ANTIBACTERIAL ACTIVITY OF AROMATIC PLANTS ESSENTIAL OILS FROM SERBIA AGAINST THE LISTERIA MONOCYTOGENES

The purpose of this study was to examine the effectiveness of selected essential oils for the control of the growth and survival of pathogenic bacteria Listeria monocytogenes ATCC 19112 and Listeria monocytogenes ATCC 19115, which are of significant importance in food hygiene. Essential oils extracted from Salvia officinalis L., Rosmarinus officinalis L., Majorana hortensis Moench., Thymus vulgaris L., Carum carvi L., Pimpinella anisum L. and Coriandrum sativum L. were evaluated. Antibacterial activity was done by the disk diffusion method in the presence of pure essential oils and four suspensions in aclcohol. The best results obtained with Thymus vulgaris and Majorana hortensis essential oils, which were acting microbicidaly on both observed strains of Listeria monocytogenes, even in the small concentration. Because some of the essential oils were highly inhibitory even in small quantities to selected pathogenic bacteria, they may provide alternatives to conventional antimicrobial additives in foods.


I n t r o d u c t i o n
Listeria monocytogenes is a Gram-positive, rod-shaped bacteria, found in soil and water.Vegetables can become contaminated from the soil or from manure used as fertilizer.Animals can carry the bacteria without appearing ill and can contaminate foods of animal origin such as meats and dairy products.The bacteria can be found in a variety of raw foods, such as uncooked meats and vegetables, as well as in processed foods that become contaminated after processing, such as soft cheeses and cold cuts at the deli counter.Potential sources of listeriosis are ready-to-eat processed foods such as meat products and unpasteurized dairy products that are stored for long periods at refrigerator temperature (4 o C) (M a d i g a n at al., 2000.).Both unpasteurized (raw) and pasteurized milk may contain the bacteria, too (X).Listeria monocytogenes is the agent of listeriosis, a serious infection caused by eating food contaminated with the bacteria.Listeriosis is a serious disease for humans; the overt form of the disease has a mortality greater than 25 percent.The two main clinical manifestations are sepsis and meningitis.Meningitis is often complicated by encephalitis, a pathology that is unusual for bacterial infections.
Medicinal, aromatic and herb spices from family Apiaceae like Caraway (Carum carvi L.), Anise (Pimpinella anisum L.), Coriander (Coriandrum sativum L.) and from family Lamiaceae like Sage (Salvia officinalis L.), Rosemary (Rosmarinus officinalis L.), Marjoram (Majorana hortensis Moench.) and Thymi (Thymus vulgaris L.) are widely distributed in Serbia growing as wild or cultivated species (S a r i ć , 1989.).These plants are used like stomachic, spasmolitic, carminative and expectorant in traditional folk medicine and also in official medicine.Essential oils extracted from Apiaceae and Lamiaceae family plants can contribute the quality of food with better odor and flavor what is considerd as very important quality parametar in food manufacturing (K o v a č e v i ć , 2001).Other benefites could be application of essential oils in therapeutics purposes due to their antimicrobial effects on some pathogen microorganisms (S t e f a n i n i at al., 2001., K l a u s et al, 2007.).
Essential oils of Carvi aetheroleum, Anisi aetheroleum and Coriandri aetheroleum are usualy obtained from the ground, mature and dry fruits.The fruit of Carvi fructus contains 2-7% of essential oils.The major ingredients of essential oils are monoterpens, ketone-carvone (50-80%) and carbohydrates d-limonen (15-35%), which are well known to contribute to the carminative effect (M i c k l e f i e l d et al., 2000.).
The fruit of Anise (Anisi fructus) contains 1.5-5% of essential oils.The major ingredient being anethole (80-90%).With characteristic odour and taste it is often used in the food processing industry to enhance the odour and the taste of food commodities (K u b e c z k a et al., 1986.).It is also used in medicine as expectorant and carminative.
The fruit of Coriander (Coriandri fructus) contains up to 1% of essential oils.Monoterpens are the major ingredients of this oil with limonen (40-60%) as predominate constituent.The essential oils of Coriander has stomachic, spasmolitic and carminative effect.It also has bactericidal and fungicidal effect (B a r a t t a et al., 1998.).It is used in the food processing industry as a spice and aroma enhancer.
The folium of Rosemary (Rosmarinus officinalis L.) contains up to 2% of essential oil.The major ingredients of essential oil of Rosmarini aetheroleum are cineol (35%), pinens, camphor and borneol.It is used in the food processing industry as a spice and aroma enhancer (A n g i o n i at al., 2004.).
The herba of Thymi (Thymus vulgaris L.) contains 1-2% of essential oil.Monoterpens are the major ingredients of this oil with timol (40%) as predominate contstituent and caracrvol, which are well known like antimicrobial substances.The essential oils of Thymi has spasmolitic and expectorative effects (M a r i n o at al., 1999.).
The herba of Marjoram (Majorana hortensis Moench.)contains 1-2% of essential oil (terpinen-4-ol, borneol).It is also used in folk medicine as stomahic and as a spice and aroma enhacer in food processing industry (B i o n d i at al., 1993.).
The aim of this work was to investigate antibacterial activity of these essential oils on bacteria Listeria monocytogenes ATCC 19112 and Listeria monocytogenes ATCC 19115.

