Pelagia Research Library. European Journal of Experimental Biology, 2014, 4(1):

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1 Available online at European Journal of Experimental Biology, 2014, 4(1): ISSN: CODEN (USA): EJEBAU The antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis extracts and essential oil against Lactococcus garvieae in laboratory conditions on rainbow trout Ahmad Rafiee Pour 1, Seyed Saeed Mirzargar *1, Mehdi Soltani 1, Hossein Ali Ebrahimzadeh Mousavi 1 and Seyed Ali Mostafavi 2 1 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 2 Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran ABSTRACT The aim of this study were to investigate the antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis essential oil and extracts against Lactococcus garvieae in laboratory conditions on rainbow trout. The study also aimed to identify the minimum inhibitory concentration (MIC) of Cuminum cyminum L. and Rosmarinus officinalis essential oil against this strain (Lactococcus garvieae). In this experiment, 11 strains of Lactococcus garvieae were collected from different provinces of Iran. In addition, we has been evaluated the antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis extracts and essential oils. After the laboratory cultured Lactococcus garvieae strains, the different dilutions of Cuminum cyminum L. and Rosmarinus officinalis essential oils were added to the culture medium at three different temperatures (20, 30 and 37 C). Differences between extracts and essential oil of C. cyminum L. and Rosmarinus officinalis were tested with analysis of variance. Results of this experiment showed that the extracts of Cuminum cyminum L. and Rosmarinus officinalis against Lactococcus garvieae has not effects. The minimum inhibitory concentration of C. cyminum L. essential oil in the culture medium of strains L. garvieae at three different temperatures were effective. From the results of the present study it can be concluded that Cuminum cyminum L. and Rosmarinus officinalis essential oils could be effective for the inhibition of Lactococcus garvieae in rainbow trout. Key words: Essential oils, Lactococcus garvieae, Minimum inhibitory concentration (MIC), Rainbow trout. INTRODUCTION Control of plant bacterial diseases remains difficult due to the limited availability of bactericides. Only a few chemical products are available, and their use is hampered by limited efficacy in the field but mainly for their potential negative effects either in the environment or with human and animal health [10]. The use of antibiotics in plant protection is limited because of the possibility to select pathogen populations resistant to bactericides and the potential transfer of resistant genes to animal and human pathogenic bacteria [13]. As a consequence, measures to control plant bacterial diseases are mostly limited to prevention. Agronomic practices that minimize initial infection and dissemination of bacterial pathogens between plants and fields are very useful although poorly effective under high disease pressure [10]. The availability of new and ecocompatible bactericides may be very useful for bacterial 456

