means (P < 0.05). DIZ, diameter of the inhibition zone. h NT, not tested.

with the digital imaging software MegaViewG2 (Olympus, Muenster, Germany).

Statistical analysis. One-way analysis o f variance and Duncan’s multiple range tests were carried out to determine significant differences (P < 0.05) between the means, using Data Processing System (DPS, Ruifeng Information Technology Co., Hangzhou, China) and Excel (Microsoft, Redmond, WA). RESULTS Chemical composition of the essential oil. The chemical composition of the essential oil was analyzed by GC and GC-MS, and the results are presented in Table 1. In total, 34 components were identified in the essential oil of A. kravanh fruits, representing 95.84% of the total amount, and 1,8-cineole (68.42%) was found to be the main component, followed by a-pinene (5.71%), a-terpinene (2.63%), (3-pinene (2.41%), and other minor components. DIZs, MICs, and MBCs of the essential oil. The DIZs, MICs, and MBCs of the essential oil from A. kravanh fruits are presented in Table 2. The results showed that the essential oil had antibacterial activity against all of the foodborne pathogens tested, including both gram-positive and gram-negative bacteria. The DIZs for all bacterial strains tested were in the range of 10.8 to 18.2 mm. S. enterica had the maximum DIZ, followed by S. dysenteriae, E. coli, and B. suhtilis. The MICs and MBCs for the bacterial strains ranged from 1.25 to 5.0 mg/ml and 2.5 to 5.0 mg/ml, respectively. The MICs and MBCs of the essential oil were above the maximum concentration tested for S. aureus. Among the gram-positive bacteria, the essential oil possessed the largest DIZ and lowest MIC and MBC against B. suhtilis, which indicated that it was a more effective bacterial inhibitor against B. suhtilis. Among the gram-negative bacteria, there were slight differences in DIZs and MICs. Therefore, the antibacterial properties and mechanism of action of essential oil from A. kravanh fruits against B. suhtilis and E. coli were investigated further in this study.

ANTIBACTERIAL ACTIVITY OF THE ESSENTIAL OIL FROM A. KRAVANH

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0

Time (h)

FIGURE 1. Effects of the essential oil from Amomum kravanh fruits on the growth of B. subtilis (A) and E. coli (B) at 37 °C in nutrient broth. MIC =2.5 mglml.

Growth as monitored by OD over time. Based on the sensitivities of the foodbome pathogens tested, one gram­ positive bacterium (B. subtilis ATCC 6051) and one gram­ negative bacterium (E. coli ATCC 25922) were selected to study the effects of the essential oil from A. kravanh fruits on their growth as monitored by OD over time. The effects of the essential oil on the OD values of B. subtilis and E. coli at 600 nm are shown in Figure 1. As observed by the results in Figure 1A, the susceptible B. subtilis treated with the essential oil at 0.5 x MIC showed a slow increase in the trend of OD values over the 12-h period of the test compared to the growth of the control, while in treatments at 1 x MIC, the OD values of the B. subtilis cultures did not change during 12 h of incubation, which indicated that concentra­ tions of the essential oil lower than the MIC did not completely inhibit the growth of B. subtilis. As observed by the results in Figure IB, the essential oil had the same influence on the trend of OD values of E. coli. These results confirmed the antibacterial activity of essential oil from A. kravanh fruits and showed a severe effect on the growth rate of surviving B. subtilis and E. coli cells, supporting the results stated above, and showed that the treatment time and concentration of the essential oil had great influences on its antibacterial effects. Cell membrane permeability. The results in Figure 2 show the effects of essential oil from A. kravanh fruits on the membrane permeability of B. subtilis and E. coli. For B. subtilis, there was little change in the relative electrical conductivity of the control during the first 60-min period of the test, and then an increase in the relative electrical conductivity was found (Fig. 2A), which may be due to

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---------*-------- !---------*---------‘-------- ‘---------*-------- 1--------

0

15

30

45 60 120 240 360 Time (min)