Materials and Methods
Antibacterial effects of essential oils extracted from Apiaceae and Lamiaceae family plants on Gram-positive bacterial strains Listeria monocytogenes ATCC-19115 and Listeria monocytogenes ATCC-19112 were investigated.ATCC cultures were taken from American Type Culture Collection, Rockville, Maryland.
The cultures were adjusted to approximately 10 5 CFU/ml with sterile saline solution.Petri dishes were inoculated with 0.2ml suspension of microorganisms and overlaid with 20ml of the medium.For cultivating bacteria Listeria monocytogenes TSA-YE were used.Three filter disks (Sigma-Aldrich's Whatman ® Schleicher & Schuell, 6mm in diameter) were placed on every agar and diffusion method was performed by adding 10μl of appropriate suspension on every disk.Blind probe contained only 96% C 2 H 5 OH, without any ethereal oils.Bacteria were incubated at 37 o C for 24 h and after the incubation period the zone of inhibition was measured.Small sectors from the zone of inhibition were taken and inoculated in TSB-YE for cultivating Listeria monocytogenes.Sectors in TSB-YE were incubated at 37 o C for 24 h to see if the effect of essential oils were microbicidal or microbistatic (K l a u s et al., 2007.).Studies were performed in triplicate, and mean value was calculated.

Results and Discussion
After 24 h incubation at 37 o C the zone of inhibition around the filter disks were measured and results are presented in Table 1.If TSB-YE with sectors taken from the zone of inhibition showed turbidity after incubation, influence of applied essential oils were regarded as microbistatic.In the cases when TSB-YE with sectors taken from the zone of inhibition stayed clear after incubation, influence of applied essential oil were microbicide.Blind probe showed that 96% C 2 H 5 OH had no antimicrobial influence on investigated microorganisms.
Tab. 1. -The zone of inhibition procured by presence of pure essential oils and in concentrations 2:1 (2 ml essential oil: 1 ml 96% C 2 H 5 OH) and 1:1 (1 ml essential oil : 1 ml 96% C 2 H 5 OH) -with no influence on the growth In the case of the bacteria Listeria monocytogenes ATCC 19115 all concentrations and pure essential oil obtained from Majorana hortensis showed microbicide effect (inhibitory zone 57 mm with suspension 2:1, Fig. 1; 51.67 mm with suspension 1:1 and 38 mm in the presence of pure oil).In the presence of Thymus vulgaris essential oil microbicide effect appeared (inhibitory zone 20.67 mm with pure oil and 16 mm with suspension 2:1, while suspension 1:1 had no effect on the growth of bacteria).Pure oils from Carum carvi and Coriandrum sativum showed microbistatic effect on Listeria monocytogenes ATCC 19115 with inhibitory zone of 13 mm.Suspension 2:1 of essential oil extracted from Coriandrum sativum made inhibitory zone of 17 mm.In the presence of Carum carvi essential oil microbistatic effect appeared with inhibitory zone of 8 mm.When essential oil from Rosmarinus officinalis were applied microbistatic effect appeared (inhibitory zone 8.67 mm with pure oil, inhibitory zone 4.67 mm with suspension 2:1, 5.67 mm with suspension 1:1).Essential oil from Salvia officinalis showed microbistatic effect (inhibitory zone 6.67mm with pure oil, inhibitory zone 4.67 mm with suspension 2:1 and suspension 1:1; Tab.1.).
In the case of the bacteria Listeria monocytogenes ATCC 19112 the best results appeared in the presence of suspensions and pure oil from Majorana hortensis.Suspensions 1:1 (with inhibitory zone of 36.33 mm), 2:1 (with inhibitory zone of 32 mm) and pure oil (with inhibitory zone of 31 mm) showed microbicide effect.In the presence of Thymus vulgaris essential oil microbicide effect appeared (inhibitory zone 18.67 mm with pure oil and 22.67 mm with suspension 2:1, while suspension 1:1 had no effect on the growth of bacteria).Essential oil extracted from Coriandrum sativum showed microbistatic effect on the Listeria monocytogenes ATCC 19112 (inhibitory zone of 11 mm with suspension 2:1, inhibitory zone of 8 mm with suspension 1:1 and inhibitory zone of 6 mm with pure oil).In the presence of Salvia officinalis essential oil microbistatic effect appeared (inhibitory zone 9.67 mm with pure oil, inhibitory zone 5.67 mm with suspension 2:1 and suspension 1:1).Pure oils extracted from Rosmarinus officinalis (inhibitory zone 9 mm), Carum carvi and Coriandrum sativum (inhibitory zone 6 mm) and Pimpinella anisum (inhibitory zone 4 mm) showed microbistatic effect (Tab.1.).