2 control of diseases in the field and for seed treatments. Essential oils are known for their antimicrobial capability and have the potential to control plant diseases caused by bacteria and, in particular, eradicate bacteria from seeds [5]. Essential oils have already found a considerable range of applications. The majority of them are used as fragrances in perfumery as well as in food and beverages industry. The recent years have also witnessed a revival of traditional natural products in medicine and in food and cosmetics preservation [12]. Despite the development of antibiotics, bacterial and fungal infections are still a major issue in medicine, and the presence of numerous drugresistant strains poses a new challenge. Herbal drugs have been extensively used in this field for many centuries. Recently, there has been a growing interest in natural products due to their availability, fewer side effects or toxicity as well as better biodegrability as compared to the available antibiotics and preservatives [12]. The exploration of naturally-occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food products and a growing concern of microbial resistance toward conventional preservatives [8]. It is well known that clean sanitation is essential in keeping the microbial population to a minimum, because storage life is shorter with high initial microbial loads [3]. Nychas [16] reported antimicrobial activity of essential oils from oregano, thyme, sage, rosemary, clove, coriander, garlic and onion against both bacteria and fungi. Phenolic components, present in essential oils, have been known to possess antimicrobial activity and some are classified as generally recognized as safe substances and therefore could be used to prevent post-harvest growth of native and contaminant bacteria [11, 21]. In this study, 11 strains of Lactococcus garvieae were used. Lactococcus garvieae is a known fish pathogen that causes lesions in the vascular endothelium, leading to hemorrhages and petechias at the surface of internal organs. As few as 10 bacterial cells per fish can cause an infection [24]. L. garvieae is usually identified within aquatic species. However, it has also been found in subclinical intramammary infections in cows, subclinical mastitis in water buffalos, poultry meat, raw cow's milk, meat products, porcine blood from industrial abattoirs and from cat and dog tonsils. L. garvieae was first discovered in rainbow trout raised on a Japanese fish farm in the 1950s. In 1988, L. garvieae was identified in the rainbow trout from Spanish fish farms as well. In later years, L. garvieae was isolated from several septicemic processes in fish and phenotypical and molecular taxonomic studies confirmed the same agent as E. seriolicida. This species was reclassified as a junior synonym of L. garvieae [23]. In this regard, plant essential oils may offer a great potential and hope. Therefore, their composition and antimicrobial activities have been thoroughly and systematically studied. The antimicrobial properties of essential oils and their constituents have already been reviewed [7]. Cumin (Cuminum cyminum L.) and caraway (Rosmarinus officinalis ) are aromatic plants included in the Apiaceae family and are used to flavor foods, added to fragrances, and for medical preparations. The results obtained by GC and GC MS analyses of the essential oils of C. cyminum and R. officinalis are presented in Tables 1 and 2. (Source: Gachkar et al., 2007). Rosemary, Rosmarinus officinalis L. (Labiatae) is an evergreen perennial shrub grown in many parts of the world. It has been reported to possess a number of therapeutic applications in folk medicines in curing or managing of a wide range of diseases. It is well known that the activity of rosemary extracts in medicine and food industry due to the presence of some important antioxidant oil and phenolic components, to prevent oxidative degradation of oil and lipid containing foods. Rosemary has long been recognized as having antioxidant molecules, such as rosmarinic acid, carnasol, rosmaridiphenol and these have found in ethanol-soluble fraction [2]. Cumin (Cuminum cyminum L.) is a aromatic plant that have a variety of purposes and demonstrate antioxidant and antimicrobial properties. Methanolic and acetonic seed extracts of this plants was able to neutralize free radicals and carried antioxidant properties. Seed extracts of cumin showed slightly higher neutralization ability than caraway seed extracts (57.0 and 52.4% vs and 39.5%, respectively). In addition, Antioxidant properties of Cumin seed plant may be useful in pharmacologic preparations [16]. However, on the basis of current knowledge there is no much information on the antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis essential oil against Lactococcus garvieae. Therefore, the aim of this study was to investigate the antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis essential oil against Lactococcus garvieae in laboratory conditions on Rainbow Trout. 457

3 Table 1: Chemical composition of Cumminum cyminum essential oil No. Compounds RI % 1 Isobutyl isobutyrate α-thujene α-pinene Sabinene Myrcene δ-3-carene p-cymene Limonene ,8-Cineole (E)-Ocimene γ-terpinene Terpinolene Linalool α-campholenal trans-pinocarveole δ-terpineole Terpinene-4-ol α-terpineole trans-carveole cis-carveole Geraniol Linalyl acetate Methyl geranate α-terpinyl acetate Neryl acetate Methyl eugenol β-caryophyllene α-humulene Spathulenol Caryophylleneb epoxide Humulene epoxide II Acetocyclohexane dione (2) Source: Gachkar et al., 2007 Table 2: Chemical composition of Rosmarinus officinalis L. essential oil No. Compounds RI % 1 α-pinene Camphene Octanone Sabinene Myrcene O-Cymene ,8-Cineole Linalool Myrcenol Camphor Borneol Terpinen-4-ol α-terpineol Verbinone Piperitone Bornyl acetate β-caryophyllene cis-β-farnesene Germacrene D α-bisabolol Source: Gachkar et al., 2007 MATERIALS AND METHODS This study was conducted in the department of Aquatic Animals Health, Faculty of Veterinary Medicine, University of Tehran, Iran from April 2012 to April In this experiment, 11 strains of Lactococcus garvieae were 458