FIGURE 2. Effects of the essential oil from Amomum kravanh fruits on the permeability of cell membrane of tested B. subtilis (A) and E. coli (B). normal lysis and death of bacteria, resulting in a rise in the relative electrical conductivity. Compared to that of the control, the relative electrical conductivity of the suspension increased immediately after the addition of essential oil at a concentration greater than or equal to the MIC, and it also increased rapidly with increasing treatment time and the concentration of essential oil. As observed by the results in Figure 2B, the essential oil had the same influence on the trend of the relative electrical conductivity of E. coli. The permeability of the bacterial membrane would have increased correspondingly, causing the leakage of intracel­ lular ingredients, especially losses of electrolytes, including K + , Ca2+, Na + , and so on. Cell constituent release. Table 3 shows the results for the release of cell constituents, including proteins and reducing sugar, and the absorbance at 260 nm of the supernatants of tested bacteria when B. subtilis and E. coli were treated with different concentrations of essential oil from A. kravanh fruits for 4 h. Compared to the results for the control, the release of cell constituents by the two strains treated with essential oil at the two concentrations tested increased after incubation under all conditions. In addition, the release of cell constituents increased visibly with the increased concentration of the essential oil. After treatment with 1 x MIC of the essential oil, the concentrations of protein, reducing sugars, and cell constituents (OD26o) in suspensions of B. subtilis increased significantly (P < 0.05), by 8.24, 2.63, and 11.56 times, respectively, while they increased (P < 0.05) by 13.18, 4.68, and 16.87 times, respectively, after treatment at 2 x MIC. Similar results

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TABLE 3. Effect of the essential oil on release o f cell constituents by B. subtilis and E. coli" Protein (|ig/ml)

Cell constituents (OD260)

Reducing sugar (gg/ml)

Concn of oil

B. subtilis

E. coli

B. subtilis

E. coli

Control 1 x MIC 2 x MIC

9.7 ± 0.9 c 89.7 ± 8.9 b 137.6 ± 1.2 a

13.2 ± 0.7 c 91.2 + 8 . 6 b 141.3 ± 9.9 a

16.5 ± 1.3 c 59.9 ± 5.1 b 93.7 ± 7.2 a

17.0 ± 1.1 c 61.6 ± 4.7 b 91.8 + 5.9 a

B. subtilis

E. coli

0.023 ± 0.006 c 0.034 + 0.011 c 0.289 ± 0.018 b 0.277 ± 0.014 b 0.411 + 0.026 a 0.501 ± 0.020 a

° Values represent means of three independent replicates + standard deviations. Different letters within a column indicate statistically significant differences between the means (P < 0.05).

were found for E. coli; the concentrations of proteins, reducing sugars, and cell constituents (OD26o) in the suspensions increased significantly (P < 0.05), by 5.90, 2.62, and 7.14 times at 1 x MIC and 9.07, 4.40, and 13.74 times at 2 x MIC. These results implied that irreversible damage to the cytoplasmic membranes might have occurred, leading to the loss of cell constituents, such as proteins and some essential molecules, and to cell death. Electron microscope observation. Figure 3 shows TEM images of B. subtilis and E. coli cells after treatment with the essential oil at 1 x MIC for 4 h. It was observed from the TEM photographs that untreated B. subtilis and E. coli bacteria remained intact and had clearly discernible cell membranes with uniformly distributed cytochylema and electron densities inside the cells after cultivation for 14 h (Fig. 3A and B). However, cells treated with the essential oil were damaged to different degrees (Fig. 3A and B). Some of the exposed cells changed from the normal rod shape into irregular cell morphology, and the electron FIGURE 3. Transmission electron micro­ photographs o/B . subtilis and E. coli. (A0 and B0) Untreated E. coli and B. subtilis bacteria, respectively. (Aj and By) E. coli and B. subtilis bacteria, respectively, treated with the essential oil at 1 x MIC.

densities inside the bacterial cells became uneven. The cell walls were broken, and the cytoplasmic membranes were thin and even hard to distinguish, which could give rise to the leaching out of cell contents, supporting the results of the cell membrane permeability and cell constituent release assays. DISCUSSION Essential oils are volatile and odorous principles of plant secondary metabolism which have wide applications in the food flavoring and preservation industries (2). In this study, 1,8-cineole was found to be the major compound of essential oil from A. kravanh, which was supported by the previous results (28,33). However, Zeng et al. (33) reported a higher value for 1,8-cineole (85.88%), and Wu et al. (28) reported that essential oils of A. kravanh extracted by GCMS-extracted ion chromatography and GC-MS-extracted multi-ion chromatography showed 1,8-cineole (59.70%), a-terpinenyl (6.34%), and aceatae (5.09%) as the main components, but there were significant differences between

