Rosemary oil, clove oil, and a mix of thyme-orange essential oils inhibit Salmonella and Campylobacter in vitro

Transcription

1 2014 Poultry Science Association, Inc. Rosemary oil, clove oil, and a mix of thyme-orange essential oils inhibit and Campylobacter in vitro R. Thanissery,* S. Kathariou, and D. P. Smith * 1 * Prestage Department of Poultry Science, and Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh Primary Audience: Researchers, Quality Assurance, Research and Development SUMMARY The demand for foods that are free of pathogens and chemical residues has increased interest in the use of plant-based products as natural antimicrobials. Essential oils (Eo) from plants are natural compounds that have been shown to have antimicrobial properties against food-borne pathogens. The objective of the current study was to determine the ability of various concentrations of 4 selected EO to inhibit enterica (3 different serovars and a cocktail of all 3) and Campylobacter (2 strains of Campylobacter jejuni, one strain of Campylobacter coli, and a cocktail of all 3). The disc diffusion method was used to screen the oils of thyme, orange, rosemary, and clove oil. The minimum inhibitory concentration or minimum bactericidal concentration of the Eo was determined using a 2-fold broth dilution method at concentrations ranging from to 1.000% (vol/vol). Two independent experiments were performed. Zones of inhibition (ZI) were expressed in millimeters and concentrations were expressed in percentages. All the oils demonstrated antibacterial activity against the strains tested. However, thyme oil demonstrated the strongest inhibitory activity than other oils against (ZI of 18.5 mm). In general, Campylobacter was more susceptible to the antibacterial activity of Eo, with plates containing thyme or clove oil showing no growth. orange oil was also highly effective on Campylobacter, with a mean ZI of 17.5 mm. The least expensive treatment effective against both and Campylobacter was a combination of 100% concentrations of thyme and orange oil combined on a 50:50 proportion. Tested on the same strains of bacteria, the thyme-orange combination (ToC) had a mean ZI of 20.5 mm for and 21.3 mm for Campylobacter. Thyme-orange combination demonstrated a synergetic effect against, but no such effect was noticed for Campylobacter. on average, 0.14% ToC was required to inhibit both pathogens. Hence, ToC can be considered as a potential antimicrobial for future studies on food systems. Key words: thyme oil, orange oil,, Campylobacter, essential oil 2014 J. Appl. Poult. Res. 23 : /japr DESCRIPTION OF PROBLEM and Campylobacter continue to be leading causes of human food-borne illness worldwide. In the United States alone, an estimated one million cases of and 800,000 cases of Campylobacter are reported annually [1]. Contaminated poultry and poultry 1 Corresponding author: doug.smith@ncsu.edu

2 222 JAPR: Research Report products are major vehicles for the transmission of both pathogens to humans [2, 3]. Prevalence in broiler meat at retail in the United States ranges from 4 to 61% for and up to 76% for Campylobacter [4 6]. Postharvest pathogen reduction is achieved primarily using chlorinated water or spraying with broad spectrum antimicrobial agents, such as peroxyacetic acid, trisodium phosphate, acidified calcium sulfate, organic acids, and cetyl pyridinium chloride [7]. To comply with recent food safety enhancement efforts and stricter performance standards, processors have increased the rinse water volume or the use of chemicals such as those listed above. However, organic producers have restrictions in the use of chemical interventions [8]. With the increasing demands for foods that are free of pathogens and chemical residues, plant-based natural antimicrobials are becoming popular. Plant essential oils (EO) are naturally derived compounds known historically for their antiseptic properties. However, their use in foods has primarily been limited to flavor additives in soft drinks and sweets, and more recently as preservatives [9 12]. These oils are classified as generally recognized as safe food additives in the United States [9]. Thyme oil is derived from Thymus vulgaris, and its major components are thymol and carvacrol. Sweet orange oil is derived from Citrus sinensis, and its major component is limonene. The acute toxicity by oral intake is very low, with the lethal dose 50 (determined in rats, representing the lethal dose of oil per unit of weight for killing 50% of the population of test animals) of 