Innovative Food Science and Emerging Technologies 13 (2012) 169–177
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Bactericidal action of lemon grass oil vapors and negative air ions Amit K. Tyagi a, b, Anushree Malik a,⁎ a b
Applied Microbiology laboratory, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi-110016, India Dipartimento di Scienze degli Alimenti, Università degli Studi di Bologna, Sede di Cesena, Piazza G. Goidanich, 60, 47023 Cesena, Italy
a r t i c l e
i n f o
Article history: Received 23 January 2011 Accepted 23 September 2011 Keywords: Lemon grass oil Vapor-phase antimicrobial activity Negative air ions SPME-GC-MS TEM E. coli
a b s t r a c t In this study, bactericidal efficacy and mechanism of action of lemon grass oil vapors against Escherichia coli were investigated. Next, in order to develop the application of the vapor as room/surface disinfectant and to study its integration with another antimicrobial agent i.e. negative air ion (NAI), a special set-up was designed and kill time assays were conducted. Zone of inhibition (56 mm) due to the vapor phase antimicrobial activity evaluated using disk volatilization assay was compared with direct assay (well diffusion assay) in liquid phase (i.e. 20 mm for the same dose of oil). The Chemical analysis of the Essential oil vapor has been done by SPME GC-MS and -Myrcene (3.5%), Limonene (30.3%), Camphene (6.5%), α-Citral (17.6%), β-Citral (11.3%), 6-methyl hepten-2-one (14.6%) and linalool (1.5%) were recorded as major components. The morphological and ultrastructural alterations in vapor treated E. coli cells were studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. Results of the kill time assays demonstrated that the combination of NAI with lemon grass oil vapors has a greater bactericidal effect (100% reduction in viability) than NAI alone (42%) or vapors alone (78%) within 8 h exposure. Present results indicate that lemon grass oil is highly effective in vapor phase and its efficacy can further be enhanced by integration with Negative air ion (NAI) for reducing the viable microbial load. The integration described here offers a novel technique for reducing the concentration of E. coli on surfaces/indoor spaces. © 2011 Elsevier Ltd. All rights reserved.
Industrial relevance: Negative air ion (NAI) generators offer a low cost, simple and sustainable source of NAI, which possess substantial antibacterial activity but have certain limitations like requirement of very high concentrations of air ions and accumulation of potentially infectious particles. Nevertheless, the integration of the essential oil vapours with negative air ion generators could yield better results in a relatively simple set up. However, no study describes such integration of lemon grass oil vapour and NAI exposure. Therefore in the present study, detailed investigations on the mechanism and efficacy of lemon grass essential oil vapours against E. coli were conducted through petri-plate bioassay. Secondly, a set-up was developed to facilitate the integration of lemon grass oil vapours (through EOV generator) and NAI (through NAI generator) to establish the kill time. The integration described here offers an innovative method for reducing the concentration of bacteria on surfaces/indoor spaces in food processing environments and makes a novel contribution in the research field. 1. Introduction Novel or emerging non-thermal food processing technologies including natural antimicrobials are gaining increasing importance in the recent era (Knorr, 1998). Plant derived essential oils have been used through ages in perfume preparation, aromatherapy and several ⁎ Corresponding author. Tel.: + 91 11 26591158; fax: +91 11 26591121. E-mail addresses:
[email protected],
[email protected] (A. Malik). 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.09.007
other applications (Burt, 2004; El-Shafei, El-Saidb, Attia, & Mohammed, 2010). However, antimicrobial investigations have evaluated the effect only when there is direct contact between microorganism and essential oil (Goncalves, Cruzb, Cavaleiroa, Lopesb, & Salgueiroa, 2010). Recent studies suggest that essential oils in vapor phase could be highly effective against surface pathogens and food spoilage bacteria at relatively lower concentrations (Lopez, Sanchez, Batlle, & Nerin, 2005). Further, essential oil volatiles have the advantage that they can treat large areas without requiring direct contacts with surfaces. This can make it suitable for use as disinfectant of rooms/surfaces and as a component for exposure of the perishable harvested/processed commodities. The application of volatiles could also generate eco-friendly solutions for preventing decay of natural materials (like bamboo) in green buildings, which are being promoted extensively (Sudhakar, Gupta, Korde, Bhalla, & Satya, 2007). However, despite several advantages, recent investigations suggest that there are some hidden risks associated with the use of essential oils that should be carefully considered (Chiang, Chiu, Lai, Chen, & Chiang, 2010). The exposure to essential oils or perfume can cause respiratory problems, especially in high-risk populations (Hammer, Carson, Riley, & Nielsen, 2006; Millqvist, Bengtsson, & Lowhagen, 1999). Hence, ventilation should be enhanced during the indoor use of these essential oils. On the other hand, some investigations suggest that Lemon Grass Oil vapor could be an effective control agent for respiratory tract pathogens (Inouye, Takizawa, & Yamaguchi, 2001). High volatility of lemon grass oil makes it an excellent antimicrobial agent in vapor phase (Tyagi & Malik, 2010a) that could easily be integrated