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the extracted ion chromatography and extracted multi-ion chromatography results; Hamdan et al. (12) reported 21 components and found that [l-pinene, cineole, linalool, a-terpinyle, and terpinyle acetate were the major com­ pounds in the essential oil from A. kravanh. These differences in the components of the essential oil from A. kravanh may be due to the geographical origins (33) of the samples and the extraction methods (28) and conditions of analysis of the essential oil. The results from the Oxford cup method, followed by the measurements of MICs and MBCs, indicated that the essential oil from A. kravanh had some inhibitory effect against all of the foodbome pathogens tested, including both gram-positive and gram-negative bacteria, although, on a concentration basis, it is not a highly effective essential oil antimicrobial. Li et al. (16) found that A. kravanh essential oils exhibit an inhibitory effect against S. aureus and E. coli, which supports our results in the present study. Further­ more, the effects of the essential oil on the growth of B. subtilis and E. coli were investigated, and the results revealed that exposure to the essential oil had an inhibitory effect on the growth of the bacterial pathogens tested. Similar to our findings, other essential oils from plants also exhibited inhibitory effects against various foodbome pathogens (1, 3). However, it is well known that the inoculum size influences the antibacterial capability of bacterial inhibitors, and therefore, further research on the effects of different cell loads on the MIC and MBC of the essential oil from A. kravanh is still necessary. Some studies reported that the active components of the essential oil might bind to the cell surface and then penetrate to the phospholipid bilayer of the cytoplasmic membrane and membrane-bound enzymes. Subsequently, the active components can lead to disruption of the synthesis of DNA, RNA, protein, and polysaccharides, causing the death of the cells (23, 29). Some authors suggested that the distortion of the cell wall and cytoplasmic membrane would cause the expansion and destabilization of the membrane and increase membrane permeability, resulting in leakage of various vital intracellular constituents and leading to cell death (13, 17, 23). In this study, variation of the cell morphology and disruption of the cell wall were observed, which indicated that the cell wall might be one of the targets of the essential oil of A. kravanh fruits. The increases in cell permeability and rapid losses of cell constituents also implied that damage occurred to cell membranes, which was supported by the results of TEM. But it is still a mystery where the damage takes place, whether to the lipopolysaccharides or membrane proteins in the cell wall; this needs to be studied further. The TEM micrographs of B. subtilis and E. coli cells treated with the essential oil of A. kravanh fruits showed that severe morphological alterations appeared in the cell wall and membrane, which have also been observed for various kinds of organisms when treated with different essential oils (3, 7). The physical and morphological changes in bacterial cells might come from the effect of essential oil on the permeability of the cell membrane, resulting in lysis of the bacterial cell wall, followed by the loss of intracellular dense

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materials at the surface of treated cells. In addition, we found that the mechanism of action of essential oil from A. kravanh fruits against B. subtilis and E. coli was likely to be similar in this regard, though some studies reported that gram-positive bacteria were more sensitive to some plant extracts than gram-negative ones because of the structural differences in the outer layers of bacteria (5, 7). However, just as Burt (4) reported, it is most likely that their antibacterial activity is not due to one specific mechanism but is a combined effect of several mechanisms because of the complicated antibacterial components of the plant extracts. Therefore, it needs to be studied further whether the essential oil influences other vital intracellular constit­ uents (DNA, RNA, protein, polysaccharides, etc.) to cause the cell death. Based on the present research, the essential oil from A. kravanh fruits possessed certain antibacterial activity against selected foodbome pathogens in this study. We conclude that one of the mechanisms of action of the essential oil from A. kravanh fruits against B. subtilis and E. coli may be described as disruption of the cell wall, leading to increased cell membrane permeability and, thus, to leakage of electrolytes as well as losses of intracellular constituents, including proteins, reducing sugars, and materials with an absorbance of 260 nm. However, because of the heteroge­ neous composition of essential oils, it seems unlikely that there is only one mechanism of action or that only one component is responsible for the antibacterial action. Therefore, further research on the mechanisms involved, including against other foodbome pathogens, as well as interactions with other food ingredients, is still necessary in order to justify the application of the essential oil from A. kravanh in food preservation. ACKNOWLEDGMENTS This work was financially supported by a Project of the Natural Science Foundation of Shanxi Province, China (Project No. 2012011031-3), and a Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi.

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Chemical composition, antibacterial activity, and mechanism of action of the essential oil from Amomum kravanh.

Amomum kravanh is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistill...
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