Romanian Biotechnological Letters Vol. 16, No.6, 2011, Supplement Copyright 2011 University of Bucharest Printed in Romania.

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Antimicrobial activity of essential oils against food-borne bacteria evaluated by two preliminary methods

ORIGINAL PAPER

Received for publication, September 19, 2011 Accepted, November 21, 2011

1, ALINA ALEXANDRA VALERIA 2 , NICULITA 3 GAGIU PETRU University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, 59 Marasti Blv, 011464, Bucharest, Romania. (E-mail: dobrealinaa@yahoo.com) 2 National Institute of Research & Development for Food Bioresources IBA, 6 Dinu Vintila Street, RO 021102, Bucharest, Romania, Tel/Fax: +40-21-2109128, 3 University of Agricultural Sciences and Veterinary Medicine, Biotechnology Faculty, 59 Marasti Blv, 011464, Bucharest, Romania 1 DOBRE

Abstrac t

The purpose of this research was to evaluate the in vitro antimicrobial activity of seven essential oils against two Grampositive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778) and two Gramnegative bacteria (Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076) using two preliminary methods: agar disc diffusion method and disc volatilization method. Results showed that all seven essential oils presented antibacterial activity against all the test strains in direct contact method. On the other hand, only two EOs presented significant antibacterial effect through volatilization method against test bacteria. Oregano oil, clove bud oil and white thyme oil showed maximum activity against all the bacteria tested in direct contact method, having a greater inhibition diameter than the reference control (streptomycin 50 mg/ml). The results support the high efficacy of oregano, white thyme and clove bud essential oils to control pathogenic microorganisms and their use in developing new systems to prevent bacterial growth, extend the shelf life and increase the safety of the processed food.

Key words : essential oils, food-borne pathogens, antibacterial activity, agar diffusion method, vapor phase activity

Food safety is a known problem worldwide, affecting hundreds of millions of people that suffer from contaminated food. World Health Organization (WHO) defines this issue as one of the most widespread health problems and a major cause of the reduction in economic productivity [21]. Nowadays, consumers are continuously concerned with the growing number of illness caused by some pathogenic and spoilage microorganisms in food and also for the safety of foods containing synthetic preservatives. Thus, it shows a growing interest about the replacement of synthetic preservatives with natural, effective and nontoxic antimicrobialThere is growing interest in using natural antimicrobial compounds, such compounds. as extracts and essential oils (EOs) of spices and herbs, for food conservation. Essential oils are volatile oily liquids obtained from different plant parts and widely used as food flavors [3]. are variable mixtures of essential terpenoids, especially monoterpenes (C10) They and sesquiterpenes (C15), although diterpenes (C20) may also be present, and of a variety a low molecular weight aliphatic hydrocarbons, acids, alcohol, aldehydes, phenolic compounds, or lactones [19]. The composition, structure, as well as functional groups of the acyclic esters, oils play an important role in determining their antimicrobial activity. It has been demonstrated that the essential oils exercises their antimicrobial activity by causing structural and functional damages to the bacterial cell membrane. It is also indicated that the optimum hydrophobicity is involved in the toxicity of the essential oils range of [11].
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Introductio n

ALINA ALEXANDRA DOBRE, VALERIA GAGIU, NICULITA PETRU

Essential oils from spices and herbs are the most promising natural becauseantimicrobials, they do not cause microbial resistance due to the diversity of mechanisms of action.They have a GRAS status given by the U.S. Food and Drug Administration [10], meaning that they are generally recognized as safe for human consumption without limitations on intake and commonly accepted by consumers. purpose of this research was to cre ate directly comparable, qualitative data on the The capacity of seven essential oils to prevent a diverse range of foodborne and spoilage microorganisms strains based on two preliminary test methods: agar disc diffusion method volatilization method. These two methods were used to screen essential oils and disc for antimicrobial activity, generating the preliminary, qualitative data only, and permitting the selection of the most active essential oils for further evaluation.

Materials methods

and

ESSENTIAL OILS this research seven high purity essential oils were Within cinnamon leaf oil used: (Cinnamomum CAS No: 8015-91- , garlic ( Allium CAS No: zeylanicum, 16)CAS No: 8002-72- oil 78-0), onion oil (Allium , white sativum, Thymus8000thyme ( CA oil S No: 8007-46-3) , cepa, oregano oil ( 0) Thymus CAS No: 8007-11-vulgaris, , basil (Ocimu oil CAS m No: basilicum CAS No: 8015-73- capitatus, bud oil 2)Eugenia and clove ( , 4) caryophyllata, 34-8), obtained by steam distillation, purchased from Sigma Aldrich, 8000Germany. Essential oils quality parameters (appearance, color, purity, odor, density at 0 C and -20 refraction index at 0 C) were described in an accompanying technical These oils -20 selected based on literature survey with documented antimicrobial report. were activity.The oils were dissolved in DMSO (Dimethyl sulfoxide, >= 99,0%, CAS No: 67-685, Sigma Aldrich, Germany) 1:2 (v/v) to give stock solutions after which they were mixed for total solubility at 180 rpm for 10 minutes. For the bioassay, the stock solutions of essential oils were sterilized by filtration using sterile membrane filters (Millex GP, pore size 0.22 m). Until subsequent use, stock solutions of essential oils were stored in a refrigerator at +4 0 C. TEST ORGANISMS In this research were used pure references strains of food-borne microorganisms involved in food toxi-infections. The selected test organisms used to evaluate the antimicrobial activity of the essential oils were as follows: Gram positive Bacillus ( cereus ATCC Staphylococcus ATCC 25923) and Gram negative Salmonell 11778, ATCC aureus Escherichia ( ATCC enteritidi 13076, 25922) all purchased a from s coli MicroBioLogics, SUA. The source of bacteria strains was ATCC, American Type Culture Collection. bacterial and fungal strains used in this research were a part of the collection All the of reference strains of Microbiology ELISA Laboratory from National Institute of Research &evelopment for Food Bioresources IBA, D Bucharest. PREPARATION OF TEST 0 C (Plate Count ORGANISMS test organisms were maintained in agar slants at The cultures of Agar +4 PCA; Biokar Diagnostics, France) and used as stock cultures. acterial inoculums were obtained from reference stock culture inoculated in B 0 C for 18-24 hours. From the fresh grown TSB medium, which was incubated at 6 CFU/mL, 37 cultures decimal dilutions were made in sterile PS, up to the concentration of used 10 for testing essential oils. IN VITRO ANTIMICROBIAL ACTIVITY TESTING Because it appears that no standardized test has been developed for evaluating the antimicrobial activity of possible preservatives against food-related microorganisms, the CLSI
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Antimicrobial activity of essential oils against food-borne bacteria evaluated by two preliminary methods

(Clinical and Laboratory Standards Institute) method for antimicrobial susceptibility testing modified for testing essential oils. We used two preliminary methods: agar has been disc diffusion method and disc volatilization method (vapor phase activity) for detecting the most efficient essential oils against test organisms. AGAR DISC DIFFUSION METHODdiffusion method is the most widespread technique of antimicrobial The agar activity assessment. This method is normally used as a preliminary check and to select between essential oils [15, 2, 13]. The appropriate solidified medium (Plate Count Agar efficient 6 CFU/mL) and spread over - CA) was inoculated with 100 l of bacterial inoculum P (10 using a sterile rod display in order to get a uniform microbial growth on both the plates control plates. After inoculum absorption by agar, sterile filter discs (Whatman no and test 1, England, 6 mm diameter) were impregnated with 10 l of stock solutions of essential oils and on the agar surface using forceps dipped in ethanol and flamed. placed Growth cultures containing essential oils were accompanied by DMSO solution as a negative control. Positive control cultures with streptomycin solution (50 /mL) w ere used to mg assess the susceptibility of tested strains and to compare with them the essential oils efficiency. Petri dishes were sealed with sterile laboratory parafilm to avoid All eventual evaporation of the essential oils. The dishes were left for 30 min at room temperature to allow the diffusion of oil, and then were incubated at 0 C for 24h. After the incubation period, 37 the mean diameter of inhibition halo where test microorganism did not grow (clearly visible inhibition zone) was measured in millimeters, for each disc and evaluated for susceptibility or resistance. DISC VOLATILIZATION [15 METHOD This method evaluates the activity of essential ] oils vapors on the same strains, technically near to disc diffusion method. It is used for defining the activity of essential oils, which are to be employed as atmospheric preservatives. Solidified medium was inoculated with 100 l of bacterial inoculums. Then, 10 l of each stock solution of essential oil were added to 6 mm diameter sterile blank filter discs and in the center of the cover of the Petri dish in which was previously cast a thin layer placed of medium to avoid adsorption of essential oils onto the plastic material of the cover. The dishesthen sealed using sterile laboratory parafilm to avoid eventual evaporation of the were essential oils, followed by incubation at 0 C for 24h. Blanks were prepared by adding 10 l of 37 DMSO solution to the filter The effectiveness of the essential oils was calculated by measuring discs. the diameter (in mm) of the zone of microorganism growth inhibition above the disc.

Results discussion

and

DIRECT CONTACT VERSUS VAPOUR PHASE METHOD Antimicrobial activity of selected essential oils, both by direct contact and vapor phase, against four bacterial species was qualitatively assessed by the presence or absence of the inhibition zone. Each assay in these experiments was repeated three times and the resultsof zone of inhibition) were expressed as average values ( standard deviation). (mm Bacteria susceptibility to the essential oils, as determined by the agar diffusion method, showed that with the highest inhibitory effects produced inhibition zones of 20-46 mm diameter. oils The antibacterial activity of the selected essential oils is summarized in Table 1.

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ALINA ALEXANDRA DOBRE, VALERIA GAGIU, NICULITA PETRU Table 1. Antibacterial activity of tested essential oils Sensitivity to essential oils -mean diameter of the inhibition zone

diameter

* diameter)

m m Direct contact Vapor phase EOs Gram positive Gram negative Gram positive Gram negative S B S E B S S . . . . . . . cereu aureu enteritidi col cereu aureu enteritidi s s s 46 i s 32 s 37 s 48.3 Whit 39.3 e thyme 35.5 0.3 0.3 0. 0. 0.34 0.1 37.7 12 5 1 1 5 Clove 2 25. 31. 8 2 2 2 bud 0.07 0.2 0.2 0.17 - - - 1 4 Orega 45. 43.5 42. 42 41.6 26.3 43 2 5 no 0.67 0.4 0.2 0.2 0.0 0.1 0.2 2 1 8 5 1 6 Cinn 19. 18. 12. amo 20 2 7 3 0.03 0.3 0.1 0.15 - - n 21.2 leaf 1 7 9.5 Onion 1.09 0.2 0.10 - - - 38.2 9.5 1 13 33 Garlic 0.21 0.1 0.2 0.15 - - 25 12.7 0 1 24. Basil 10 5 0.35 12 0.9 0.07 - - - 5 The diameter 18.7 filter paper disc (6 mm) is included. - No inhibition (< 6 mm of the
*

in

E . col i 46.3 0.2 3 27 0. 1

The results revealed that all the selected essential oils showed antibacterial with a activity activity in direct contact method and only two essential oils higher presented antibacterial effect through volatilization method against test significant bacteria. mong the essential oils, oregano, clove bud and white thyme oil presented a A higher antibacterial activity in direct contact method especially E. and B. , with against zones of 42 mm, 31.2 mm and 39.3 mm and 45.2 mm, 28 coli and 35.5 cereusB. inhibition mm , for having a greater inhibition diameter than the control sample (streptomycin 50 mg/mL).cereus Such an activity could be strictly related to their chemical composition: in fact, carvacrol, thymol and eugenol found in these oils, are able to disi ntegrate the outer membrane of gramnegative releasing lipopolysaccharides and increasing the permeability of the bacteria, cytoplasmic ATP, deteriorate the cell wall and inhibit production of amylase and proteases membrane to [3]. For the reference control results are shown in Table 2. DMSO solvent as control negative no antimicrobial activity against the tested strains in any method presented used proving that is a suitable solvent for testing oils. 2. Diameter of inhibition zones of streptomycin for the test bacteria by direct contact Table method Mean inhibition zone diameter * after 24 h of Reference (mm) Gram positive Gram incubation control negative B. cereus S. aureus S. enteritidis E. coli Streptomycin 50 mg/ml 28 0.1 33 0.1 26 0.28 25 0.17 * The diameter of the filter paper disc (6 mm) is included. The essential oil with the widest spectrum of activity was found to be oregano oil followed by white thyme oil > clove bud oil > cinnamon oil > garlic oil > onion oil > basil oil, in that order.
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Antimicrobial activity of essential oils against food-borne bacteria evaluated by two preliminary methods