4 collected from different provinces of Iran. In addition, we has been evaluated the antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis extracts and essential oils. After the laboratory cultured Lactococcus garvieae strains, the different dilutions of Cuminum cyminum L. and Rosmarinus officinalis essential oils were added to the culture medium at three different temperatures (20, 30 and 37 C). Bacterial strains and culture conditions Lyophilized culture of L. garvieae obtained from Department of Aquatic Animals Health, Faculty of Veterinary Medicine, University of Tehran, Iran, was used in this study. The lyophilized culture was grown in tube containing 10 ml of BHI broth (Merck KGaA, Darmstadt, Germany) twice consequently and incubated each time at 30 C for 18 h. Then it was followed by streaking on BHI agar (Merck KGaA) slant and incubated at 30 C for 18 h. The culture was stored at 4 C as working culture and subcultured at monthly intervals. The bacterial cells of L. garvieae grown in the broth were adjusted to an optical density (OD) of 0.02 at 600 nm using the Spectronic 20 spectrophotometer (Milton Roy Company, USA). This adjustment gave a cell concentration of CFU / ml [14]. Paper disc diffusion method The disc diffusion method was applied for the determination of antimicrobial activities of the extracts and essential oil (NCCLS, 2000a). Extracts and essential oils were dissolved in dimethyl sulfoxide (DMSO). L. garvieae were cultured in a nutrient broth (Merck, Germany) for 24 h and diluted with sterilized peptone water. Then, 100 µl of each culture (10 5 CFU) was spread onto the surface of Mueller Hinton agar (Oxoid, England) to create a bacterial lawn [19]. Sterile blank filter paper discs of 6 mm in diameter (Oxoid, England) were wetted with 20 µl crude extract of C. cyminum L. and R. officinalis (100 mg/ml) and left to dry before being placed on the microbial lawn. The plates were incubated at 37 C for 24 h. The antibacterial activity was compared with 10 µl of chloramphenicol (1 mg/ml), 20 µl of 5 % lactic acid and 20 µl of 10 % acetic acid. Antimicrobial activity was determined based on the diameter of the clear zone surrounding the paper discs [4]. Three replicate discs were prepared for each extract and essential oil in this study. Determination of minimal inhibitory concentration The estimation of the minimal inhibitory concentration (MIC) were measured by the broth microdilution method (NCCLS, 2002). The essential oils were individually dissolved in sterilized physiological saline solution (0.9% w/v) supplemented with Tween 80 (Sigma) at a final concentration of 0.5% (v/v). Serial doubling dilutions of the oils were prepared in a 96-well microtiter plate in the range 1000 to 7.8 µg/ml. Each essential oil dilution (100 µl) was dispensed into the wells of a microtiter plate; each well was then inoculated with 100 µl of the suspension. The resulting suspensions were mixed with a micro-pipettor. The final concentration of each strain was adjusted to CFU/mL. All microtiter plates against all microorganisms were incubated at 37 C for 24 h. After incubation, the wells were examined for growth of microorganisms and the MIC was determined. The MIC is defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth. Each experiment was repeated three times [6]. Differences between extracts and essential oil of C. cyminum L. and Rosmarinus officinalis were tested with analysis of variance (ANOVA). All statistical analyses were tested at a 0.05 level of probability with the SAS software (SAS User s Guide: Statistics. SAS Institute Inc., Cary, NC). RESULTS AND DISCUSSION The results of minimum inhibitory concentration of C. cyminum L. and Rosmarinus officinalis essential oil in the culture medium of strains L. garvieae at three different temperatures (20, 30 and 37 C) are presented in below tables (Table 1 to Table 6). 459

5 Table 1. Minimum inhibitory concentration (µg/ml) of C. cyminum L. essential oil in the culture medium of strains L. garvieae at 20 C 20 C C. cyminum L. Essential oil F1A A L3B L3A ALS F Table 2. Minimum inhibitory concentration (µg/ml) of C. cyminum L. essential oil in the culture medium of strains L. garvieae at 30 C 30 C C. cyminum L. Essential oil F1A A L3B F Table 3. Minimum inhibitory concentration (µg/ml) of C. cyminum L. essential oil in the culture medium of strains L. garvieae at 37 C 37 C C. cyminum L. Essential oil F1A A L3B F

6 Table 4. Minimum inhibitory concentration (µg/ml) of Rosmarinus officinalis essential oil in the culture medium of strains L. garvieae at 20 C 20 C Rosmarinus officinalis Essential oil F1A A L3B F Table 5. Minimum inhibitory concentration (µg/ml) of Rosmarinus officinalis essential oil in the culture medium of strains L. garvieae at 30 C 30 C Rosmarinus officinalis Essential oil F1A A L3B F Table 6. Minimum inhibitory concentration (µg/ml) of Rosmarinus officinalis essential oil in the culture medium of strains L. garvieae at 37 C 37 C Rosmarinus officinalis Essential oil F1A A L3B F The minimum inhibitory concentration of C. cyminum L. essential oil in the culture medium of strains L. garvieae at three different temperatures were effective. The MIC of essential oils in this study were similar to the known literature with a little difference, which could be for several reasons such as a different growing environment, different extraction methods of essential oils, and so on. Different essential oils have different antimicrobial activity because of their components [4, 20, 15]. Roomiani et al., [19] showed that the essential oil of rosemary has the strongest antimicrobial activity. In addition, this researcher demonstrated that the MIC of the R. officinalis essential oils and diameter of zone of inhibition in comparison to other essential oils showed potent antimicrobial activity and inhibitory effect. This suggested R. officinalis as an effective antimicrobial agent among 461