2 to 5 and >5 g/kg of BW for thyme and orange oils, respectively. Generally, the concentration of EO in food is kept low to minimize any sensory or flavor changes [10]. Essential oils are volatile, complex mixtures produced by aromatic plants as secondary metabolites. Most oils are made up of 20 to 60 compounds from a variety of chemical classes, predominantly terpenes, and their derivatives. Often the antimicrobial activity of the oil is due to the complex interactions among these compounds, although the bioactivity of the oil would be closely related to the main component. Usually combinations of essential oils or their purified major components targets multiple biochemical processes in the bacteria leading to a synergistic, additive, or sometimes even antagonistic effects. The possible mechanism of action of this oil blend was due to interactions between the phenolic compounds from thyme oil and the alkyl groups from the orange oil. Phenolic compounds are hydrophobic and antimicrobial. The major phenolic compounds of thyme oil are carvacrol [2-methyl-5-(1-methylethyl) phenol] and thymol (2-isopropyl-5-methylphenol). The hydrophobicity of the phenolics enables them to attach to the lipid bilayer of the cytoplasmic membrane, leading to the leakage of ions and other critical molecules, resulting in death of the cell [11 13]. The major component of orange oil is limonene [1-methyl-4-(1-methylethenyl)- cyclohexene], a type of alkyl group that is more active than other forms, such as p-cymene. Alkyl substitution of phenolic compounds renders an increased antimicrobial activity [14]. The large variety of active compounds also prevents microbes from developing resistance [15]. Previous reports of in vitro studies show that thyme, orange, rosemary, and clove oils were effective either against or Campylobacter [16 19]. Therefore, these 4 oils were selected to study the effect of various concentrations on inhibition of and Campylobacter. The purpose of the current study was to identify an inexpensive combination of EO that can be safely used in poultry food systems to reduce contamination by pathogens. MATERIALS AND METHODS Test Compounds Thyme oil, clove oil, rosemary oil [20], and orange oil [21] were obtained in certified foodgrade form. Thyme-orange combination (TOC) was a blend of undiluted thyme and orange oils at 50:50 ratios. Dimethyl sulfoxide [22] was added to increase the solubility of the EO in aqueous media. Test Bacteria. Enteritidis, Montevideo, and Heidelberg stock cultures were grown on nutrient agar slants [23]. Test strains were transferred to brain heart infusion medium (BHI) [23] before use. Each working culture was individually inoculated to fresh BHI and incubated at 37 C for 12

3 Thanissery et al.: ESSENTIAL OILS INHIBIT PATHOGENS 223 h to obtain 10 8 cfu/ml. Equal volumes of individual cultures were mixed to obtain mixtures of the 3 strains. Campylobacter. Field isolates Cambylobacter jejuni (11601MD, from the intestine of a turkey), C. jejuni (RM1221, from chicken skin), and Campylobacter coli (RM2228, a multidrug resistant isolate from chicken carcass) were used. The cultures were preserved at 80 C in BHI medium with 20% glycerol. Test strains were transferred to Muller Hinton agar [23] plates and grown under microaerobic condition before use. The test organisms were further subcultured in Muller Hinton broth and incubated at 42 C for 24 h under microaerobic conditions to obtain 10 7 cfu/ml. The individual strains were combined in equal volumes to obtain a cocktail. Disc Diffusion Method Disc diffusion is a method to initially screen chemicals to determine antimicrobial activity. The zone of inhibition indicates the relative antibacterial activity [9]. The agar media plates (brilliant green agar with sulfadiazine [24] for and charcoal cefoperazone deoxycholate agar [23] for Campylobacter) were swabbed with the bacterial suspensions ( cfu/ml). Sterile filter paper discs (6.0 mm in diameter) were soaked with 10 µl of individual undiluted EO or TOC (5 µl of thyme oil and 5 µl of orange oil) and placed on the surface of inoculated agar plates. All plates were left at room temperature for 30 min to allow diffusion of oil before inverting the plates for incubation. plates were incubated at 37 C for 24 h, and Campylobacter plates under microaerobic conditions at 42 C for 48 h. Control plates lacked exposure to any EO. Two plates containing 2 discs each were used for each oil bacteria combination. Two independent experiments were performed and zones of inhibition (ZI; in