Antibacterial Activities of the Extracts of Some Herbs Used in Turkish Herby Cheese Against Listeria Monocytogenes Serovars

Abstract

In this study, antimicrobial activities of the four different extracts (in methanol, ethanol, n‐hexane and water) of some herbs including Allium vineale, Chaerophyllum macropodum , and Prangos ferulacea were investigated against Listeria monocytogenes serovars 1/2b, 4b, and 4ab. For many centuries, these herbs were used in food production in Turkey. While water extracts of all the plants did not show any antibacterial activity, the other extracts of these plants showed variable degrees of antibacterial activity against L. monocytogenes serovars tested. The extracts of Allium vineale showed higher antibacterial activity than the other plants. The methanol extracts of all the plants exhibited higher activity than the ethanol and n-hexane extracts. L. monocytogenes 4ab was the most sensitive serovar to extracts of these plants. The results indicated that the methanol and ethanol extracts of these plants displayed remarkable activity against L. monocytogenes serovars and therefore, they could be used as natural anti-Listeria monocytogenes additives in herby cheese.

Keywords:

• Listeria monocytogenes
• Antilisterial
• Herbs

INTRODUCTION

Listeria monocytogenes is a Gram-positive, short, motile, psychrotropic rod that can cause serious illness such as septicemia and meningitis in humans. It is a widely spread environmental microorganism found in sewage, silage, soil, fertilizer, vegetable matter, and many foods.[1] This pathogen is relatively resistant to various environmental conditions. It can grow at temperatures of 0–45°C, at pH values of 4.3–9.5 and survive for long periods in frozen or dried foods and high salt concentrations.[2] Food-associated outbreaks of human listeriosis involving many deaths[3–5] have led to an awakening of interest in controlling its diffusion in foodstuffs. Food industry at present uses chemical preservatives like sodium acetate and lactate to prevent the growth of L. monocytogenes in food products. However, due to the consumer's concerns over the safety of some chemical preservatives, a lot of attention has been paid to natural products. This situation forced the scientists to search new antimicrobial substances from various sources like medicinal plants.[6,7] Although many herbs and their derivatives have been studied in detail against several bacteria,[8–14] there are lacking researches on antibacterial activities of Allium vineale, Chaerophyllum macropodum, and Prangos ferulacea. Herbs like A. vineale, C. macropodum, and P. ferulacea are used as food ingredients e.g., Van herby cheese, to offer aroma and flavor. In addition, it is believed by local people that these plants have antibacterial activity. Therefore, the lack of documented knowledge of its claims prompted us to examine the effects of extracts of these plants against L. monocytogenes. The aim of this study was to investigate the anti-Listeria monocytogenes activity of methanol, ethanol, n-hexane, and water extracts of Allium vineale, Chaerophyllum macropodum and Prangos ferulacea.

MATERIALS AND METHODS

Plant Material

Allium vineale L. (Liliaceae), Chaerophyllum macropodum Boiss. (Apiaceae), and Prangos ferulacea (L.) Lindl. (Apiaceae) were collected from Alacabuk (Pelli) Mountain (Bitlis, Turkey) in August 2003, with assistance of knowledgeable villagers. They were identified by Dr. Fevzi Ozgokce, Department of Biology, University of Yuzuncu Yil (Van-Turkey) where the voucher specimens have been deposited (F 11 178, F 11 176, and F 11 175, respectively, VANF). The plants were separately dried in shade, pulverized by a mechanical grinder, and stored in airtight glass containers in the dark until extraction.

Preparation of Extracts

Four solvents, methanol, ethanol, n-hexane, and water were used at herb extractions. Thirty grams of the dried and powdered plant materials were extracted with 300 ml of solvents by using Soxhlet apparatus for 10 h at a temperature not exceeding the boiling point of the solvents.[15] The extracts were filtered using Whatman No. 1 filter paper and then concentrated under vacuum at 40°C using a rotary evaporator. The resulting materials were stored in a desiccator until further use.

Listeria Monocytogenes Serovars

The three L. monocytogenes serovars, L. monocytogenes RSKK 472 (serovar 1/2b), L. monocytogenes RSKK 475 (serovar 4b), obtained from Refik Saydam National Type Culture Collection (RSKK) in Turkey and L. monocytogenes serovar 4ab, obtained from Central Veterinary Control and Research Institute in Ankara in Turkey were used in these experiments. Individual stock cultures were maintained at 4°C on tryptone soya agar (Oxoid) slants through bimonthly transfers.