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with the existing room conditioning devices with proper safe guards to ease the application. However, to the best of authors' knowledge, no such study has been reported in the literature. In a recent study from Mexico, fecal coliforms (FC) were found in dust samples collected from both indoor and outdoor environments (Rosasa et al., 2006). Nevertheless, FC were more abundant in indoor samples, and Escherichia coli was found in all FC-positive indoor samples. Another very recent study reported about the exposure of hog producers to aerosolized human pathogens and tetracycline-resistant bacteria (that can contaminate the nasal flora) in swine confinement buildings (Létourneau et al., 2010). Such evidences suggest that the home as well as working environment could be an important source of infectious diseases and necessitates the development of suitable disinfection techniques (Larson & Gomez-Duarte, 2001). In recent years, new air purification and disinfection technologies (Plasma Cluster Ion technology) have been developed based on the application of positive and negative ions (Digel et al., 2005; Nishikawa & Nojima, 2001; Seo, Mitchell, Holt, & Gast, 2001). There is a great potential for utilization of NAI generators in preventing decay of fruits and vegetables (Hildebrand, Song, Forney, Renderos, & Ryan, 2001), reducing the ambient air microbial load (Tanimura, Nakatsugawa, Ota, & Hirotsugi, 1997) and hospital acquired infections in clinics (Gabby, Bergerson, Levim, Brenner, & Eli, 1990; Kerr et al., 2006), as well as preventing the spread of diseases in animal houses (Estola, Makela, & Hovi, 1979). However, normally very high concentrations of air ions are required to produce the antimicrobial effects and different microbial species could have variable responses to the system (Arnold, Boothe, & Mitchell, 2004). Further, NAI generators cause accumulation of potentially infectious particles onto adjacent surfaces or grounded parts of the ionizer itself, as suggested by the localized outbreak of TB-infected animals following ionizer cleaning (Escombe, Oeser, Gilman, Navincopa, & Ticona, 2007). Recently, the relative antimicrobial effects of diffuse ionized gaseous species (generated through burning candle) and volatile bactericidal compounds in the vapor phase have also been studied. There is synergism demonstrated between the ionized species generated by a candle and active volatiles such as orange oil and thyme oil, which on their own demonstrates limited antibacterial effects (Gaunt, Higgins, & Hughes, 2005). It was demonstrated that the combination of an ionizing source, such as a candle flame, with a bactericidal volatile has a greater effect on surface-borne bacteria than either treatment alone (Gaunt et al., 2005). NAI generators offer a low cost, simple and sustainable source of NAI, which possess substantial antibacterial activity (Tyagi & Malik, 2010b; Tyagi, Nirala, Malik, & Singh, 2008). The integration of the essential oil vapors with an ionizing source (negative air ion generators) could yield better results in a relatively simple set up. Nevertheless, and to the best of our knowledge, there is no published report regarding the antimicrobial effectiveness of combinations of lemon grass oil vapor with NAI exposure. Also, systematic investigations in terms of the chemical characterization of lemon grass oil vapors and morphological changes induced in treated cell have not been reported. Such studies shall establish the mechanism of action and provide deeper insight for better antimicrobial application development. Therefore in the present study, detailed investigations on the mechanism (through SPME GC-MS analysis for composition of vapors and electron microscopy of vapor treated cells) and efficacy of lemon grass essential oil vapors against E. coli were conducted through petri-plate bioassay. Secondly, a set-up was designed to facilitate the integration of lemon grass oil vapors (generated through essential oil vapor (EOV) generator) and NAI (through NAI generator) to establish the kill time. 2. Material and methods 2.1. Materials and bacterial culture preparation Lemon grass oil was procured from Natural Aromatics Private Limited, New Delhi (India). E coli DH5α and E. coli ATCC 25922 strains
collected from the central microbial culture facility, Department of Biotechnology & Biochemical Engineering, Indian Institute of Technology Delhi, New Delhi (India), and Himedia Pvt. Ltd. (India), respectively were grown in Muller Hinton broth (MHB) medium at 30 °C, 180 rpm for 24 h. Cells were harvested by centrifugation, suspended in sterile distilled water and used immediately. 2.2. Antimicrobial assays Although the major thrust of the study was to evaluate the antimicrobial activity in vapor phase, for comparison of the results, antimicrobial assays were conducted in both liquid phase (well diffusion method) and vapor phase (disk volatilization method). 