7 all kinds of tested herbs. The essential oil of rosemary was the most effective as an antibacterial agent. The antibacterial activity has been attributed to the presence of some active constituents in the oils. Sensitivity of Gram-positive bacteria to essential oil of rosemary is in line with an earlier study [17, 12, 18, 21]. Zaouali et al. [22] showed that R.officinalis have more inhibitory effects on Gram- positive bactria than Gram-negative bacteria, which is probably because of the different cell wall membrane structures between Gram-positive and Gram-negative bacteria. Although antibiotics have been used to control bacterial infections in fish for many years, several antibiotics have been banned from use in aquaculture due to their adverse effects. In addition, products from aquaculture facilities using antibiotics are not accepted by many countries [18]. Therefore, finding an alternative antimicrobial substance to replace antibiotics has become an issue of interest of many research groups. In cultured fishes, lactococcus infections often develop into lethal septicaemic conditions. Fish in both freshwater and marine environments may be affected, and farms in many parts of the world have consequently suffered serious economic losses [19]. In particular, such information would be valuable for operation planning, and for the control and prevention of such fish pathogens entering into water column [1]. Plant-derived essential oils due to their antimicrobial content possess potential significance as naturally occurring agents for food preservation. Many volatile compounds naturally occurring in various essential oils possess strong antibacterial activities, thereby considered as natural antibacterial agents to inhibit the growth of food-borne pathogens [19]. The renewal of interest in food science and technology, and increasing consumer demand for effective natural products means that quantitative data on plant based essential oils is required. The use of essential oils may improve food safety and overall microbial quality. If essential oils were to be more widely applied as antibacterials in foods, the organoleptic impact would be important [19]. For practical addition of essential oils in food, it is important to know if oils at very low concentrations, have efficient antimicrobial effects without affecting sensory qualities [9]. CONCLUSION From the results of the present study it can be concluded that Cuminum cyminum L. and Rosmarinus officinalis essential oils could be effective for the inhibition of Lactococcus garvieae in rainbow trout. Further studies should evaluate the safety and toxicity of Cuminum cyminum L. and Rosmarinus officinalis extracts and essential oils to human consumption before considering their use for food preservation or medicinal purposes. Acknowledgements This Study was funded by the research council of University of Tehran (Grant No:7505/6/5).The authors are grateful to Mr. Hadi bagheri, staff of Aquatic Animal Health Lab., Faculty of Veterinary Medicine for his kind helps. Also we would like to have a special thanks to the Dr. Seyed Ali Mostafavi (Manager of Kerman Alzahra Rosewater Company) and Maryam Ghodratnama for providing a friendly environment and research facilities. REFERENCES [1] AL-Bulushi, M., Poole, S., Barlow, R., Deeth, H. and Dykes, G. International Journal of Food Microbiology. 2010, 138: [2] Bakırel, T., Bakırel, U., Keles, O., Gu nes, S., Lgen, U. and Yardibi, H. Journal of Ethnopharmacology. 2008, [3] Bolin, H. R., Stafford, A. E., King Jr., & Huxsoll, C. C. Journal of Food Science, 1977, 42, [4] Celiktas, O., Kocabas, E., Bedir, E., Sukan, F., Ozek, T. and Baser, K. Food Chemistry. 2007, 100: [5] Cowan, M. M. Clin. Microbiol. ReV. 1999, 12, [6] Fu, Y., Zu, Y., Chen, L., Shi, X,G., Wang, Z., Sun, S. and Efferth, T. Phytotherapy Research Phytotherapy Research. 2007, 21, [7] Gachkar L, Yadegari D, Rezaei M, Taghizadeh M, Alipoor Astaneh SH, Rasooli I. Food Chemistry, 2007, [8] Gould, G. W. Food preservation by moisture control (pp ). Lancaster: Techromic Publishing Co., Inc [9] Holley, R. and Patel, D., Food Microbiology. 2005, 22: [10] Iacobellis, N, Pietro C, Francesco C, Felice S. J. Agric. Food Chem. 2005, 53, [11] Kabara, J. J. Food preservatives (pp. 200). London: Blackie [12] Kalemba D, Kunicka A. Current Medicinal Chemistry, 2003, 10, [13] Kaur, G. and Arora, D. BMC Complementary and Alternative Medicine. doi: /

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