In Vitro Antimicrobial Activity of Essential Oils from Aromatic Plants against Selected Foodborne Pathogens

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1 5 Journal of Food Protection, Vol. 67, No. 6, 00, Pages 5 56 Copyright q, International Association for Food Protection Research Note In Vitro Antimicrobial Activity of Essential Oils from Aromatic Plants against Selected Foodborne Pathogens C. ROTA, * J. J. CARRAMIÑANA, J. BURILLO, AND A. HERRERA Departamento de Producción Animal y Ciencia de los Alimentos, Unidad Higiene, Inspección, Control y Microbiolog a de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet, 77 Zaragoza, Spain; and Servicio de Investigación Agraria, P.O. Box 77, Zaragoza, Spain MS 0-: Received 0 July 00/Accepted 0 January 00 ABSTRACT The purpose of this study was to examine the effectiveness of selected essential oils for the control of growth and survival of pathogenic microorganisms of signi cant importance in food hygiene and to determine whether the antimicrobial effect was due to the major compounds of the oils. MIC and MBC were determined by the tube dilution method. Essential oils from Thymus vulgaris from Spain and France, Salvia sclarea, Salvia of cinalis, Salvia lavandulifolia,lavandula latifolia, Lavandula angustifolia, three hybrids of Lavandula latifolia Lavandula angustifolia (Lavandin Super, Lavandin Abrialis, and Lavandin Grosso ), Rosmarinus of cinalis, Hissopus of cinalis, and Satureja montana were evaluated. Inhibition ranged from the strong activity of Satureja montana and Thymus vulgaris (France) to no inhibition with Salvia sclarea and Hissopus of cinalis for each of the test strains: Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli O57:H7, Yersinia enterocolitica, Shigella exneri, Listeria monocytogenes serovar b, and Staphylococcus aureus. Because some of the essential oils were highly inhibitory in small quantities to selected pathogenic microorganisms, they may provide alternatives to conventional antimicrobial additives in foods. Food safety researchers and regulatory agencies have been increasingly concerned with the growing number of foodborne illness outbreaks caused by some pathogens (6). The increasing antibiotic resistance of some pathogens that are associated with foodborne illnesses is another concern (, 5). The antimicrobial properties of volatile oils and their constituents from a wide variety of plants have been assessed (, 5, ). These studies have demonstrated that these secondary plant metabolites have potential uses in medical procedures and in the pharmaceutical and food industries (,, 7). Biological activity of essential oils depends on their chemical composition, which is determined by the plant geneotype and is greatly in uenced by several factors such as geographical origin and environmental and agronomic conditions (5). Plant volatile oils are variable mixtures of principally terpenoids, speci cally monoterpenes (C0) and sesquiterpenes (C5), although diterpenes (C0) may also be present, and a variety a low-molecular-weight aliphatic hydrocarbons, acids, alcohols, aldehydes, phenolic compounds, acyclic esters, or lactones. Terpenes are among the chemicals responsible for the medicinal, culinary, and fragrance uses of aromatic plants (6). This study was undertaken to investigate the in vitro effects of different essential oils on the survival and growth of selected foodborne pathogens and to elucidate which * Author for correspondence. Tel: ; Fax: ; crota@unizar.es. components are likely to contribute to the antimicrobial activity according to gas chromatography characterization of the essential oil. Previous research has shown that many essential oils are antimicrobial, but there is little quantitative data (MIC or MBC) on the antimicrobial activity against foodborne pathogens of essential oils isolated from plants cultured in different geographical areas of Aragon (Spain). MATERIALS AND METHODS Plant material. Plants were collected from experimental elds of the Servicio de Investigación Agraria of Zaragoza located in four different geographical areas of Aragon, a region in northeastern Spain with a dry continental climate. The aromatic plants were cultivated in four different elds: Ibieca (Huesca), Trasobares (Zaragoza), Alacón (Teruel), and Calamocha (Teruel). The airdried plant material (aerial parts) was pulverized, and the essential oils were isolated after h of hydrodistillation (7). The essential oils investigated were obtained from Thymus vulgaris local (L), Thymus vulgaris French (F), Salvia sclarea, Salvia of cinalis, Salvia lavandulifolia, Lavandula latifolia, Lavandula angustifolia, three hybrids of Lavandula latifolia Lavandula angustifolia (Lavandin Super, Lavandin Abrialis, and Lavandin Grosso ), Rosmarinus of cinalis, Hissopus of cinalis, and Satureja montana. Determination of MIC and MBC. The essential oils were preliminarily screened for antimicrobial activity using the agar diffusion technique (6) against seven microorganisms of significant importance. All strains were obtained from the Spanish Collection of Type Cultures (CECT) and maintained in skim milk

2 J. Food Prot., Vol. 67, No. 6 AIMICROBIAL ESSEIAL OILS AGAINST FOODBORNE PATHOGENS 5 (Difco, Becton Dickinson, Sparks, Md.) at 08C. The gram-negative bacterial strains used to assess the antimicrobial properties of the test samples were Salmonella Enteritidis (CECT 55), Salmonella Typhimurium (CECT ), Escherichia coli O57:H7 (CECT 67), Yersinia enterocolitica serotype O:8; biotype (CECT 5), and Shigella exneri serovar a (CECT 585). The gram-positive strains were Listeria monocytogenes serovar b (CECT 95) and Staphylococcus aureus (CECT 9). Bacterial strains were grown in Trypticase soy broth (TSB; Difco) and incubated at 78C (Salmonella Enteritidis, Salmonella Typhimurium, E. coli O57:H7, L. monocytogenes, S. exneri, and S. aureus) or at 98C (Y. enterocolitica). The essential oils that showed antimicrobial activity (.- mm zone of inhibition) were tested for determination of MIC and MBC by the tube dilution method against the same seven bacterial strains. Well-isolated single bacterial colonies from plates incubated overnight were transferred into TSB, and the appropriate amount of the essential oil dissolved in 95% ethanol was added to the broth. Triplicate serial dilutions were prepared from this broth and were then inoculated with the test strains to a nal concentration of 0 6 cells per ml. The highest concentration tested was 9% (vol/ vol), although for some strains 5% (vol/vol) was used. The lowest concentration tested was 0.% (vol/vol). The growth of each culture was monitored by plating suitable 00-ml aliquots of the culture (viable counts) on Trypticase soy agar (TSA) at speci c time intervals. In each experiment, control broths containing % (vol/vol) ethanol were also prepared. After overnight incubation at the appropriate temperature, the MIC and MBC were calculated. The MIC was the lowest concentration at which bacteria failed to grow in TSB, but bacterial growth was observed after transferring 00 ml to TSA. Similarly, the MBC was the lowest concentration at which bacteria failed to grow in TSB and after the subsequent transfer to TSA (). The evaluation of MIC and MBC was carried out in triplicate. Analyses of essential oils. The gas chromatography characterization of essential oils obtained from aromatic plants has been reported elsewhere (9, 0,, 9). RESULTS AND DISCUSSION A preliminary screening of the in vitro antimicrobial activity of 5 essential oils from aromatic plants was studied against seven pathogenic microorganisms using a disk diffusion assay. After this study, seven essential oils (from Salvia sclarea and Hissopus of cinalis) were discarded because of their low inhibitory activity against the strains under investigation. More precise data on the antimicrobial properties were obtained through the determination of bacteriostatic (MIC) and bactericidal (MBC) concentrations. The MIC (ml essential oil per ml medium) and the MBC (ml essential oil per ml medium) of 8 essential oils from aromatic plants cultivated in four different areas were then obtained against seven microorganisms (Table ). The MBC and MIC tests revealed that the oil of Satureja montana and Thymus vulgaris (F) were the most inhibitory oils examined; both exhibited inhibitory activity (MBC of # ml/ml) against all seven bacterial strains tested (Table ). The MIC was lower for the gram-positive bacteria (L. monocytogenes and S. aureus) than for the gram-negative bacteria (Salmonella Enteritidis, Salmonella Typhimurium, E. coli O57:H7, Y. enterocolitica, and Shigella exneri). For most strains, MBC and MIC were of the same order, but the MBC was higher than the MIC for the two gram-positive strains. Thymus vulgaris (L) inhibited the growth of only ve bacteria (Salmonella Typhimurium, Y. enterocolitica, S. exneri, L. monocytogenes, and S. aureus) with MIC of # ml/ml, whereas the MBC was #ml/ ml for three gram-negative bacteria: to 5 ml/ml for S. aureus and 9 to 0 ml/ml for L. monocytogenes. These results are in agreement with those reported by Marino et al. (). The main components of the essential oils of S. montana are carvacrol and the phenolic precursors p-cymene and g-terpinene, and T. vulgaris (F) is rich in the phenolic compounds carvacrol and thymol. The bacteriostatic properties of S. montana oils are supposed to be associated with the high levels of carvacrol, which has been reported to have signi cant antibiotic activity (). The genus Thymus has numerous species and varieties, and their essential oil composition has been studied (0). From Thymus vulgaris grown in France and in other countries (0), six chemotypes have been described; from T. vulgaris grown in Spain, another chemotype with,8-cineole as the main component has been described. This chemotype also contains linalool, b-pineno, a-terpineol, camphor, borneol, and traces of thymol and carvacrol (). However, Marino et al. () studied the chemical composition of four essential oils from Thymus vulgaris cultivated in Italy and extracted at different stages of plant growth and found thymol, g-terpinene, and p-cymene. The percentage of these compounds varied slightly across the different collection periods, but the sum of the relative percentages of the compounds remained almost contant. The composition of T. vulgaris (F) and T. vulgaris (L) essential oils explains the differences in inhibitory activity against the bacterial strains studied. Rosmarinus of cinalis exhibited inhibitory activity against L. monocytogenes (MIC # ml/ml and MBC to 6 ml/ml), Salmonella Typhimurium and Y. enterocolitica (MIC and MBC # ml/ml), S. exneri (MIC and MBC ml/ml), and S. aureus (MIC to 5 ml/ml and MBC to 5 ml/ml). Piccaglia and Marotti (9) reported the quali-quantitative composition of Lavandin Abrialis, Lavandin Grosso, and Lavandin Super oils. Seventy-three compounds were separated, and 60 were identi ed. The main components were,8-cineole, linalol, camphor, and lynalil acetate. In our study, Lavandin Grosso had a higher level of antimicrobial activity than did Abrialis and Super because of the different quantitative composition. Lavandin Grosso exhibited inhibitory activity against ve bacteria: Salmonella Typhimurium, Y. enterocolitica, and S. exneri (MIC and MBC # ml/ml), L. monocytogenes (MIC # ml/ml and MBC # ml/ml), and S. aureus (MIC ml/ml and MBC ml/ml). The lowest MIC of Grosso was against E. coli O57:H7. Super and Abrialis exhibited inhibitory activity against four bacteria, although Abrialis was most effective, with lower MIC and MBC than Super. Preliminary screening of the antimicrobial activity of

3 5 ROTA ET AL. J. Food Prot., Vol. 67, No. 6 TABLE. MIC and MBC of selected plant volatile oils Inhibitory activity (ml/ml) against bacterial strains b Oils/ location a S. E. S. T. E.c. L.m. Y.e. S.f. S.a. Thymus vulgaris (L) c Thymus vulgaris (F) 0. Salvia of cinalis Salvia lavandulifolia 0. Lavandula latifolia Lavandula angustifolia Lavandin Super Lavandin Abrialis

4 J. Food Prot., Vol. 67, No. 6 AIMICROBIAL ESSEIAL OILS AGAINST FOODBORNE PATHOGENS 55 TABLE. Continued Inhibitory activity (ml/ml) against bacterial strains b S.a. S.f. Y.e. L.m. E.c. S. T. S. E. Oils/ location a.5.5 Lavandin Grosso Rosmarinus of cinalis ,0.,0.,0.,0. Satureja montana a Location:, Ibieca (Huesca);, Trasobares (Zaragoza);, Alacón (Teruel);, Calamocha (Teruel). b S. E., Salmonella Enteritidis; S. T., Salmonella Typhimurium; E.c., E. coli O57:H7; L.m., L. monocytogenes (b); Y.e., Y. enterocolitica; S.f., S. exneri; S.a., S. aureus. c, not tested. essential oils of S. lavanducifolia, S. of cinalis, and L. angustifolia against gram-negative bacteria produced negative results. Essential oils of S. of cinalis and L. angustifolia inhibited the growth of the gram-positive S. aureus and essential oils of S. lavanducifolia and S. of cinalis inhibited the growth of L monocytogenes. L. monocytogenes and S. aureus were the most susceptible organisms; their growth was inhibited by of the 8 different essential oils tested. R. of cinalis had the lowest activity against S. aureus, with MBCs between and 5 ml/ml (Table ). S. of cinalis, S. lavanducifolia, and L. latifolia had the least activity against L. monocytogenes, with MBCs from 7 to ml/ml. The main components of essential oils of S. lavanducifolia and S. of cinalis are camphor,,8-cineole, and thujone. L. monocytogenes is a pathogen of great concern to the food industry, especially in foods stored under refrigerated conditions where, unlike most foodborne pathogens, L. monocytogenes is able to multiply (). Consequently, refrigeration should not be relied upon as the sole method for the control of L. monocytogenes but should be incorporated with another means of preservation. One possible option is the use of plant essential oils. Based on the data from the present study (Table ), the most effective essential oils would be those from S. montana and T. vulgaris (F), which had low values for MIC and MBC. Of all the pathogens tested, Salmonella Enteritidis and E. coli O57:H7 appeared to be least sensitive to the essential oils, with the oils of only Satureja montana and T. vulgaris (F) showing a bactericidal effect at a concentration of, ml/ml. Friedman et al. (8) evaluated the bactericidal activity of 96 essential oils and observed that the essential oils most active against Salmonella Enteritidis were thyme, oregano, and cinnamon. However, our results indicate that Salmonella Typhimurium was more sensitive than Salmonella Enteritidis; Salmonella Typhimurium was inhibited by six essential oils at MICs of,0. to ml/ml. T. vulgaris (F) and S. montana were the most effective essential oils against Y. enterocolitica and S. exneri, followed by T. vulgaris (L), Lavindin Grosso, Lavindin Abrialis, L. latifolia, and Lavindin Super. The determination of bacteriostatic (MIC) and bactericidal (MBC) concentrations is more sensitive than the agar disc diffusion technique, which was used only as a screening tool to eliminate those oils with no signi cant inhibitory properties against the seven pathogens. The MIC tests revealed that the gram-positive S. aureus and L. monocytogenes were more sensitive to plant essential oils than were the ve gram-negative bacteria, Salmonella Enteritidis, Salmonella Typhimurium, E. coli O57:H7, Y. enterocolitica, and S. exneri. It is unclear why gram-negative bacteria were less susceptible to the essential oils, but the difference in susceptibility may be related to the outer membrane, which makes the bacterial surface strongly hydrophilic and acts as a strong permeability barrier (8). Comparison of the data obtained in this study with previously published results should be made with great caution. The composition of plant oils and extracts varies according to local climatic and environmental conditions (),

5 56 ROTA ET AL. J. Food Prot., Vol. 67, No. 6 and some oils with the same common name may be derived from different plant species (). In this study, volatile oils from Satureja montana and Thymus vulgaris (F) had signi cant activity against the growth of food pathogens such as Salmonella Enteritidis, Salmonella Typhimurium, E. coli O57:H7, Y. enterocolitica, Shigella exneri, L. monocytogenes b, and Staphylococcus aureus. All the essential oils from the plants studied contained main components that were biologically active, i.e., thymol, carvacrol,,8-cineole, p-cymene, g-terpinene, camphor, linalool, thujone, and limonene (7). It is dif cult to establish a relationship between oil composition and biological activity because of the synergistic action between certain components. The low MICs and MBCs for some plant essential oils against some of the most important causes of bacterial food poisoning provides an exciting potential for future interventions, especially in the light of the shift away from arti cial preservatives and toward natural alternatives. These results con rm the potential use of plant essential oils in the food industry for the preservation of foodstuffs against bacteria and to increase the shelf life of foodstuffs. Studies involving the incorporation of these essential oils into minced meat and skim/whole milk are currently in progress in our laboratory. ACKNOWLEDGME Funding for this research was provided by the University of Zaragoza, Spain (Service of Gestion of the Investigation, Research fellowship 00/07). REFERENCES. Baratta, T. M., D. H. J. Dorman, and S. G. Deans Chemical composition antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano, and coriander essential oils. J. Essent. Oils Res. 0: Baratta, T. M., D. 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