2.2.1. Determination of minimum inhibitory concentration (MIC) Minimum Inhibitory Concentration (MIC) of essential oils was determined by agar dilution assay. The agar plates were prepared using Muellar Hinton Agar (15 ml per petri dish) amended with various concentrations of Lemon grass oil (i.e. 0.27–18 mg/ml). For enhancing the oil solubility, Tween-80, 0.5% (v/v) was added. These plates were inoculated with one ml cell suspension (10 6 cfu/ml), of each E.coli DH5α and E.coli ATCC 25922. All the plates were incubated in duplicate for each concentration at 30 °C for 24 h. Plates with Tween-80 but without Lemon grass oil were used as control. Observation of the plates (Bacterial growth) was done at 12 h and number of colonies was counted after 24 h of incubation. 2.2.2. Well diffusion method Well diffusion method was employed for the determination of antimicrobial activities of the essential oil (Baratta et al., 1998; NCCLS, 1999). Essential oil was dissolved in 0.5% dimethylsulphoxide (DMSO) and filter-sterilized using a 0.45 μm membrane filter. Each test strain was suspended in sterile double distilled water and serially diluted to 10 6 cfu/ml concentration. A 100 μl portion of each suspension was spread over the surface of Muller Hinton Agar (MHA) plate and allowed to dry. The wells (8 mm in diameter) were cut from the agar and different doses (10, 20, 30 and 40 μl) of essential oil solutions mixed with requisite amount of diethyl ether (to make the volume 50 μl) were delivered into them. After incubation for 24 h at 30 °C, all plates were examined for any zones of growth inhibition, and the diameters of these zones were measured in millimeters. All tests were performed in triplicate. 2.2.3. Disk volatilization method The standard experimental set-up (Lopez et al., 2005) was used, as follows: a 100 μl portion of each suspension containing approximately 10 6 cfu/ml was spread over the surface of MHA plate and allowed to dry. A paper disk (diameter 6 mm, Sigma Aldrich) was laid on the inside surface of the upper lid and 10 μl essential oil was placed on each disk. The plate inoculated with microorganisms were immediately inverted on top of the lid and sealed with parafilm to prevent leakage of essential oil vapor. Plates were incubated at 30 °C for 24 h and the diameter of the resulting inhibition zone in the bacterial lawn was measured. Volume of essential oils tested was varied (20, 40 or 60 μl) by using appropriate number of sterile disks. 2.3. Sample preparation for scanning electron microscopy and transmission electron microscopy E. coli cell suspension (10 6 CFU/ml) was inoculated on MHA plates and the plates were incubated at 30 °C for 15 h. These pre-grown cells were treated with essential oil vapors at room temperature for 4 h. Control plates were also held under similar conditions but without any vapor pre-treatment. The treated and untreated cells were scrubbed gently with the help of brush from the petri plate and collected in separate test tubes. The cells were harvested by centrifugation
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and were prefixed with a 2.5% glutaraldehyde solution overnight at 4 °C. After this, the cells were again harvested by centrifugation and washed three times with 0.1 M sodium phosphate buffer solution (pH 7.2). Now each resuspension was serially dehydrated with 25, 50, 75, 90, and 100% ethanol, respectively. Then, cells were dried at “critical point”. For SEM, a thin film of cells was smeared on a silver stub. The samples were gold-covered by cathodic spraying (Polaron gold). Finally, morphology of the E. coli cells was observed on a scanning electronic microscope (ZEISS EVO 50). The SEM observation was done under the following analytical condition: EHT = 20.00kv, WD = 10 mm, Signal A = SE1. For TEM, the pellet was post fixed in 1% osmium tetraoxide for 30 min, washed with phosphate buffer solution (pH 7.2), serially dehydrated in ethanol as mentioned above and embedded in Epon–Araldite resin for making the blocks of the cells pellet. Ultra thin sections of the cells were stained with uranyl acetate and lead citrate and observed under a Philips transmission electron microscope (CM-10) at 100 eV and direct magnification of 50.0 k.
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continuous concentration of about 10 6 NAI/ml (monitored by an Air Ion Counter, Alpha Lab, Inc., Salt Lake City, UT), was maintained. For EOV/NAI exposure, appropriate serial dilution of the culture (to obtain 100–300 cfu) was plated on MHA plates. The plates were opened inside the chamber and fixed at different locations with the help of double-sided tape. After a particular time period (0.5, 1, 2, 4, 6, 8 and 12 h) the plates were detached, closed and incubated at 30 °C for 18–20 h. All the plates were used in triplicate. For studying the effect of Negative air ion (NAI), only NAI generator was switched on and it was ensured that the chamber did not contain any residual vapor. For investigating the effect of LGO vapor alone, only EOV generator was switched on. For studying the effect of combination of Lemon grass oil vapor with NAI, both EOV and NAI generator were switched on (Fig. 1B). The control plates were kept closed under similar conditions outside the chamber during the exposure period. For each exposure duration of the treated plates, control plates were run in triplicate and subsequently incubated at 30 °C for 18–20 h along with the treated plates. The results were represented as percentage reduction in viability in treated plates in relation to the untreated control plates.
