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ANTIBACTERIAL OF Kaempferia pandurata Roxb ESSENTIAL OIL TUBERS ON Listeria monocytogenes INCLUDING

MECHANISM AND APPLICATION

Miksusanti Jurusan Kimia FMIPA UNSRI

ati_mik@yahoo.com

ABSTRACT The objective of this research are to analyze antibacterial activity of Temu Kunci essential oil and use the oil as antibacterial agent in sago starch edible film. Compound of essential oil which is trapped in sago starch edible film and were analyzed with GC-MS. MIC and MBC temu kunci essential oil were 0.009 (v/v) and 0.04(v/v) respectively. Incorporation of the oil into sago starch film can turn the film to be antibacterial edible film. The qualitative test for antibacterial activity of the films were performed with disk inhibition zone assays. The film showed positive contact area with concentration oil start from 0.1 (ml/g). Inhibitory zone were showed when the concentration of oil in the film was 0.7 (ml/g). Key words: Listeria monocytogenes, MIC, MBC, antibacterial edible film, sago starch. Introduction Listeria monocytogenes, a facultative bacterial pathogen, causes disease in high risk groups, including pregnant women, neonates, immunocompromised adults and occasionally, people who have no predisposing underlying condition. Listeriosis is associated with height mortality rate, of nearly 30% . L. monocytogenes is widely distributed throughout the natural environment, and may also colonize the environment of food processing facilities and contaminate processed food stuffs (Arques et al. 2005).

L. monocytogenes is psychotropic food-borne pathogens. Many effort has been done to fulfill consumer demands for “natural” foods by using minimally processed, such as vacuum-packaged, and refrigerated product. However, the microbial safety of these products still being questioned because of psychotropic bacteria such as L. monocytogenes.

Because of its ubiquitous distribution and its association with domestic livestock, L. monocytogenes is likely to occur in raw meats. Its prevalence both at the slaughterhouse and in the processing environment increases the potential for post processing contamination. The incidence and growth of L. monocytogenes in processed meat products are well documented (Johnson et al. 1990). Its prevalence ranges from 5 to 13 % in ready to eat meat product, in which typical plate counts ranged from < 10 to 1,000 CFU/g. Although most cases of human

listeriosis associated with the consumption of ready to-eat meat products appear to be sporadic, the high mortality rate has led the Food and Drug Administration to impose a zero-tolerance policy for L. monocytogenes in ready-to-eat meat products ( Winskowsky et al. 1993).

Antibacterial agents, including food preservatives have been used to inhibit this kind of food borne bacteria and extend the self life of processed food. Many naturally accruing extracts like essential oils from edible and medicinal plants, herbs and spices have been shown to possess Antibacterial functions and could serve as source for Antibacterial agents against food spoilage and pathogens. More particularly, essential oils and their components are known to be active against a wide variety of microorganisms, including gram-positive bacteria such as L. monocytogenes (Oussalah et al. 2006). The purpose of this study was to validate the use of essential oil from Temu Kunci (TK) tubers (Kaempferia pandurata Roxb) to inhibit L. monocytogenes. The present study to examine effect of essential oils from Kaempferia pandurata Roxb on L. monocytogenes cell. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the leakage of cell, and the morphology change were determined using a range method. Materials and Methods Extraction of the essential oils Air-drying of temu kunci tuber was performed in a shady place at room temperature for 10 days. The tuber were

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M irsen Kamphor Sineol Osimen Linalol Borneol Terpineol Geraniol AsamSinamat