C o n c l u s i o n
Listeria monocytogenes, the cause of listeriosis, is emerging as an important foodborne pathogen.This is acid-tolerant, psychrotolerant (coldtolerant) and salt-tolerant bacterium, which can make difficult the preservation of food.It can contaminates the food at any stage during food growth or processing and method such as refrigeration, which ordinarily slow microbial growth, are ineffective in limiting growth of the organism.
This investigation showed possibility of some essential oils, especially those extracted from Thymus vulgaris and Majorana hortensis, to act microbicidaly on Listeria monocytogenes, even in the small concentration.Suspensions of Thymus vulgaris and Majorana hortensis essential oils in 96% C 2 H 5 OH in small concentrations, 1:2 (1ml essential oil:2ml 96% C 2 H 5 OH) and 1:3 (1ml essential oil:3ml 96% C 2 H 5 OH) were acting microbicidaly in contact with both investigated strains of Listeria monocytogenes.This could be very important regarding the fact that all microorganisms become resistant on numerous antibiotics.By adding some herbs or their essential oils in food in precisely defined concentration, by taking care of harmonizing with flavor, taste and odor of the particular groceries, it is possible to upgrade the quality of food and also, to protect the groceries from unwanted Listeria monocytogenes.a obezbede zamenu za konvencionalne antimikrobne aditive u hrani.

Fig. 1 .
Fig. 1. -Inhibitory effect of Fig. 2. -Inhibitory effect of Majorana hortensis essential oil Thymus vulgaris essential oil (ratio 2:1) on the growth of Listeria (ratio 1:3) on the growth of Listeria Monocytogenes ATCC 19115 monocytogenes ATCC 19112 1.A n g i o n i , A., B a r r a , A., C e r e t i , E., B a r i l e , D., C o ï s s o n , J.,D., A r l o r i o , M, D e s s i , S., C o r o n e o , V., C a b r a s , P. (2004.):Chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of Rosmarinus officinalis L., Agric.Food Chemistry, 52 (11), pp.3530-3535.2. B a r a t t a , M.T., D o r m a n , H.J.D., D e a n s , S.G.B i o n d i D.M. (1998.):Chemical composition, antibacterial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils, Journal of essential oil research, 10, 618-627.3..B i o n d i , D., C i a n c i , P., G e r a c i , C., R u b e r t o , G., P i a t t e l l i , M., (1993.):Antimicrobial activity and chemical composition of essential oils from sicilian aromatic plants.Flavour and Fragrance Journal, Vol. 8, Issue 6, pages 331-337.4. K l a u s , A, B e a t o v i ć , D., N i k š i ć , M., J e l a č i ć , S., N e d o v i ć , V., P e t r o v i ć , T. (2007): Influence of ethereal oils extracted from Apiaceae family plants of some pathogen microorganisms.5 th International Congress on Food Tehnology, "Consumer protetcion trought Food Procees Improvement  Innovation in the Real World", 9-11.March 2007, Thessaloniki, Greece, Proceedings, Volume 3, pages 421-428. 5. K l a u s , A., B e a t o v i ć , D., N i k š i ć , M., J e l a č i ć , S., N e d o v i ć ,V., P e t r o v i ć , T., (2008.):Influence of ethereal oils extracted from Lamiaceae family plants on some pathogen microorganisms, Matica Srpska Proceedings for Natural Sciences, Zbornik Matice srpske za prirodne nauke, 115, in press, Novi Sad. 6.K o v a č e v i ć , N. (2000.):Osnovi farmakognozije, (prvo izdanje), Farmaceutski fakultet Beograd.