2.4. SPME GC-MS analysis of Lemon grass oil vapors 2.6. Statistical analyses SPME optimisation analyses were carried out using a Shimadzu 2010 GC instrument equipped with a data processor and Shimadzu GCMS-QP2010 Plus. An AB-Innowax 7031428 capillary column (60.0 m × 0.25 mm i.d., 0.25 μm film thickness) was used for the separation of the sample components. Lemon grass oil volatiles from petri plates were extracted using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber. The fiber was conditioned in a GC-MS injector port as indicated by manufacturer (Supelco, France). The injector was maintained at 260 °C and operated in splitless injection mode with the split valve closed for 1 min. Helium gas was used as the carrier gas at a constant pressure of 136.3 kPa. The column oven was initially maintained at 40 °C for 2 min, raised to 180 °C at 8 °C/min, then to 230 °C at 4 °C/min. The interface temperature was 260 °C and the ionization mode was electron impact (70 eV). The mass selective detector was operated in the scan mode between 20 and 700 m/z. Data acquisition was started 4.0 min after injection. MS parameters used were; Ionization Voltage (EI) 70 eV, peak width 2 s, mass range 40–600 amu and detector voltage 1.5 V. Peak identification was carried out by comparison of the mass spectra with mass spectra available on database of NIST05, WILEY8 libraries and those of pure compounds. The compound identification was finally confirmed by comparison of their relative retention indices with literature values (Davies, 1990). 2.5. Exposure regimes To validate the results of the disk volatilization assay in a larger set-up (which simulated an indoor environment) and to investigate the integration of vapors with another antimicrobial agent i.e. negative air ion (NAI), a set-up was designed. It was used to study the antimicrobial efficacy of NAI alone, lemon grass oil vapor alone and combination of NAI and lemon grass oil vapors. A compact chamber made up of acrylic material (size 50 cm × 50 cm; W × L) was used for this purpose (Fig. 1A). The height of the chamber was 50 cm on the back side and 25 cm at the front side. The total volume of the chamber was 0.09375 m 3 (93.75 l). The front side of the chamber had gloves through which the things inside the chamber could be handled without opening the chamber. Prior to exposure, the chamber was cleaned with ethanol and UV sterilized. Two essential oil vapor (EOV) generators (evaporation rate = 0.50 ml/h) and two negative air ion (NAI) generators were fixed in this chamber. A negative air ion generator (Electronic Airpurifier, Escort) which contains several needle shaped electrodes to provide the negative charge to the air ions, was used. The NAI generators were positioned such that a
All the experiments were done in triplicate and the data presented here represents the mean of three replicates with standard deviation. Data related to the zone of inhibition due to the lemon grass oil and oil vapor were subjected to analysis of variance (one way ANOVA) in Duncan multiple range test using SPSS (version 10) statistical software. The differences with p b 0.05 were considered significant. 3. Result and discussion 3.1. Determination of MIC and MBC of Lemon grass oil against E. coli MIC of the Lemon grass oil was determined against E. coli strains. The oils exhibited concentration-dependent inhibition of growth. A 0.288 mg/ml concentration of Lemon grass oil was enough for complete growth inhibition of both the strains of E. coli. 3.2. Zone of inhibition Antibacterial potential of the lemon grass oil was observed in terms of zone of inhibition generated by the diffusion of the essential oil components into the microorganisms inoculated agar plate. The zone of inhibition increased from 12 mm to 20 mm with the increasing concentration (i.e. 10, 20, 30 and 40 μl) of lemon grass oil in each well (Fig. 2A). Nevertheless, no significant differences were noticed in the zone of inhibition in case of E. coli DH5α and E. coli ATCC 25922 at various concentrations of lemon grass oil. The MIC determined by various methods (data not shown) also indicated that both the E. coli strains had similar susceptibility towards lemon grass oil. Previously, antimicrobial activity of methanol extracts from the bark and shoot of Cinnamomum cassia Blume against various E. coli strains has been studied (Kim, Kim, & Ahn, 2004). Burt and Reinders (2003) analyzed the antibacterial properties of essential oils against E. coli O157:H7 using the disk diffusion method where oregano and thyme essential oils were found to exhibit stronger antimicrobial properties than clove and bay. In our study, the antimicrobial activity of lemon grass oil was similar to that of the clove oil while it was higher than the C. cassia extract. The zone of inhibition resulting from the exposure to lemon grass oil vapors varied from 44 mm to 90 mm with increasing dose (20– 60 μl) of the oil corresponding to 0.364–1.069 μl/ml vapor concentration (Fig. 2B). The zones of inhibition due the vapor increased significantly with increase in concentration and a significant difference among the two strains was also noticed at lower concentrations (i.e.
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A (1)
(2)
(3)
(4)
(5) (6)
B Essential oil vapours & Negative air ions
Exposure time, 8 h
78.1 % Reduction in viability
=
Exposure time, 8 h
NAI Generator
Negative air ions
=
100 % Reduction in viability
Essential oil vapours
=
EOV Generator
Exposure time, 8 h
40 % Reduction in viability
Fig. 1. Exposure regimes for kill time assay. (A) Line diagram of the experimental set up showing (1) UV lamp, (2) Petri-plates, (3) Essential oil vapor (EOV) generator, (4) Gloved openings, (5) Negative air ion (NAI) generator, (6) Electrical sockets. (B) Results of the Kill time assay under different exposure regimes.