millimeters, including the 6 mm of the disk) were expressed as mean values. Macro-Broth Dilution Technique Minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of thyme, orange, clove, rosemary, and TOC was determined using a macro-broth dilution technique. Serial 2-fold dilutions of the EO were made in tubes using BHI broth for, and Muller Hinton broth for Campylobacter, in a total volume of 10 ml of dimethyl sulfoxide was added to increase the solubility of the EO. The concentration of the oils in the test tubes ranged from to 1.000% (vol/vol). Overnight broth cultures (0.5 ml of cfu/ ml) of or Campylobacter were then added to the tubes. Positive controls contained no EO or DMSO. Negative controls were tubes containing DMSO but no EO. The tubes were incubated at 37 C for 24 h, and Campylobacter tubes at 42 C under microaerobic conditions for 48 h. Turbidity was visually determined for tubes containing, and the least concentration of the oil at which no visible growth was recorded as the MIC. The turbidity of orange oil impaired visual determination of MIC, and, therefore, MBC was determined by plating 100 µl from each dilution on to brilliant green agar plates. Minimum bactericidal concentration was defined as the lowest concentration at which no growth was observed in the plates. Minimum bactericidal concentration was determined for all oils tested for inhibition of Campylobacter. This is because the turbidity that developed from Campylobacter growth in broth was not sufficient enough to be differentiated from the turbidity of the oil. Results are reported as mean values from 2 replicate trials with each dilution used. Statistical Analysis Differences in ZI between different treatments were tested by one-way ANOVA using the GLM procedure of SAS [25]. The antibacterial effects of the oils were compared with the different strains and the cocktail of stains for and different strains, species, and the cocktail of strains and species for Campylobacter. Data was pooled across trials, as no significant trial treatment interaction was observed. Hence, the N value was 8 for each oil bacteria combination, except for a few unreadable plates where N = 6. Statements of statistical significance were based upon P < Tukey s test was applied for means separation. Data are presented as means ± SEM. Minimum bactericidal concentration and MIC are expressed in percent-

4 224 JAPR: Research Report Table 1. Zones of inhibition of serotypes in pure and mixed culture by essential oils determined by disc diffusion assay 1,2 Oil (mm) Heidelberg Montevideo Enteritidis Strain cocktail Thyme 17 b 19 b 19 b 19 a Orange 0 e 7 d 7 d 0 d Rosemary 9 d 7 d 8 d 7 c Clove 13 c 15 c 14 c 13 b TOC 3 19 a 23 a 21 a 19 a SEM a d Within columns, means without common subscripts are significantly different (P < 0.05). 1 Zone of inhibition is expressed as diameter in millimeters. 2 Values represent means of inhibition zones of 2 independent experiments; n = 8. Individual undiluted essential oils were used at the rate of 10 µl per disk. 3 TOC = thyme-orange combination at the rate of 5 µl of undiluted thyme oil and 5 µl of undiluted orange oil. ages and they are average values of 2 separate trials. RESULTS AND DISCUSSION In the present study, the EO had varying degrees of growth inhibition against the microorganisms tested. The disc diffusion results for are summarized in Table 1. The oils of thyme, clove, and rosemary, used at the rate of 10 µl/disc, had ZI against all strains of. Thyme oil had the widest ZI (P < 0.05), at an average of 18.5 mm, followed by clove (13.8) and rosemary (7.8). Similar properties have been observed by other researchers. The ZI recorded for thyme, clove, and rosemary oils against Enteritidis were 11.1, 11.1, and 9.3 mm, respectively [26]. In another report, the authors found a ZI of 26 mm for thyme, 16 mm for clove, and 5 mm for rosemary oil against Pullorum [15]. Orange oil did not have any effect on Heidelberg and the cocktail of strains, and exhibited the lowest antibacterial effect on Montevideo and Enteritidis, with a mean ZI of 7 mm. Low activity of orange oil on has also been reported by others [15, 26]. The cost of thyme oil is $0.60/mL, whereas orange oil is available for only $0.12/mL. Therefore, these EO were combined to arrive at a least cost treatment. In the present study, an interesting observation was made when the combined activity of thyme and orange oils were tested against. The TOC produced an average ZI of 20.5 mm, which was greater than (P < 0.05) the effects of individual