White thyme essential oil presented a higher activity against gram-negative bacteria like and E. S. , both direct contact and vapor phase method, while oregano essential enteritidis coli in oil was most efficient against gram-positive bacteria B. and S. ). Clove ( aureus bud essential oil demonstrated antibacterial activity against cereus food-associated bacteria all the with of growth inhibition ranging from 22 mm to 31.2 mm, but only in direct contact zone method. The maximum zone of inhibition showed by this oil was showed against E. coli (31.2 mm) followed B. (28 S. (25.2 mm) S. (22 by cereus also enteritidis and aureus mm). Activity was mm), shown by cinnamon leaf oil and basil oil against the same bacterial strains and with the same amount (10 l/paper disc), with inhibition zones fewer than 30 mm as those presented by white thyme and oregano essential oils. Cinnamon leaf oil and basil oil expressed a moderate action against tested strains in direct contact method while in vapor method only cinnamon leaf oil showed antibacterial activity phase B. with a against growth inhibition diameter of 12.3 mm. Cinnamon leaf oil presented acereus greater antibacterial activity against gram-positive bacteria than basil oil. Basil oil was more active against gram- bacteria E.col (diameter of inhibition of 24.5 negative i In direct contact mm). method, onion essential oil inhibited weakly the development of gram-negative bacteria; both bacteria tested expressed the same sensitivity against onion oil. the tested gram-positive B. On was strongly inhibited by onion and bacteria, oils, with a diameter of the inhibition zone of 38.2 mm and 25 mm, respectively. cereus garlic essential The phase of onion and garlic essential oils presented no inhibition against the test vapor strains,garlic essential oil that was active against B. except with a growth diameter cereus inhibition larger than that in direct contact.The zone of inhibition resulting from the exposure to selected essential oil vapors varied maybe because of the presence of different volatile chemical components. Only oregano and white thyme essential oils vapors exhibited antibacterial activity against all the strains, activity higher than that present in direct contact method. Zone of inhibition test due to vapors generated by 10l white thyme essential oil was higher (i.e. 48.3 mm S against ) than oregano oil (43 mm also S. enteritidi ). The antimicrobial activity . s essential oils in vaporr phase is closely associated with their chemical composition. In against enteritidis of the the direct contact assays for liquid phase, the activity depends upon the diffusibility and solubility of the essential oil compounds into the agar while the antimicrobial activity of the vapor assay depends upon the volatility of each compound [11].
E. coli

B. cereus

S. aureus S. enteritidis

Figure Antibacterial activity (zones of inhibition) of white thyme essential oils in direct contact method 1. (1) and volatilization method (2)

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Conclusion
The investigations on antimicrobial activity of seven essential oils against foodborne bacteria confirmed the potential of plant volatile oils to be used in food conservation as alternative to che mical preservatives. suggested that the evaluation techniques described could be used as a The results preliminary, qualitative step, which can determine the sensitivity of many microorganisms to essential oils and select the oils with the best antimicrobial activity, in order to use them for further evaluations. Disc volatilization method proved to be a useful method for simple screening of antimicrobial activity of vapor phase of essential oils.In our study, selected essential oils exhibit inhibitory effects against the selected bacterial strains, both in liquid form and volatile vapors. The result of the direct contact method that white thyme, oregano and clove bud oil were the most active essential showed oils against foodborne bacteria. Gram-positive B. andS. , as a mean bacteria, against all essential oils tested, were less sensitive thanaureus cereus sensitivity the Gram-negative bacteria andS. E. , but the difference in susceptibility were not that evident. By coli enteritidis disc volatilization method only two essential oils represented by white thyme and oregano oil were found highly effective.importance of these preliminary results is that pathogenic bacteria can be The controlled using plant essential oils. Further investigations include quantitative tests in order to determine the concentration of essential oils (minimum inhibitory concentration) needed to exhibit antimicrobial activity against food related microorganims in order to use them natural antimicrobial agents to extend the shelf life and increase the safety of the as processed food.

Acknowledgment s
This research was supported by the Sectorial Operational Programme Human Resources Development 2007-2013 under the project number POSDRU/88/1.5/S/52614 increase the quality of training young researchers in the PhD scholarships to field of agronomy and veterinary medicine .

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