20 and 40 μl). Also, the zone of inhibition resulting from the exposure to lemon grass oil vapors was significantly larger than that due to the same concentration (20 and 40 μl) of essential oil in liquid phase (Fig. 2A, B). As shown in Fig. 2B, the zone of inhibition due to 40 μl essential oil vapor was 60 mm for E. coli ATCC25922 and 56 mm for E. coli DH5α while the corresponding zone of inhibition in presence of oil was around 20 mm for both the strains (Fig. 2A). This might be due to the different composition as well as the different mode of contact of the antimicrobial agents in both assays as discussed later. In the well diffusion method, the activity depends on the diffusibility of lemon grass oil compounds (which is low), and it is produced by the activity of more hydrophilic and less volatile substances. The antimicrobial activity of the vapor depends on the volatility of each compound and the high volatility of active compounds of essential oils makes them a better antimicrobial agent in vapor phase. Earlier, we had noticed that in spite of increased monoterpene hydrocarbons in the Eucalyptus oil vapor, it was more effective against several food spoilage microorganisms as compared to the Eucalyptus oil itself (Tyagi & Malik, 2011). Pibiri (2006) has also demonstrated that certain essential oils in gaseous phase had a lethal effect on Staphylococcus aureus and Pseudomonas aeruginosa, even in small doses. Hence, it can be inferred that significant antimicrobial activity of the different
essential oil vapors can be achieved at lesser amount than essential oil in liquid phase. To establish this deeper scientific investigations are required. However, to the best of author's knowledge, systematic investigations on the efficacy of lemon grass oil vapors in terms of its chemical characterization and morphological changes induced in cells, has not been reported. Such studies shall establish the mechanism of action and provide deeper insight for better antimicrobial application development. 3.3. Morphological and ultrastructural alteration in E. coli Bacterial cells treated with lemon grass oil and its vapor underwent considerable morphological alterations in comparison to the control when observed by a Scanning Electron Microscope (Fig.3). Control E. coli cells appeared intact, rod shaped, separated from each other, turgid and whole with smooth surface (Fig. 3A). The cells appeared to be completely destroyed when exposed to lemon grass oil vapor (Fig.3B). Only the ghost cells were left (shown by arrows) with apparent cellular debris (Fig. 3B) while shrinkage and deformation was observed in the lemon grass oil treated cells (Fig.3C). The observations clearly demonstrate that the damage caused by vapors is much more than that caused by lemon grass
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Zone of inhibition (mm)
A 50 45 40 35 30 25 20 15 10 5 0
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A 10 µl 20µl 30 µl 40 µl
E. coli DH5 α
E. coli ATCC25922
Bacterial strains
Zone of inhibition (mm)
B 100 90 80 70 60 50 40 30 20 10 0
B 20 µl 40 µl 60 µl
E. coli DH5 α
E. coli ATCC 25922
Bacterial strains Fig. 2. Zone of inhibition due to different concentrations of (A) Lemon grass oil evaluated by well diffusion assay, (B) Lemon grass oil vapors evaluated by disk volatilization assay. Error bars represent the standard deviation at n = 3.
oil. No previous reports comparing the influence of lemon grass oil and its vapor on E. coli cell morphology are available. Nevertheless, Inouye et al. (2006a) reported that the vapor of oregano oil also induced lysis of the Trichophyton mentagrophytes mycelia. Morphological examination by scanning electron microscope (SEM) revealed that the cell membrane and cell wall were damaged in a dose- and timedependent manner by the action of oregano vapor, causing rupture and peeling of the cell wall, with small bulges coming from the cell membrane. To obtain more information on ultra-structural alterations in vapor treated cells, TEM was used. Untreated cells were also studied as a control to ensure that the observed differences (shown by the arrows) between control and the treated bacterial cells were indeed due to the effect of lemon grass oil vapors and not to the preparation method. TEM photomicrographs of untreated E. coli cells show a regular outlined cell wall, plasma lemma lying closely to the cell wall and some dense bodies, regularly distributed over the cytoplasm (Fig. 4A, B). Electron microscopy revealed that some of the vapor treated cells still retained a cell wall structure similar to untreated cells, however, in majority of the cells, cell wall thickness varied and occasionally it appeared disrupted (Fig. 4C, D). Besides, extensive internal damage and several abnormalities were observed in the vapor treated cells (Fig. 4C, D, E, F). As shown in Fig. 4, plasma lemma damaged and became irregular in the treated cells (Fig. 4E, F). Periplasmic space was altered and it became larger and irregular. Intra-cytoplasmic changes were noticed and the cytoplasm appeared very dense at certain locations and hence asymmetrically distributed in the cell (Fig. 4C, D, E, F). Mostly, coagulated material accumulated close to the cell wall and near the apical ends (Fig. 4C, D). Earlier, Goldberg and Goff
C
Fig. 3. Scanning electron micrographs of untreated and treated (4 h) E. coli cells: (A) Untreated cells (10.00 K), (B) Lemon grass oil vapor treated sample showing deformed and ghost (indicated by arrows) cells (10.00 K), (C) Lemon grass oil treated sample showing deformed cells (10.00 K).
(1986) suggested that this type of material is due to the precipitation of abnormal proteins. The formation of extra-cellular electron dense blebs (in Fig. 4C) on the surface of treated cells was also observed. It may represent collections of coagulated membrane and cytoplasmic constituents, which have pushed through holes produced in the cell wall by lemon grass oil vapors. These effects are believed to be caused by the ability of these substances to disrupt membrane structure (Koyama, Yamaguchi, Tanaka, & Motoyashiya, 1997). Similar observations have been reported by Rasooli, Rezaei, and Allameh (2006) for Listeria monocytogenes treated with thymus and for E. coli treated with tea tree oil (Gustafson et al., 1998) and cinnamon and oregano (Becerril, Gómez-Lus, Goñi, López, & Nerín, 2007). Wide range of morphological and ultrastructural alterations indicate that there are several targets in the cells and most likely the
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A
B
C
D
E
F
Fig. 4. TEM micrographs (×50,000) of E. coli (A, B) Untreated cells, (C–F) Lemon grass oil vapor treated cells (4 h) Arrows show the varying thickness of cell wall in lemon grass oil vapor treated cells.
antibacterial activity of lemon grass oil vapors is not attributable to one specific mechanism. To further establish the mechanism of action, it is necessary to identify the individual compounds and the overall chemical composition of lemon grass oil vapors.
3.4. Chemical characterisation of the lemon grass oil and its vapors Detailed chemical characterization of lemon grass oil vapors was done by SPME GC-MS, and compared with the chemical characterization
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of lemon grass oil done previously (Tyagi & Malik, 2010a) by GC-MS. In lemon grass oil, 37 components were identified, which represented about 94.5% of the total detected constituents. The essential oil contains a complex mixture consisting mainly of monoterpene hydrocarbons (7.9%), oxygenated monoterpenes (78.2%), sesquiterpene hydrocarbons (3.8%) and oxygenated sesquiterpenes (1.6%). The major constituents of the essential oil were α-citral or geranial (36.2%), β-citral or neral (26.5%), Nerol (5.1%), limonene (4.19%), Neryl acetate (4%) and 5hepten-2-one (2.9%) (Tyagi & Malik, 2010a). SPME GC-MS is a very sensitive and useful technique, which has been used recently for determining the headspace volatile composition of Michelia champaca flowers while still attached to the branch of the tree and soon after plucking (Rout, Naik, & Rao, 2006). SPME is performed by exposing a fiber coated with single or multiple polymers to the headspace of a sample matrix until equilibrium is reached between the volatile compounds partitioned in the fiber coating and the volatile compounds in the sample matrix. The amount of the volatile compounds absorbed onto the fiber is linearly proportional to its initial concentration in the sample matrix. The polymeric film on the fiber concentrates organic compounds on its surface through either adsorption or absorption. Qualitative and quantitative analysis of the lemon grass oil vapor listed in Table 1 shows that 13 compounds constituting 93.8% of the vapor could be identified. D-limonene (30.3%) was the major component, followed by α-citral (17.6%), β-citral (11.3%), 6-methyl hepten-2-one (14.6%), Camphene (6.5%), Nonane (4.1%), β-myrcene (3.5%), Linalool (1.5%), 2 propyl 1-pentanol (1.2%). Other components were present in amounts lesser than 1%. There are no previous reports available on the detailed composition of lemon grass oil vapor for comparison of these results. However the high antimicrobial efficacy could be attributed to the presence of oxygenated monoterpenes (48.5%) represented by citral compounds. Citral is a mixture of two isomers, geranial and neral, which are acyclic α, βunsaturated monoterpene aldehydes and are known to possess significant antimicrobial activity (Tzortzakis & Economakis, 2007). Earlier Inouye, Uchida, and Abe (2006b) studied the vapor activity of 72 essential oils against a T. mentagrophytes and observed that the vapor phase efficacy of oils could be readily correlated to the major component with the following trend; oils with phenolN aldehyde N alcohol N ketone, ester, and ether/oxide (lactones)N hydrocarbon. The same tendency was observed with the pure major components themselves as phenols and aldehydes exhibited the highest vapor activity, followed by alcohols, ketone, ester, ether/oxide (lactones), and hydrocarbon. Nevertheless, lemon grass oil vapor had higher antimicrobial activity than pure citral
Table 1 Chemical composition of lemon grass oil vapors. RT (min)
Compound
Percentage
RI
9.4 12.3 13.5 15.2 17.7 18 20.2 22.8 24.7 28.9 30.4 30.9 33.4
Camphene β-myrcene D-limonene β-ocimene Nonane 6-methyl hepten-2-one Verbenone 2 propyl 1-pentanol Linalool β-citral α-citral Neryl acetate Nerol Monoterpene hydrocarbons Oxygenated monoterpenes Total of identified compound
6.5 3.5 30.3 0.9 4.1 14.6 0.6 1.2 1.5 11.3 17.6 0.8 0.9 45.3% 48.5% 93.8%
1066 1156 1206 1228 – 1562 1733 – 1506 1680 1730 1735 1757
Retention Indices on AB-Innowax column. Relative area percentage without using the FID response correction factor. RT: Retention Time (min). (Results are based on GC-FID; MS acquisition started after 4 min).