thyme and orange oils, indicating a synergistic effect. Although the disc diffusion study was done for the initial screening of EO, one limitation was the difference in solubility of the different oils in the agar media. Hence, the macro-broth dilution method was used to confirm the findings of the disc diffusion assay and to determine the inhibitory concentrations. Essential oils are not soluble in water, and it was therefore necessary to add DMSO as an emulsifier. The negative controls with DMSO showed no antibacterial activity. The MIC and MBC values for are listed in Tables 2 and 3. The Table 2. Minimum inhibitory concentration of essential oils on selected strains of in pure and mixed culture 1 Oil (%) Heidelberg Montevideo Enteritidis Strain cocktail Thyme Rosemary Clove Inhibitory concentrations are average values of 2 independent trials. Each value is an average of 2 observations. The minimum inhibitory concentration was run in a total volume of 10 ml.

5 Thanissery et al.: ESSENTIAL OILS INHIBIT PATHOGENS 225 Table 3. Minimum bactericidal concentration of essential oils on selected strains of in pure and mixed culture 1 Oil (%) Heidelberg Montevideo Enteritidis Cocktail Orange >1 >1 >1 >1 TOC Inhibitory concentrations are average values of 2 independent trials. Each value is an average of 2 observations. The minimum bactericidal concentration was run in a total volume of 10 ml. 2 TOC = thyme-orange combination at the rate of 5 µl of undiluted thyme oil and 5 µl of undiluted orange oil. average MIC values for the oils of thyme, clove, and rosemary were 0.06, 0.11, and 0.88%, respectively. A higher concentration of orange oil (MBC > 1%) was required to inhibit. The average concentration of TOC required for a similar bactericidal effect was 0.14%. The inhibitory concentrations reported in other essential oil studies varied from 0.04 to 2% (thyme), 0.04 to 2% (clove), >1 to 2 (rosemary), and >2% (orange) [16, 26]. The synergistic effect of the major components of thyme oil, thymol, and carvacrol with the other essential oil components, such as eugenol, cymene, linalool, and cinnamaldehyde, has been reported [27 30]. In some studies, researchers have suggested that the whole EO has higher antibacterial effect than the individual components, suggesting that the minor components in the oil are also crucial for the observed activity [31, 32]. Hence, in the current study we used a combination of whole oils. To our knowledge, this is the first report on anti- effects from the blend of thyme and orange oils. Campylobacter was found to be relatively sensitive to all the oils tested. The results of disc diffusion assay for Campylobacter are presented in Table 4. Oils of thyme and clove showed the strongest antibacterial activity (resulting in no visible growth on the plates), followed by rosemary and orange oils (ZI of 13.5 and 17.5, respectively). Because testing was conducted to screen multiple oils, additional testing at other dilutions was not conducted for thyme or clove oil. The orange oil in the TOC was found to lessen the activity of thyme; however, TOC produced a ZI of 22 for C. jejuni and 21 for C. coli. No growth was noticed even at the highest dilution of oil tested in the present study. However, growth was observed in the control and DMSO tubes. Therefore, the exact MIC or MBC could not be determined. In other studies, C. jejuni was found to be 5- to 10-fold more sensitive to many EO than other genera of bacteria [18, 33], possibly reflecting the fastidious growth requirements of the organism. In a study conducted by Friedman et al. [18], to 0.034% Table 4. Zones of inhibition of different strains of Campylobacter jejuni and Campylobacter coli in pure and mixed culture by essential oils determined by disc diffusion assay 1,2 Oil (mm) C. jejuni 11601MD C. jejuni RM1221 C. coli RM2228 Strain cocktail Thyme NG 3 NG NG NG Orange 18 b 16 b 19 b 17 b Rosemary 17 b 15 b 11 c 11 c Clove NG NG NG NG TOC 4 22 a 22 a 21 a 20 a SEM SEM a c Within columns, means without common subscripts are significantly different (P < 0.05). 1 Zone of inhibition is expressed as diameter in millimeters. 2 Values represent means of inhibition zones of 2 independent experiments; n = 8; n = 6 for orange treatment. Individual undiluted essential oils were used at the rate of 10 µl per disk. 3 NG = no visible growth on the plate. 4 TOC = thyme-orange combination at the rate of 5 µl of undiluted thyme oil and 5 µl of undiluted orange oil. 5 SEM when n = 8. 6 SEM when n = 6 (as a few plates were not readable).