Disc Diffusion Method

The antibacterial activity of the extracts was determined using disc diffusion method.[9,10] The residues were dissolved in their extracting solvents to yield the final concentration of 50 mg/ml, sterilized by membrane filter (pore size 0.45 μm). For the preparation of the inoculation, the bacteria were cultured in tryptone soya broth (Oxoid) at 35°C for 24 hours and standardized for the same absorbency, number 0.5 of the McFarland Nephelometer, which corresponds to the order of 10⁸ cfu/ml.[16] One hundred microliters of prepared cultures were inoculated on surface of Mueller-Hinton agar (Oxoid). Sterile filter paper discs (Whatman No. 3, diameter 6 mm) were soaked with extracts of 50 mg/ml and placed on the culture medium. Negative control discs were soaked with the same solvents employed to dissolve the plant extracts. Antibiotic discs of ampicillin (10 μg) (Oxoid) were also used as positive control. The plates were kept at ambient temperature for 30 min to enable diffusion of extracts and then incubated at 35°C for 48 hours. The antibacterial activity was evaluated by measuring the diameter of inhibition zone. Each experiment was repeated at three times and the mean of the diameter of the inhibition zones was calculated.

RESULTS AND DISCUSSION

The solvents used in the study showed no activity against any serovars of L. monocytogenes studied (Table 1). The anti-Listeria monocytogenes activities of Allium vineale, Chaerophyllum macropodum and Prangos ferulacea using four different solvents are presented in Table 1. While the methanol and ethanol extracts of all the plants species had antibacterial activities against all the L. monocytogenes serovars, the n-hexane extract of Allium vineale was active also against only serovar 4ab. However, the water extracts of these plants showed no activity against any of L. monocytogenes serovars tested. These results are similar to those reported by other workers, where water extracts of the plants do not have much activity against bacteria.[17,18] Ozcan[19] reported that methanol was the most effective solvent for plant extraction; our findings were similar to this report. There were differences among the three serovars in term of the antibacterial activity. The most susceptible and resistant serovars to the antibacterial activity of the tested extracts were L. monocytogenes 4ab and L. monocytogenes 4b, respectively. Kim et al.[20] noted that bacterial serovars respond differently to the action of various essential oils. Some researchers announced that L. monocytogenes was highly inhibited by essential oil of conifers.[21] Similarly, Wen et al.,[22] Baydar et al.,[13] Kumar et al.,[23] and Mytle et al.[24] reported that phenolic acids, the essential oils of oregano, wild oregano, thyme and savory, black cumin and clove oils were active against L. monocytogenes, respectively. Regardless of the plant source, the methanol extracts showed relatively the best antibacterial activity against L. monocytogenes serovars. These results are in accordance with those from previous screenings of plants for antimicrobial activity, where it was reported that methanol is a better solvent for extraction of antibacterial substances from plants than other solvents.[25,26,27] It is known that the organic solvents usually provide a higher efficiency in extracting compounds of antimicrobial activities, compared with water extraction.[28] Most of the identified components with antimicrobial activity extracted from plants are aromatic or saturated organic compounds and they are more soluble in methanol and ethanol.[29] The components concerning with antibacterial activity in the extracts were not determined in this study. However, it might be suggested that antimicrobial components from studied plants could be extracted by using methanol better than other solvents studied for antibacterial analyses as previous studies.[19] Methanol is a toxic solvent, so it must not be used in food systems. Allium vineale exhibited the best antibacterial activity against L. monocytogenes serovars compared to Chaerophyllum macropodum and Prangos ferulacea. Previous studies showed the high antibacterial activity of sulfur and other numerous phenolic compounds found in Allium plants.[30,31] Therefore, the higher antibacterial activity of methanol extract from Allium vineale may be due to the higher phenolic content of this plant. Extracts from the genus Prangos were found to be rich in coumarin and terpenoids.[32,33] The coumarin derivatives have been reported to have a slight antibacterial activity against Escherichia coli and Candida albicans.[34,35] Terpenes have also been found in Chaerophyllum plants and they have been shown to be useful in the control of L. monocytogenes.[35,36]

Table 1 Antibacterial activities of herb extracts on L. monocytogenes serovars ^a .

		Inhibition zone diameter (mm)
Plant species	Extracts	Lm 1/2b	Lm 4b	Lm 4ab
Allium vineale	Methanol	13	11	15
	Ethanol	9	8	14
	n-hexane	– ^b	–	9
	Water	–	–	–
Chaerophyllum macropodum	Methanol	10	7	13
	Ethanol	8	7	11
	n-hexane	–	–	–
	Water	–	–	–
Prangos ferulacea	Methanol	10	8	13
	Ethanol	9	9	11
	n-hexane	–	–	–
	Water	–	–	–
Negative control (Absolute solvents)	Methanol	–	–	–
	Ethanol	–	–	–
	n-hexane	–	–	–
	Water	–	–	–
Positive control, (Ampicillin, 10 μg/disc)		17	15	18

CONCLUSION

The results obtained from the present study indicated that the methanol and ethanol extracts of studied plants showed antibacterial activity against L. monocytogenes serovars. Among the plants tested, Allium vineale showed very promising and significant antibacterial activity against L. monocytogenes serovars. This study is a preliminary evaluation of antibacterial activity of the plants studied. Further phytochemical studies are required to elucidate the components responsible for antibacterial activity of these extracts against L. monocytogenes.

Notes

^aBacteria: Lm 1/2b, Listeria monocytogenes serovar 1/2b; Lm 4b, Listeria monocytogenes serovar 4b; Lm 4ab, Listeria monocytogenes serovar 4ab.

^b–: inactivity.