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vapor, indicating the synergistic role of other vapor components such as linalool and limonene. Lemon grass oil vapor in the present study also contained monoterpene hydrocarbon (45.3%) such as D-limonene (30.3%), Camphene (6.5%) and myrcene (3.5%). Bakkali, Averbeck, Averbeck, and Idaomar (2008), reported that compounds, such as and limonene and linalool also have substantial antimicrobial activities. Hence, these compounds could also have shown the synergistic or additive effect in enhancing the antimicrobial activity of the main components. Apart from the chemical composition, the mode of action of the lemon grass oil and its vapor may also influence their antimicrobial efficacy. In the direct contact assays for liquid phase, the activity depends upon the diffusability and solubility of the essential oil compounds into the agar while the antimicrobial activity of the vapor assay depends upon the volatility of each compound (Goni et al., 2009). Since active compound of essential oils are highly volatile, therefore, essential oils possess high antimicrobial activity in vapor phase. Further, presence in gaseous form can better facilitate the solubilization of lipophilic monoterpenes in cell membranes thereby inflicting higher damages as compared to the liquid phase (Inouye et al., 2006a). On the basis of the microscopic analyses of the Candida cells, we have previously observed that lemon grass oil vapors are more potent than lemon grass oil for causing irreparable damage to Candida cells, probably due to better penetration and contact (Tyagi & Malik, 2010a). Some studies also suggest that antimicrobial activity of volatile compounds results from the combined effect of direct vapor absorption on microorganisms and indirect effect through the medium that absorbed the vapor (Inouye et al., 2001; Inouye et al., 2006b). A significant contribution of the volatile compounds through agar absorption was reported for E. coli (Gocho, 1991). Inouye et al. (2001) reported that when certain microbial strains on blood agar were exposed to lemon grass oil vapors, highest amounts of geranial and neral accumulated in the agar. On the other hand, the amount of DLimonene accumulated in the agar was very low. The authors suggested that absorption is governed by the hydrophobicity, volatility and stability of the volatile compounds. Although the agar and bacterial accumulation of volatile compounds have not been analyzed in the present study, it seems quite possible that the citral would have accumulated in the agar/microbial cells leading to higher toxicity while Limonene remained more in the vapor phase. In the present study, SEM/TEM evidence/alterations correlate well with the ability of monoterpenes (as per the SPME results) to interact with hydrophobic structures like bacterial membranes and guides us to the probable mechanism of action. With this evidence and data it is possible to suggest that lemon grass oil vapor kills microorganisms in a fashion similar to membrane active disinfectants. In order to develop the application of the lemon grass oil vapors, it is important to design an appropriate model equipped with a slowrelease device for vapor generation and to validate these results through kill time assays under this simulated environment. 3.5. Exposure regimes Further studies had twin objectives directed towards development of the application of lemon grass oil in vapor phase. Firstly, validation of the petriplate bioassay results on lemon grass oil vapors in a larger set-up which simulated an indoor environment. Secondly, to investigate the integration of vapors with another antimicrobial agent i.e. negative air ions (NAI). To accomplish this, a set-up as detailed out in methodology was designed and used (Fig. 1). Briefly, it facilitated the simultaneous generation of lemon grass oil vapors (through EOV generator) and NAI (through NAI generator) as well as each of these individually. Kill time assays were conducted in this set-up to establish the lag time required by E. coli ATCC 25922 to be altered by the active agents. Kill time assays were done by exposing the
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Reduction in viability (%)
inoculated plates to NAI alone, lemon grass oil vapor alone, and combination of lemon grass oil vapor and NAI in the closed airtight chamber for 12 h. Result of this study are shown in Fig. 5 in terms of percentage reduction in viability. NAI alone resulted in maximum 42% reduction in viability of E. coli after 12 h. These results are close to our previous observation of 33% reduction in viability of E. coli DH5α following 4 h exposure to similar concentration of NAI (Tyagi et al., 2008). Further, in the present study, the increase in killing beyond 4 h was almost negligible (Fig. 5) indicating that longer exposures may not materialize in increasing the efficacy of NAI. Moreover, it has been reported that normally very high concentrations of air ions are required to produce the antimicrobial effects and different microbial species could have variable responses to the system (Arnold et al., 2004). Kerr et al. (2006) studied the effect of six negative air ionizers installed in the Intensive Care Unit in a hospital. Ionizers caused no change in MRSA colonization/infection but Acinetobacter cases were significantly reduced. Therefore, to ensure better and reliable performance, NAI may be integrated with another antimicrobial agent such as lemon grass oil vapors. During the exposure to lemon grass oil vapor alone, the killing increased from 20.3% (0.5 h) to 87.5% (12 h) with increase in exposure duration. Based on the evaporation rate of the EOV and the chamber volume, the lemon grass oil vapor concentration during this period changed from 5.333 × 10 −3 μl/ml (at 0.5 h) to 0.128 μl/ml (at 12 h). Hence, these results are consistent with the results of disk volatilization assay, where a considerable increase in zone of inhibition was observed with increase in the lemon grass oil dosage (20–60 μl) corresponding to 0.364–1.091 μl/ml vapor dose. These results are very encouraging in suggesting that scale-up of the working volume from petri-plate bioassay to larger chamber did not decrease the killing effect of lemon grass oil vapors. Also, the slope of the graph (Fig. 5) suggests that extension of exposure time would have increased the killing further. It is also interesting to note that during the initial period (0.5 h), almost similar reduction in viability of E. coli due to the lemon grass oil vapor alone (20.3%) and NAI alone (20.0%) was observed. However, subsequently the rate of reduction in viability due to the lemon grass oil vapors alone was higher than NAI alone. As a result, after 12 h exposure to vapor alone (87.5%) and NAI alone (42%), significant variation in killing was observed. This can be explained through the facts that vapor concentration in the chamber increased gradually with time while the NAI concentration remained almost constant (N10 6 ions/ml). NAI are short lived with a typical lifetime of 100– 1000 s in clean air (Gaunt et al., 2005) and therefore their average concentration does not change. Further, the mode of action and
120 110 100 90 80 70 60 50 40 30 20 10 0
LGOV NAI LGOV + NAI
0
2
4
6
8
10
12
14
Time (h) Fig. 5. Kill time assay; percentage reduction in viability of E. coli cells due to pre-incubation exposure to negative air ions alone (NAI), lemon grass oil vapors alone (LGOV) and combination of both (LGOV+ NAI) for different time durations. Error bars represent the standard deviation at n= 3.