6 226 JAPR: Research Report of various EO was required to cause a 50% decrease in C. jejuni relative to a buffer control. Conversely, Smith-Palmer et al. [26] reported Campylobacter to be the most resistant organism among those they investigated. Differences in activity between studies could be due to the differences in the composition of the oils. In general, the antimicrobial activity of the EO depends on their chemical composition, which is determined by the genotype of the source plants, region, and environmental conditions in which they are grown [34, 35]. CONCLUSIONS AND APPLICATIONS 1. The antibacterial activity of thyme, orange, clove, and rosemary EO against and Campylobacter has been reported in various publications. These findings were confirmed in the present investigation. 2. Camplobacter was found to be more sensitive to the essential oils tested than. 3. Thyme-orange oil combination when used at 0.14% was found to be effective against both and Campylobacter grown in laboratory media. Hence, TOC can be considered as a potential antimicrobial for future studies on poultry food systems. REFERENCES AND NOTES 1. Scallan, E., P. M. Angulo, F. J. Tauxe, R. V. Hoekstra, and M. Robert Foodborne illness acquired in the United States Unspecified agents. Emerg. Infect. Dis. 17: Bryan, F. L., and M. P. Doyle Health risks and consequences of and Campylobacter jejuni in raw poultry. J. Food Prot. 58: Doyle, M., C. Kaspar, J. Archer, and R. Klos White paper on human illness caused by from all food and non-food vectors. FRI Briefings 24. Food Research Institute, University of Wisconsin-Madison. Accessed July Human_Illness_6_09.pdf. 4. Bokanyi, R. P., J. Stephens, and D. Foster Isolation and characterization of from broiler carcasses or parts. Poult. Sci. 69: Cui, S., B. Ge, J. Zheng, and J. Meng Prevalence and antimicrobial resistance of Campylobacter spp. and serovars in organic chickens from Maryland retail stores. Appl. Environ. Microbiol. 71: Zhou, F., B. Ji, H. Zhang, H. Jiang, Z. Yang, J. Li, J. Li, and W. Yan The antibacterial effect of cinnamaldehyde, thymol, carvacrol and their combinations against the foodborne pathogen Typhimurium. J. Food Saf. 27: Oyarzabal, O. A Reduction of Campylobacter spp. by commercial antimicrobials applied during the processing of broiler chickens: A review from the United States perspective. J. Food Prot. 68: O Bryan, C. A., P. G. Crandall, and S. C. Ricke Organic poultry pathogen control from farm to fork. Foodborne Pathog. Dis. 5: Kim, J., M. R. Marshall, and C. Wei Antibacterial activity of some essential oil components against five foodborne pathogens. J. Agric. Food Chem. 43: Burt, S Essential oils: Their antibacterial properties and potential applications in foods A review. Int. J. Food Microbiol. 94: Helander, I. M., H. Alakomi, K. Latva-Kala, T. Mattila-Sandholm, I. Pol, E. J. Smid, L. G. Gorris, and A. von Wright Characterization of the action of selected essential oil components on Gram-negative bacteria. J. Agric. Food Chem. 46: Dorman, H. J., and S. Deans Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88: Trombetta, D., F. Castelli, M. G. Sarpietro, V. Venuti, M. Cristani, C. Daniele, A. Saija, G. Mazzanti, and G. Bisignano Mechanisms of antibacterial action of three monoterpenes. Antimicrob. Agents Chemother. 