Inhibition of Listeria monocytogenes by natural antimicrobial

The aim of the study was to evaluate antimicrobial activity of essential oil from plants as natural antimicrobial to inhibit Listeria monocytogenes. A total of 6 essential oil extracted from galangal (Alpinia galanga), ginger (Zingiber officinale), lemongrass (Cymbopogon citratus), orange skin (Citrus sinensis), rosemary (Rosmarinus officinalis) and turmeric (Curcuma longa). By using Minimum Inhibitory Concentration (MIC), the highest and lowest antimicrobial activity of essential oil to inhibit the growth of L. monocytogenes was found on lemongrass oil (0.32 % ± 0.12 %) and turmeric (7.46 % ± 2.79%), respectively. By using disc diffusion assay, the highest antimicrobial activity to against L. monocytogenes was shown by lemongrass oil (7.46 ± 2.79 mm). There is no antimicrobial activity observed in orange peel oil. Out of the essential oil tested, lemongrass oil showed the most promising natural antimicrobial to inhibit L. monocytogenes.


Introduction
Essential oils, the natural antimicrobial, have been known for decades and, with increasing demand from changes in food consumer trends and isolation of antibiotic resistant microorganisms, being other to chemical based antimicrobial which introduce the accumulative residue and give an adverse human health. Essential oil are composed by bioactive compounds which known as generally recognized as safe (GRAS) to food and might be required by food industries for ensuring food safety. Xie et al. [1] revealed that the biological agents of essential oils including phenolics and polypenols have to be high activity against pathogenic bacteria [1,2].
L. monocytogenes is a Gram-positive bacteria and become the main causal agent of listeriosis [3,4], the rare but lethal food-borne disease, especially for susceptible group [5]. This group was categorized as young, old, pregnant, and immunocompromised (YOPI) [6]. The contamination of L. monocytogenes may occur during food chain and could be spread to food processing equipments and may contaminate food products [7,8].
The study of essential oils, natural antimicrobials, extracted from Indonesia plants has to be investigated further. To do so, this study was aiming to evaluate antimicrobial activity of essential oil from plants as natural antimicrobial to inhibit Listeria monocytogenes. Nevertheless, it becomes a need to get the effective antimicrobial and safe to human health.

. The strains of microorganism
Pure culture of L.monocytogenes (Nusaroma, Gajah Mada University, Yogyakarta) was cultured on Agar Listeria Ottavani and Agosti (ALOA) (Himedia, India) with supplement of ALOA (Himedia, India) for 24 until 48 hours at 37°C. The colonies of L. monocytogenes were appeared as blue colonies with halo surround the colonies. By using streaking method, a single colony was purified onto culture media namely tryptic soya agar (TSA) (Himedia, India).

Minimum Inhibitory Concentration (MIC)
MIC was measured as done by Budiati et al. [2]. Cultures were grown at 37 o C for 16 hours in Luria Broth (LB, Himedia, India) in shaking incubator. The cultures were diluted and compared the turbidity. This has to be set at 0.5 of McFarland scale. A serial concentration was prepared on Mueller Hinton Broth (Oxoid, United Kingdom). A total of 20 μL of the aliquot was artificially contaminated to the serial dilution. These were incubated at a temperature of 37 o C for 24 hours. MIC value (%) describes the lowest concentration of the bacteria might grow. These values are calculated as mean values of three measurements. The experiment was done in triplicate. A 15 µl aliquot of 10% DMSO was dropped onto a serial dilution as negative control. Penicillin G (10 unit/ml) was used as positive control.

Disc diffusion assay
By using disc diffusion assay, the antimicrobial activity was measured as the method of Bauer [9]. Cultures were grown at 37 o C for 16 hours in Luria Broth (LB, Himedia, India) in shaking incubator. The cultures were diluted and the turbidity was set at 0.5 of McFarland scale. By using a sterilized cotton swab, the cultures were swabbed onto Mueller Hinton Agar (Oxoid, United Kingdom) and were let about 30 min. Twenty microliter essential oil was dropped on sterilized disc. This disc was then placed onto the agar. This was incubated for 18-24 h at 37 °C. The inhibition zones was measured in four different directions after the incubation period. This was recorded as milimeter. The experiment was done in triplicate. Fifteen microliter aliquot of 10% DMSO was dropped onto a sterile paper disc as negative control. Penicillin G (10 unit/ml) was used as positive control.

Statistical Analysis
By using one-way ANOVA (SPSS version 13.0), the differences of antimicrobial activity among essential oils to inhibit the growth of L. monocytogenes were determined at a significance level of P < 0.05.

Results and Discussion
Wide-spectrum of antimicrobial extracted from plant namely essential oils to against Gram-positive bacteria have been well documented [10,11]. L. monocytogenes is a Gram-positive bacteria that can be inhibited by essential oil [12,13,14]. By using disc diffusion assay (inhibition zone) and MIC, the antimicrobial activity of six different essential oils extracted from plant in Indonesia to against L. monocytogenes was studied. This present study found that the highest antimicrobial activity to inhibit L. monocytogenes was observed on lemongrass as shown in table 1. This is similar to other study that reported that lemongrass was effective to inhibit Gram-positive bacteria such as L. monocytogenes [15]. L. monocytogenes was composed by peptidoglycan which was disrupted due to the exposure of citrate and other terpene in lemongrass. The devastating effect in the microbial cell by disrupting the cell membrane integrity by this compound may inhibit the growth of L. monocytogenes [16,17,18]. This may disrupt to the fatty acid biosynthesis and peptidoglycan biosynthesis [19]. Hadjilouka et al. [19] also reported that the gene expression in L. monocytogenes was observed due to the exposure of lemongrass essential oil. The genes were accP, accA and fapR that involved in fatty acid biosynthesis. Moreover the expression genes of murR and pbpB was also reported to be downregulated [19] to cause the disruption of peptidoglycan biosynthesis.
This present study observed that the MIC value of ginger oil, galangal oil, orange peel oil, rosemary oil and turmeric oil were significant different for each other (table 1). However, inhibition zone of ginger oil, galangal oil, orange peel oil, rosemary oil and turmeric oil were not significant different. It indicated that all those essential oil were not effective to inhibit L. monocytogenes compared to lemongrass oil. The lowest antimicrobial activity was observed on turmeric oil by using MIC. The majority compound of turmeric was curcurmin and other compounds such as camphor, terpenes, lactones, alkaloids, and phenols [21]. The presence of those compounds indicated to be not effective to against L. monocytogenes. This is similar to other studies. Thongson et al. [22] reported that turmeric had no antimicrobial effect against L. monocytogenes. There is no antimicrobial activity was observed on orange peel oil. The compound of orange peel oil was dominated by limonene [23]. In contrary, the study of Fisher and Philips [23] reported that orange peel had antimicrobial effect to L. monocytogenes.

Conclusion
Lemongrass oil showed the highest antimicrobial effect to against the growth of L. monocytogenes. These essential oil seem to be a potential natural antimicrobial for inhibiting L. monocytogenes which was safe to food product. This was also a potential natural antimicrobial that could be used as disinfectant to clean food processing equipments which was vulnerable for the growth of L. monocytogenes as biofilms.

Acknowledgment
The financial support was provided by PNPB research scheme -Politeknik Negeri Jember is gratefully acknowledged.