penetration in the cells may also be different in each case. Multiple mechanisms of action of NAI are reported such as the disruption of plasma membrane due to strong electrical field, secondary electrochemical effects such as generation of ozone or other reactive species (Fletcher et al., 2007) and reduction in airborne dust levels. The latter mechanism is most likely a physical effect of charged airborne particulates being strongly attracted to grounded surfaces such as walls and floors. Gast, Mitchell, and Holt (1999) reported that presence of negative air ionizers was associated with a 77.7% reduction in mean airborne dust concentrations in comparison to the control cabinets housing chicks and this was correlated with potential impact on the likelihood of airborne transmission of Salmonella enteritidis in this experimental setting. This offers a very useful synergy since essential oil vapors can't induce such effect but may be effective in deactivating the pathogen-bearing dust particles aggregated on selective surfaces. In fact, accumulation of potentially infectious particles onto adjacent surfaces or grounded parts of the ionizer itself, as suggested by the localized outbreak of TB-infected animals following ionizer cleaning (Escombe et al., 2007), is a great disadvantage to negative air ionization. In this regard, coupling of essential oil vapors with ionizers can result in deactivation of the accumulated pathogens. In the initial 2 h, the combined effect of lemon grass oil vapor and NAI (38.1%) on E. coli was higher than the NAI alone (23.0%) but slightly lesser than the lemon grass oil vapor alone (43.7%). However, after 4 h exposure, the rate of bacterial killing due to combined effect of lemon grass oil vapor and NAI increased over either of these alone and 100% reduction in viability was observed within 8 h exposure. These results indicate that in the initial phase antagonistic interaction between the two agents occurred but in the later phase NAI enhanced the efficacy of lemon grass oil vapors significantly. The reaction between the ionic species and the volatile molecules leads to the production of ionic gaseous species that have an enhanced antimicrobial activity (Gaunt et al., 2005). Volatile compound becomes attached to ions produced by the ionizer, and these charged particles have a stronger membrane permeability and antibacterial effect. Additionally the charged particle will have greater mobility and attraction to surfaces that an uncharged volatile molecule would not. Membrane damage is believed to be responsible for the death of bacterial cells from both ionic exposure regimes (Setti & Micetich, 1998) and essential oils (Fan, Song, Hildbrad, & Flourey, 2003; Hildebrand et al., 2001). Our microscopic studies confirm that lemon grass oil vapors cause loss of cytoplasmic material due to membrane damage. It therefore seems probable that these treatments complement each other in challenging bacterial membrane integrity. Gaunt et al. (2005) also reported that the combination of an ionizing source, such as a corona discharge or a candle flame, with a bactericidal volatile (β-pinene) has a greater effect on E. coli than either treatment alone. Earlier studies have demonstrated a strong synergism between ozone and NAI on bacterial cell death. Tanimura, Hirotsuji, and Tanaka (1998) reported that ozone and NAI were more effective in combination than either alone. Song, Fan, Hildebrand, and Forney (2000) showed that surface mold of onions could be significantly reduced in commercial storage if they were exposed to an atmosphere of ozone and NAI. Nevertheless, E. coli has been found to be most resistant organism requiring 17 hour exposure to a combination of ozone (100 ± 5 ml/l) and NAI (10 6/ml) for complete kill (Fan et al., 2003). Hence, an integration of lemon grass oil vapors with NAI that ensures complete kill within 8 h in the present study shows better efficiency. 4. Conclusion On the basis of above results it can be stated that lemon grass oil is highly effective in vapor phase and its efficacy can further be enhanced by integration with NAI for reducing the viable microbial load. Future research on chemical species generated during NAI-
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