Molekul Penyusun Minyak Atsiri

used for the analysis of essential oil composition. A portion (2 kg) of temu kunci tuber was submitted for 3 h to hot water-distillation, using a Clevenger-type apparatus. The obtained essential oil (EO) was dried over anhydrous calcium sulphate and 2 μl was used for GC -MS measurements GC-MS analysis conditions The analyses of the volatile compounds were carried out on a Hewlett-Packard GC-MS system (GC 5890 Series II; MSD 5971A) according to Velasco et al ( 2003). Preparation of Listeria monocytogenes. L. monocytogenes FNCC 156 were used as a pool. Preparation of L. monocytogenes were conducted by the protocol that had been published by Harrison et al (1990). Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The MIC and MBC of tested essential oils were determined using the agar dilution methods. The protocol of this procedure were Schillinger et al. (1998). Leakage of cellular metabolites The experiments of assessing potassium and calcium ion leakage were conducted according to Lin Chia-Min et al (2000), and Hong Seok-In et al. (1999). Incorporation Essential Oil of Kaempferia pandurata Incorporation this essential oil in sago starch edible film were conducted with protocol that had been published by Pranoto et al (2005) Statistical analysis of data Analysis of variance and Duncan’s multiple-range test were employed to analyze data collected. Differences between means were considered significant when P< 0.05. This experiment was conducted twice with four plates for each essential oil at each concentration. Result Composition of the essential oils Dried aerate of temu kunci tuber were subjected to hydro distillation and the obtained essential oil were analyzed by GC/MS. Overall 34 compounds were characterized in this oil. The main components were identified as monoterpene

hydrocarbon (Myrcene 4.58%, ocimene 20.18%,champhene 4.58% ), oxygenated monoterpene (β-linalool 2.42%, cineole 9.58%, camphor 20.39%, borneol 1.07%, terpineol 1.52%, Geraniol 22.28%, cinamic acid 6.6% ). Others compound are not mentioned because exist less than 0.01%. The diagram below shows the resolution intensity of HP-20M column, standard deviation of yields oil range, and concentration compounds in essential oil (% w/w). Fig 1. Main component of essential oil from temu kunci tuber (Kaempferia pandurata Roxb) Ocimene geraniol and cineole are found as the main component in tuber of temu kunci oil (Kaempferia pandurata Roxb) but, some species and cemotypes may contain mainly camphor and ocimene (Chairul et al. 2000). Antibacterial activity of temu kunci oil Antibacterial activity of temu kunci oil, was investigated in terms of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) compared with some other natural antibacterial. MIC and MBC temu kunci oil in ethanol solvent were 0.009% (v/v) and 0.04% (v/v). It’s MIC is much lower than those other natural antibacterial agent such as essential oil from Eucalyptus Cilantro is 0.02% (v/v) and essential oil from Eucalyptus coriander 0.47% (v/v) for it’MIC against Listeria monocytogenes (Delaquis et al . 2006). Leakage of potassium and Sodium ions Temu kunci oil at 1 MIC, and 2 MIC induced leakage of potassium ions from L. monocytogenes (Fig 2). The data, which was representatives of triplicate experiments that gave similar results, shows that leakage from L. monocytogenes cells commenced immediately upon addition of

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temu kunci oil and the extent approached 100% of total celuler after 1 hour. Fig 2. Effect of temu kunci oil on potassium leakage and calcium leakage of Listeria monocytogenes Increases in the temu kunci oil concentration in bacterial suspension caused increases in celuler leakage of potassium and calcium ion. All treatment gave significantly different in the leakage of potassium and calcium ion (P<0.0001;F value=19714.5). Duncan analysis showed that each concentration of ion were in certain group (A=MBC;B=2MIC;C=1MIC;D=0). Application of Essential Oil in Sago Starch Edible Film Essential oil of Kaempferia pandurata were incorporated in sago starch edible film to make antibacterial wrapping film for food preservative. At 0.05% (v/b) essential oil, the film did not showed antibacterial properties. Antibacterial film start at concentration of oil 0.1% (v/b), but showed no inhibition zone around the film. The inhibition zone were observed start at 0.7% (v/b) essential oil in incorporating in the sago starch edible film.

Discussion Major component of the essential

oil from temu kunci tuber (Kaempferia pandurata Roxb) are oxygenated monoterpene which mainly consist of conjugated hydroxyl group such as linalool, geraniol, and cinamic acid. The presence of hydroxyl group at the conjugated molecule, make it potential as antibacterial. A characteristic feature of conjugated hydroxyl group is significantly greater acidity than that of non conjugated aliphatic hydroxyl group (Fessenden 2000). We proposed that linalool, Geraniol and cinamic acid acts as a trans-membrane carrier of monovalent

cation by exchanging its hydroxyl proton for another ion such as a potassium ion. Undissociated molecule diffuses through the cytoplasmic membrane towards the cytoplasm and dissociates by releasing its proton to the cytoplasm. It may then return undissociated by carrying a potassium ion (or other cation) from the cytoplasm which is transported through the cytoplasmic membrane to the external environment. A proton is taken up, and the compound in the protonated forms diffuses again through the cytoplasmic membrane and dissociated by releasing a proton to the cytoplasm. This hypothesis is supported by the observed efflux of K+, and influx of H+ in B.cereus (gram positive bacteria) during exposure to carvacrol (compound with have conjugated hydroxyl) (Cox et al. 2000, Ultee et al. 2002). This phenomena could be associated with L. monocytogenes (which is also a gram positive bacteria) treated with essential oil from temu kunci. In this essential oil, we have cinamic acid which is more acid than acetate acid but still a weak acid. The mechanism of this compound can be resemble as weak acid to inhibit L. monocytogenes. Other compounds in temu kunci essential oil which is not have conjugated hydroxyl, act as cyclic hydrocarbons in inhibiting microbial growth. Many cyclic hydrocarbons, e.g. aromatic, cycloalkanes, and terpenes are toxic to microorganisms. Due to the hydrophobic character of cyclic hydrocarbons, the primary site of their toxicity is the membrane. Hydrocarbons accumulate in the lipid bilayer according to a partition coefficient that is specific for the compound. Accumulation of compounds in the membrane may lead to alteration of the membrane structure and function. An important change is the apparent increase in surface area of the membrane upon accumulation of liphopilic compounds. The expansion observed with hydrocarbons was more than two times higher than the expansion by alcohols. This variation is probably due to differences in type of hydrophobic interaction and part of the membrane where liphopilic compound reside (Sikkema et al. 1994). The conjugated hydroxyl compounds in temu kunci essential oil (cinamic acid, geraniol, linalool) play important rule in leakaging the potassium ion, calcium ion and absorbing material at 260 nm as well as at 280 nm to L. monocytogenes.

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The cyclic hydrocarbons in temu kunci essential oil (champhen, Myrcene, ocimene, camphor, borneol, terpineol) can act as toxic to L. monocytogenes due to its lipophilic character. These cyclic hydrocarbons interact hydrophobically with L. monocytogenes membrane and accumulate in this membranes. The effect can bee seen in scanning electron microscopy picture. From that picture, L. monocytogenes become larger unsymmetrical after treating with essential oil of temu kunci tuber. The result of this accumulation can effect to the glucose-dependent respiration, morphology and ability to exclude certain molecules. The accumulation cyclic hydrocarbon in the membrane can lower the activity of cytochrome c oxidase and increase proton (ion) permeability. Both effects act synergically on the magnitude of the ΔpH and Δø generated by cytochrome c oxidase. Since a 50% reductions of cytocrome c oxidase activity only causes a small drop in the ΔpH and Δø. At the end this process can cause swelling and lysis (Sikkema et al. 1994, Carson et al. 2006). This fact can be analyzed with MBC data. At minimal inhibitory concentration (MBC), all L. monocytogenes cell can be eliminated. In conclusion, our observations confirms that Antibacterial activity essential oil of temu kunci tuber results from its ability to disrupt the permeability barrier of microbial membrane structures of L. monocytogenes. The essential oil of Kaempferia pandurata can be incorporated in sago starch film. The content of essential oil in film can turn the film to be antibacterial film. The antibacterial film can inhibit the growth of L. monocytogenes. This antibacterial film can be used to wrap souses in order to preserve it without using synthetic preservation. The result of hedonic test showed that panelist still can accept the souses which was wrapped by antibacterial edible film (data is not shown in this publication). Conclution The essential oil of Kaempferia pandurata has potential antibacterial properties against L. monocytogenes. The results of this study suggest the possibility of using the essential oil or some of their component as natural food preservatives, because this oil has the low concentration of MIC and MIB. Further research is needed in order to obtain information regarding the

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