49: Pauli, A., and K. Knobloch Inhibitory effects of essential oil components on growth of food-contaminating fungi. Z. Lebensm. Unters. Forsch. 185: Deans, S., and G. Ritchie Antibacterial properties of plant essential oils. Int. J. Food Microbiol. 5: Hammer, K. A., C. Carson, and T. Riley Antimicrobial activity of essential oils and other plant extracts. J. Appl. Microbiol. 86: Nannapaneni, R., V. I. Chalova, P. G. Crandall, S. C. Ricke, M. G. Johnson, and C. A. O Bryan Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions. Int. J. Food Microbiol. 129: Friedman, M., P. R. Henika, and R. E. Mandrell Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and enterica. J. Food Prot. 65: Oussalah, M., S. Caillet, L. Saucier, and M. Lacroix Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157: H7, Typhimurium, Staphylococcus aureus, and Listeria monocytogenes. Food Contr. 18: Spectrum Chemicals Manufacturing Corporation, Gardena, CA. 21. Sigma-Aldrich Co. LLC, St. Louis, MO. 22. Fisher Scientific, Hanover Park, IL. 23. Oxoid Ltd, Basingstoke, Hampshire, UK. 24. Neogen, Lansing, MI. 25. SAS Institute SAS/STAT User s Guide. Version 9.1 for Windows. SAS Inst. Inc., Cary, NC.

7 Thanissery et al.: ESSENTIAL OILS INHIBIT PATHOGENS Smith-Palmer, A., J. Stewart, and L. Fyfe Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26: Pei, R. S., F. Zhou, B. Ji, and J. Xu Evaluation of combined antibacterial effects of eugenol, cinnamaldehyde, thymol, and carvacrol against E. coli with an improved method. J. Food Sci. 74:M379 M Ultee, A., R. Slump, G. Steging, and E. Smid Antimicrobial activity of carvacrol toward Bacillus cereus on rice. J. Food Prot. 63: Bassolé, I. H., A. Lamien-Meda, B. Bayala, S. Tirogo, C. Franz, J. Novak, R. C. Nebié, and M. H. Dicko Composition and antimicrobial activities of Lippia multiflora Moldenke, Mentha piperita L. and Ocimum basilicum L. essential oils and their major monoterpene alcohols alone and in combination. Molecules 15: Zhou, F., B. Ji, H. Zhang, H. Jiang, Z. Yang, J. Li, J. Li, and W. Yan The antibacterial effect of cinnamaldehyde, thymol, carvacrol and their combinations against the foodborne pathogen typhimurium. J. Food Saf. 27: Gill, A. O., P. Delaquis, P. Russo, and R. Holley Evaluation of antilisterial action of cilantro oil on vacuum packed ham. Int. J. Food Microbiol. 73: Mourey, A., and N. Canillac Anti-Listeria monocytogenes activity of essential oils components of conifers. Food Contr. 13: Piskernik, S., A. Klančnik, C. T. Riedel, L. Brøndsted, and S. S. Možina Reduction of Campylobacter jejuni by natural antimicrobials in chicken meat-related conditions. Food Contr. 22: Rasooli, I., and S. A. Mirmostafa Bacterial susceptibility to and chemical composition of essential oils from Thymus kotschyanus and Thymus persicus. J. Agric. Food Chem. 51: Rota, M. C., A. Herrera, R. M. Martínez, J. A. Sotomayor, and M. J. Jordán Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis, and Thymus hyemalis essential oils. Food Contr. 19: