309 Journal of Food Protection, Vol. 79, No. 2, 2016, Pages 309–315 doi:10.4315/0362-028X.JFP-15-392 Copyright Q, International Association for Food Protection
Research Note
Antimicrobial Activity of Individual and Combined Essential Oils against Foodborne Pathogenic Bacteria ´ FATIMA REYES-JURADO, AURELIO LOPEZ-MALO,
AND
ENRIQUE PALOU*
Departamento de Ingenier´ıa Qu´ımica, Alimentos y Ambiental, Universidad de las Am´ericas Puebla, Cholula, Puebla 72810, M´exico MS 15-392: Received 10 September 2015/Accepted 30 October 2016
ABSTRACT The antimicrobial activities of essential oils from Mexican oregano (Lippia berlandieri Schauer), mustard (Brassica nigra), and thyme (Thymus vulgaris) were evaluated alone and in binary combinations against Listeria monocytogenes, Staphylococcus aureus, or Salmonella Enteritidis. Chemical compositions of the essential oils were analyzed by gas chromatography–mass spectrometry. The MICs of the evaluated essential oils ranged from 0.05 to 0.50% (vol/vol). Mustard essential oil was the most effective, likely due to the presence of allyl isothiocyanate, identified as its major component. Furthermore, mustard essential oil exhibited synergistic effects when combined with either Mexican oregano or thyme essential oils (fractional inhibitory concentration indices of 0.75); an additive effect was obtained by combining thyme and Mexican oregano essential oils (fractional inhibitory concentration index ¼ 1.00). These results suggest the potential of studied essential oil mixtures to inhibit microbial growth and preserve foods; however, their effect on sensory quality in selected foods compatible with their flavor needs to be assessed. Key words: Antimicrobial activity; Combinations; Essential oils; Pathogenic bacteria
Essential oils (EOs) are well known as antimicrobial agents that may be used to control food spoilage and foodborne pathogenic bacteria because they have a wide range of active volatile components (8, 19). It has been demonstrated that several spices and herbs containing EOs effectively inhibit microbial growth, although different results are observed depending on the test conditions, the type of microorganism, and the source of the antimicrobial compounds (46). In vitro studies have shown that EOs have antimicrobial properties against gram-positive bacteria, gram-negative bacteria, molds, and yeasts; among the most studied bacteria are Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, and Staphylococcus aureus (17). Cinnamon, mustard, thyme, clove, and oregano EOs are all natural antimicrobial and flavoring substances, and they are classified as generally recognized as safe (47). The primary component of mustard EO is allyl isothiocyanate (AITC), a nonphenolic volatile compound found in plants belonging to the Crucifereae. Many studies have reported that AITC effectively inhibits a variety of pathogenic microorganisms, even when used at low concentrations (11, 46). Carvacrol and thymol are components present in EOs of thyme and oregano that have demonstrated effectiveness against several pathogenic bacteria (40, 43, 45). * Author for correspondence. Tel: þ52 (222) 229-2126; Fax: þ52 (222) 229-2727; E-mail: [email protected].
If EOs are expected to be widely applied as natural antimicrobials, their sensory impact should be considered because these agents can alter the food taste or exceed acceptable flavor thresholds (23). Recent studies established that combining different EOs can lead to a broad spectrum of activity that can increase their effectiveness and decrease their sensory impact in food (41). These combinations may also control some bacteria that are known to show consistently high resistance to antimicrobials. Therefore, the aim of this study was to investigate the antimicrobial activity of mustard (Brassica nigra), thyme (Thymus vulgaris), and Mexican oregano (Lippia berlandieri Schauer) EOs when applied individually and in combination against L. monocytogenes, S. aureus, or Salmonella Enteritidis to identify possible potential uses as food antimicrobials.
MATERIALS AND METHODS EOs. Mexican oregano, mustard, and thyme EOs were purchased from Grupo TECNNAL (Jalisco, M´exico). The EOs were kept in dark stoppered flasks and under refrigerated conditions until use. Chemical analysis. The EOs were analyzed by gas chromatography–mass spectrometry, using a 6850 Series Network GC System gas chromatograph (Agilent Technologies, Santa Clara, CA) coupled to a 5975C VL mass selective detector with triple-axis detector (Agilent Technologies) and a split-splitless injector (1:10 split ratio). A fused silica HP-5MS (5% phenyl–95% polydimethylsiloxane) capillary column (30 m by 0.250 mm; film thickness, 0.25 lm) was used. The carrier gas was helium at a flow
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TABLE 1. Effect of combinations of Mexican oregano and mustard EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC Mexican oregano
0
25
50
75
100
a
Mustard
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
0.75
0.75
S. aureus
G G G G NG G G G NG NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
1.00
1.00
Salmonella Enteritidis
G G G G NG G G G NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
0.75
G, growth; NG, no growth.
rate of 1.1 ml/min. Samples were prepared by dilution of the EO at 5:100 (vol/vol) in ethanol, and the injection volume was 1 ll. The column oven temperature was programmed from 608C (4 min) to 2408C (10 min) at 48C/min. Injector and detector temperatures were set at 250 and 2808C, respectively (22). Retention indices were calculated using a homologous series of n-alkanes C8.0 to C18.0 (Sigma, St. Louis, MO). Eluted compounds were identified by comparing their retention indices from the literature and also with the mass profile of the same compounds available from the U.S. National Institute of Standard Technology library (3). Microorganisms and culture methods. Two strains of gram-positive bacteria, L. monocytogenes (Scott A) and S. aureus (ATCC 29413), and one strain of gram-negative bacteria, Salmonella Enteritidis (PT 30), were tested. Strains were obtained from the Food Microbiology Laboratory of the Universidad de las Am´ericas Puebla, M´exico. Bacteria were maintained on Trypticase soy agar (TSA; Difco, BD, Sparks, MD) slants at 48C. The strains were activated and cultured in Trypticase soy broth (TSB; Difco, BD) at 378C for 24 h (11, 44). Determination of MIC. The broth dilution method was used to determine the MIC by using different serial dilutions of EOs in TSB (32). For each test, 0.1 ml of EO at the appropriate dilution was added to capped glass tubes containing 9.7 ml of TSB, 0.1 ml of 0.2% Tween 80, and 0.1 ml of bacterial culture (to attain a final concentration of ~104 CFU/ml); then, the suspension was thoroughly mixed. Inoculated broths were incubated at 378C for
24 h. Broths containing bacterial cultures and 0.1 ml of 0.2% Tween 80 without the tested EOs were used as positive controls. Every experiment was replicated twice. The MIC at 24 h was defined as the lowest concentration of those tested that resulted in a complete inhibition of visible growth in the broth (optical density 0.05). To verify the inhibitory effect of the EOs, 0.1-ml aliquots from the tubes where growth was not observed were spread on TSA plates and incubated at 378C. Colonies were counted after 24 h of incubation (44). Determination of combined antimicrobial efficacy. The effect of EO binary combinations was also determined. In these tests, capped glass tubes with 9.6 ml of TSB, 0.1 ml of each EO MIC proportion (Tables 1 through 3), 0.1 ml of 0.2% Tween 80, and 0.1 ml of bacterial culture (to attain a final concentration of ~104 CFU/ml) were prepared (7). Broths containing bacterial cultures and 0.1 ml of 0.2% Tween 80 without EOs were used as positive controls. Samples were incubated at 378C for 24 h. Every experiment was replicated twice. After 24 h, 0.1-ml aliquots from tubes where no growth was observed were spread on TSA plates and incubated at 378C. Colonies were counted after 24 h of incubation (44). The fractional inhibitory concentrations (FICs) were calculated with the MIC of each EO by equations 1 and 2 (21): FIC A ¼
MIC of A in the presence of B MIC of A individually
ð1Þ
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TABLE 2. Effect of combinations of thyme and Mexican oregano EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC
Thyme
0
25
50
75
100
a
Mexican oregano
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G G G NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
S. aureus
1.00
G G G G NG G G G NG NG G G G NG NG G NG NG NG NG NG NG NG NG NG
1.00
FICIndex
1.00
1.00
Salmonella Enteritidis
G G G G NG G G G NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
0.75
G, growth; NG, no growth. FIC B ¼
MIC of B in the presence of A MIC of B individually
ð2Þ
where A and B represent the evaluated EOs. Once FICs were obtained, the fractional inhibitory concentration index (FICIndex) for tested EOs can be determined by equation 3: FICIndex ¼ FIC A þ FIC B
ð3Þ
Because the FICIndex is calculated based on the MIC of each EO in combination, the resulting value indicates the effect of the mixture. An FICIndex ,1.0, 1.0, or .1.0 was defined as a synergistic, additive, or antagonistic effect of the tested EO combination, respectively (21, 44).
RESULTS AND DISCUSSION Chemical composition of EOs. Main compounds present in tested EOs are reported in Table 4. In total, 7 compounds were identified by gas chromatography–mass spectrometry in mustard EO, 17 compounds in Mexican oregano EO, and 15 compounds in thyme EO. AITC was detected in great abundance in mustard EO, representing 98.42% of the compounds present in the oil. The monoterpenes q-cymene (35.5%) and carvacrol (26.9%) were identified as the major components in Mexican oregano EO; other compounds in lower proportions included a-pinene, caryophyllene, camphene, b-pinene, a-terpineol, linalool, and a-terpineol. In thyme EO, q-cymene (19.8%),
linalool (13.6%), and thymol (12.1%) were identified as major components; other compounds, such as carvacrol, bpinene, caryophyllene, isoborneol, borneol, and camphene, were also present. These results agree with previous reports identifying similar compositions (12, 42), with AITC as the major component in mustard EO. The main components reported for oregano EOs included thymol, carvacrol, qcymene, and c-terpinene (1, 2, 14). Compounds detected in the thyme EO tested herein agree with those reported by Consentino et al. (9), Baydar et al. (5), and Imelouane et al. (27), identifying linalool, thymol, carvacrol, a-pinene, and q-cymene as major components. It is well known that the chemical composition determines the antimicrobial activity of EOs and that their major components are primarily responsible for their activity; so, if there is an increased presence of these components, the antimicrobial activity will be an enhanced (26). Antimicrobial activity depends not only on the chemical composition but also on the lipophilic properties, the potency of functional groups, and aqueous solubility; thus, a mixture of compounds with different biochemical properties may increase EO efficacy.
MICs of EOs tested individually. To determine the effectiveness of EOs against the tested bacteria, selected concentrations (0.02, 0.05, 0.08, 0.10, 0.12, 0.15, 0.20, 0.30, 0.40, 0.50, and 0.60% [vol/vol]) of the EOs were tested
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TABLE 3. Effect of combinations of thyme and mustard EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC Thyme
0
25
50
75
100
a
Mustard
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G G G NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
S. aureus
1.00
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
1.00
FICIndex
0.75
0.75
Salmonella Enteritidis
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
0.75
0.75
G, growth; NG, no growth.
using the broth dilution method. The MIC of each EO is presented in Table 5. For these MICs, no colonies were observed after 24 h of further incubation when aliquots were spread plated on TSA. EOs exhibit inhibitory effects against both gram-positive and gram-negative bacteria. Results showed that antimicrobial activity varied among the studied EOs; concentrations from 0.05 to 0.10% (vol/vol) of mustard EO were required to inhibit the growth of L. monocytogenes, S. aureus, or Salmonella Enteritidis, whereas concentrations from 0.15 to 0.40% (vol/vol) of Mexican oregano EO and from 0.10 to 0.12% (vol/vol) of thyme EO were necessary to inhibit the growth of tested bacteria (Table 5). S. aureus was the most susceptible bacterial species, followed by L. monocytogenes, and Salmonella Enteritidis was the most resistant among the studied microorganisms. It has been reported that gram-positive bacteria are more susceptible to EOs than gram-negative bacteria, and this tolerance of gram-negative bacteria to EOs has been related to the presence of a hydrophilic outer membrane that blocks the penetration of hydrophobic EOs into target cell membranes (16, 29, 33). Based on the MICs, mustard EO was the most effective against the studied bacteria; its antimicrobial activity is largely due to the presence of AITC, the main active compound in mustard EO. The antimicrobial mode of action
of AITC is related to enzyme inhibition and protein alteration by oxidative cleavage of disulfide bonds (26). This compound has been shown to have strong antimicrobial activity in liquid media as well as when applied in its vapor form (31, 34, 42). According to different reports, MICs of mustard EOs varied from 0.02 to 0.2% (vol/vol) (28, 38, 42, 44). Some reports indicate that mustard EO affects the concentration of intracellular components such as ATP in bacteria and affects the pH, suggesting that the cytoplasmic membrane is involved in the antimicrobial action of mustard EO (46). In general, it is difficult to compare published results for selected EOs because some reports do not provide the chemical composition of the EOs, or different methods were used to investigate their antimicrobial activity (24, 25). In this study, the MICs obtained for the tested EOs agree with those reported in other studies. For example, Dorman and Deans (13), Elgayyar et al. (15), Nostro et al. (39), Nevas et al. (37), Becerril et al. (6), and Nedorostova et al. (36) reported MICs from 0.06 to 0.50% (vol/vol) for different species of oregano EO against some foodborne pathogenic bacteria. Likewise, different values of thyme EOs have been reported. For example, Friedman et al. (18), Burt (8), and Imelouane et al. (27) reported MICs ranging from 0.02 to 0.60% (vol/vol) for different pathogenic bacteria. In both oregano and thyme, the antimicrobial
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TABLE 4. Main compounds of Mexican oregano, mustard, and thyme essential oils (EOs) determined by gas chromatography– mass spectrometry
Compound
Retention index
Mustard EO (%)
Mexican oregano EO (%)
Thyme EO (%)
Diallyl sulfide a-Pinene Camphene b-Pinene Myrcene AITC a-Terpinene q-Cymene c-Terpinene Linalool Isoborneol Borneol a-Terpineol Pulegone Thymol Diallyl trisulfide Carvacrol Caryophyllene Caryophyllene oxide
847 933 944 974 989 1081 1015 1022 1053 1095 1154 1165 1187 1232 1290 1297 1299 1411 1583
0.1 — — — — 98.4 — — — — — — — — — 0.2 — — —
— 3.9 4.1 4.7 — — 0.3 35.5 — 3.1 — — 11.0 0.1 — — 26.9 7.5 2.3
— 7.6 3.5 1.0 2.2 — — 19.8 9.9 13.6 6.7 3.8 5.2 — 12.1 — 7.1 6.2 0.4
activities are mainly attributed to the presence of carvacrol and thymol; previous reports indicate that these compounds have the ability to alter bacterial membranes, increasing their permeability. Furthermore, this increased permeability promotes pH gradients and causes leakage of inorganic ions (30). The antimicrobial activity of the studied EOs against gram-negative and gram-positive bacteria suggests that they may be useful as antimicrobials in selected food formulations. However, the aroma of the studied EOs is quite strong; thus, their usefulness may be limited to foods where oregano, mustard, or thyme flavor would be desirable.
Effect of EO combinations. To explore the possibility of reducing undesirable sensory impacts of the studied EOs on foods, we examined the antimicrobial effect of mixtures of these EOs. The antimicrobial activities of the EO combinations were evaluated using the broth dilution method (32). According to a checkerboard design, the concentrations of the EOs were calculated based on the individual MICs to provide different proportions. Tables 1 through 3 show the percentages of MICs tested as well as the corresponding FICIndex values of EO combinations where their effect on bacterial response was no growth. For the combinations where no growth was observed, no colonies were observed after 24 h of further incubation when aliquots were spread plated on TSA. The FICIndex results indicated synergistic effects against L. monocytogenes and Salmonella Enteritidis by combining different proportions of mustard and Mexican oregano EOs (FICIndex ¼ 0.75), thereby inhibiting these microorganisms (Table 1). Also, a synergistic effect was obtained by
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TABLE 5. Individual MIC of tested EOs against studied bacteria MIC (% [vol/vol]) Bacteria
Mexican oregano
Mustard
Thyme
L. monocytogenes S. aureus Salmonella Enteritidis
0.20 0.15 0.40
0.05 0.05 0.10
0.10 0.10 0.12
combining mustard and thyme EOs against S. aureus and Salmonella Enteritidis (Table 3). When thyme EO was combined with Mexican oregano EO (Table 2), an additive effect was usually obtained, except for Salmonella Enteritidis, for which a synergistic effect was obtained (FICIndex ¼ 0.75). Bassol´e et al. (4) reported a synergistic effect against L. monocytogenes by combining carvacrol with linalool. de Azeredo et al. (10) reported a synergistic effect against L. monocytogenes by combining oregano (Origanum vulgare) EO with rosemary (Rosmarinus officinalis) EO. According to Techathuvanan et al. (44), an additive effect was obtained against L. monocytogenes by combining white mustard and olive EOs. Some authors have reported additive effects against S. aureus by combining thymol with carvacrol (30) and cinnamaldehyde with eugenol (35); however, Gallucci et al. (20) reported antagonistic effects in some combinations of carvacrol with eugenol and carvacrol with myrcene and synergistic effects when combining menthol with geraniol. Techathuvanan et al. (44) also obtained additive effects against S. aureus by combining white mustard and olive EOs. Lv et al. (33) observed synergistic effects against S. aureus by combining oregano EO with basil or bergamot EOs. Zhou et al. (48) found synergistic effects against Salmonella Typhimurium when combining thymol with carvacrol, cinnamaldehyde with carvacrol, and cinnamaldehyde with thymol. Techathuvanan et al. (44) obtained synergistic effects against Salmonella Enteritidis by combining white mustard and olive EOs. Because higher concentrations of EOs are generally required when applied individually, a challenge for practical application is to develop combinations of the EOs to maintain product safety and shelf life, while minimizing undesirable flavor and sensory changes associated with the addition of high concentrations of EOs (23). The synergistic effect of Mexican oregano and mustard EOs may be useful to control L. monocytogenes and Salmonella Enteritidis, and combining thyme and mustard EOs against S. aureus in foods may diminish their sensory impact by reducing the amount of required EOs. The tested EOs demonstrated antimicrobial effectiveness against Salmonella Enteritidis, S. aureus, or L. monocytogenes under in vitro conditions; therefore, they have great potential to be used in foods compatible with their flavor. Although the EOs individually effectively inhibited tested microorganisms, several of their combinations enhanced the antimicrobial activity; thus, these
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combinations could reduce costs and have lower impact on sensory attributes, but still maintain microbial safety and quality. Additional studies are needed to assess the effectiveness of these combinations and their effects on sensory quality in selected foods.
ACKNOWLEDGMENTS We acknowledge financial support (Project 180748) from the National Council for Science and Technology of Mexico (CONACyT) and Universidad de las Am´ericas Puebla (UDLAP). F. Reyes-Jurado greatly acknowledges financial support for her Ph.D. studies in Food Science from CONACyT and UDLAP.
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42. Suhr, K., and P. Nielsen. 2003. Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi. J. Appl. Microbiol. 94:665–674. 43. Tajkarimi, M., S. Ibrahim, and D. Cliver. 2010. Antimicrobial herb and spice compounds in food. Food Control 21:1199–1218. 44. Techathuvanan, C., F. Reyes, J. R. D. David, and P. M. Davidson. 2014. Efficacy of commercial natural antimicrobials alone and in combinations against pathogenic and spoilage microorganisms. J. Food Prot. 77:269–275. 45. Tongnuanchan, P., and S. Benjakul. 2014. Essential oils: extraction, bioactivities, and their uses for food preservation. J. Food Sci. 79:1231–1248. 46. Turgis, M., J. Han, S. Caillet, and M. Lacroix. 2009. Antimicrobial activity of mustard essential oil against Escherichia coli O157:H7 and Salmonella Typhi. J. Food Prot. 20:1073–1079. 47. Yun, J., X. Fan, and X. Li. 2013. Inactivation of Salmonella enterica serovar Typhimurium and quality maintenance of cherry tomatoes treated with gaseous essential oils. J. Food Sci. 78:458–464. 48. Zhou, F., B. Ji, H. Zhang, H. Jiang, Z. Yang, J. Li, J. Li, and W. Yan. 2007. The antibacterial effect of cinnamaldehyde, thymol, carvacrol and their combinations against the food-borne pathogen Salmonella Typhimurium. J. Food Saf. 27:124–133.
Research Note
Antimicrobial Activity of Individual and Combined Essential Oils against Foodborne Pathogenic Bacteria ´ FATIMA REYES-JURADO, AURELIO LOPEZ-MALO,
AND
ENRIQUE PALOU*
Departamento de Ingenier´ıa Qu´ımica, Alimentos y Ambiental, Universidad de las Am´ericas Puebla, Cholula, Puebla 72810, M´exico MS 15-392: Received 10 September 2015/Accepted 30 October 2016
ABSTRACT The antimicrobial activities of essential oils from Mexican oregano (Lippia berlandieri Schauer), mustard (Brassica nigra), and thyme (Thymus vulgaris) were evaluated alone and in binary combinations against Listeria monocytogenes, Staphylococcus aureus, or Salmonella Enteritidis. Chemical compositions of the essential oils were analyzed by gas chromatography–mass spectrometry. The MICs of the evaluated essential oils ranged from 0.05 to 0.50% (vol/vol). Mustard essential oil was the most effective, likely due to the presence of allyl isothiocyanate, identified as its major component. Furthermore, mustard essential oil exhibited synergistic effects when combined with either Mexican oregano or thyme essential oils (fractional inhibitory concentration indices of 0.75); an additive effect was obtained by combining thyme and Mexican oregano essential oils (fractional inhibitory concentration index ¼ 1.00). These results suggest the potential of studied essential oil mixtures to inhibit microbial growth and preserve foods; however, their effect on sensory quality in selected foods compatible with their flavor needs to be assessed. Key words: Antimicrobial activity; Combinations; Essential oils; Pathogenic bacteria
Essential oils (EOs) are well known as antimicrobial agents that may be used to control food spoilage and foodborne pathogenic bacteria because they have a wide range of active volatile components (8, 19). It has been demonstrated that several spices and herbs containing EOs effectively inhibit microbial growth, although different results are observed depending on the test conditions, the type of microorganism, and the source of the antimicrobial compounds (46). In vitro studies have shown that EOs have antimicrobial properties against gram-positive bacteria, gram-negative bacteria, molds, and yeasts; among the most studied bacteria are Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, and Staphylococcus aureus (17). Cinnamon, mustard, thyme, clove, and oregano EOs are all natural antimicrobial and flavoring substances, and they are classified as generally recognized as safe (47). The primary component of mustard EO is allyl isothiocyanate (AITC), a nonphenolic volatile compound found in plants belonging to the Crucifereae. Many studies have reported that AITC effectively inhibits a variety of pathogenic microorganisms, even when used at low concentrations (11, 46). Carvacrol and thymol are components present in EOs of thyme and oregano that have demonstrated effectiveness against several pathogenic bacteria (40, 43, 45). * Author for correspondence. Tel: þ52 (222) 229-2126; Fax: þ52 (222) 229-2727; E-mail: [email protected].
If EOs are expected to be widely applied as natural antimicrobials, their sensory impact should be considered because these agents can alter the food taste or exceed acceptable flavor thresholds (23). Recent studies established that combining different EOs can lead to a broad spectrum of activity that can increase their effectiveness and decrease their sensory impact in food (41). These combinations may also control some bacteria that are known to show consistently high resistance to antimicrobials. Therefore, the aim of this study was to investigate the antimicrobial activity of mustard (Brassica nigra), thyme (Thymus vulgaris), and Mexican oregano (Lippia berlandieri Schauer) EOs when applied individually and in combination against L. monocytogenes, S. aureus, or Salmonella Enteritidis to identify possible potential uses as food antimicrobials.
MATERIALS AND METHODS EOs. Mexican oregano, mustard, and thyme EOs were purchased from Grupo TECNNAL (Jalisco, M´exico). The EOs were kept in dark stoppered flasks and under refrigerated conditions until use. Chemical analysis. The EOs were analyzed by gas chromatography–mass spectrometry, using a 6850 Series Network GC System gas chromatograph (Agilent Technologies, Santa Clara, CA) coupled to a 5975C VL mass selective detector with triple-axis detector (Agilent Technologies) and a split-splitless injector (1:10 split ratio). A fused silica HP-5MS (5% phenyl–95% polydimethylsiloxane) capillary column (30 m by 0.250 mm; film thickness, 0.25 lm) was used. The carrier gas was helium at a flow
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TABLE 1. Effect of combinations of Mexican oregano and mustard EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC Mexican oregano
0
25
50
75
100
a
Mustard
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
0.75
0.75
S. aureus
G G G G NG G G G NG NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
1.00
1.00
Salmonella Enteritidis
G G G G NG G G G NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
0.75
G, growth; NG, no growth.
rate of 1.1 ml/min. Samples were prepared by dilution of the EO at 5:100 (vol/vol) in ethanol, and the injection volume was 1 ll. The column oven temperature was programmed from 608C (4 min) to 2408C (10 min) at 48C/min. Injector and detector temperatures were set at 250 and 2808C, respectively (22). Retention indices were calculated using a homologous series of n-alkanes C8.0 to C18.0 (Sigma, St. Louis, MO). Eluted compounds were identified by comparing their retention indices from the literature and also with the mass profile of the same compounds available from the U.S. National Institute of Standard Technology library (3). Microorganisms and culture methods. Two strains of gram-positive bacteria, L. monocytogenes (Scott A) and S. aureus (ATCC 29413), and one strain of gram-negative bacteria, Salmonella Enteritidis (PT 30), were tested. Strains were obtained from the Food Microbiology Laboratory of the Universidad de las Am´ericas Puebla, M´exico. Bacteria were maintained on Trypticase soy agar (TSA; Difco, BD, Sparks, MD) slants at 48C. The strains were activated and cultured in Trypticase soy broth (TSB; Difco, BD) at 378C for 24 h (11, 44). Determination of MIC. The broth dilution method was used to determine the MIC by using different serial dilutions of EOs in TSB (32). For each test, 0.1 ml of EO at the appropriate dilution was added to capped glass tubes containing 9.7 ml of TSB, 0.1 ml of 0.2% Tween 80, and 0.1 ml of bacterial culture (to attain a final concentration of ~104 CFU/ml); then, the suspension was thoroughly mixed. Inoculated broths were incubated at 378C for
24 h. Broths containing bacterial cultures and 0.1 ml of 0.2% Tween 80 without the tested EOs were used as positive controls. Every experiment was replicated twice. The MIC at 24 h was defined as the lowest concentration of those tested that resulted in a complete inhibition of visible growth in the broth (optical density 0.05). To verify the inhibitory effect of the EOs, 0.1-ml aliquots from the tubes where growth was not observed were spread on TSA plates and incubated at 378C. Colonies were counted after 24 h of incubation (44). Determination of combined antimicrobial efficacy. The effect of EO binary combinations was also determined. In these tests, capped glass tubes with 9.6 ml of TSB, 0.1 ml of each EO MIC proportion (Tables 1 through 3), 0.1 ml of 0.2% Tween 80, and 0.1 ml of bacterial culture (to attain a final concentration of ~104 CFU/ml) were prepared (7). Broths containing bacterial cultures and 0.1 ml of 0.2% Tween 80 without EOs were used as positive controls. Samples were incubated at 378C for 24 h. Every experiment was replicated twice. After 24 h, 0.1-ml aliquots from tubes where no growth was observed were spread on TSA plates and incubated at 378C. Colonies were counted after 24 h of incubation (44). The fractional inhibitory concentrations (FICs) were calculated with the MIC of each EO by equations 1 and 2 (21): FIC A ¼
MIC of A in the presence of B MIC of A individually
ð1Þ
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TABLE 2. Effect of combinations of thyme and Mexican oregano EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC
Thyme
0
25
50
75
100
a
Mexican oregano
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G G G NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
S. aureus
1.00
G G G G NG G G G NG NG G G G NG NG G NG NG NG NG NG NG NG NG NG
1.00
FICIndex
1.00
1.00
Salmonella Enteritidis
G G G G NG G G G NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
1.00
0.75
G, growth; NG, no growth. FIC B ¼
MIC of B in the presence of A MIC of B individually
ð2Þ
where A and B represent the evaluated EOs. Once FICs were obtained, the fractional inhibitory concentration index (FICIndex) for tested EOs can be determined by equation 3: FICIndex ¼ FIC A þ FIC B
ð3Þ
Because the FICIndex is calculated based on the MIC of each EO in combination, the resulting value indicates the effect of the mixture. An FICIndex ,1.0, 1.0, or .1.0 was defined as a synergistic, additive, or antagonistic effect of the tested EO combination, respectively (21, 44).
RESULTS AND DISCUSSION Chemical composition of EOs. Main compounds present in tested EOs are reported in Table 4. In total, 7 compounds were identified by gas chromatography–mass spectrometry in mustard EO, 17 compounds in Mexican oregano EO, and 15 compounds in thyme EO. AITC was detected in great abundance in mustard EO, representing 98.42% of the compounds present in the oil. The monoterpenes q-cymene (35.5%) and carvacrol (26.9%) were identified as the major components in Mexican oregano EO; other compounds in lower proportions included a-pinene, caryophyllene, camphene, b-pinene, a-terpineol, linalool, and a-terpineol. In thyme EO, q-cymene (19.8%),
linalool (13.6%), and thymol (12.1%) were identified as major components; other compounds, such as carvacrol, bpinene, caryophyllene, isoborneol, borneol, and camphene, were also present. These results agree with previous reports identifying similar compositions (12, 42), with AITC as the major component in mustard EO. The main components reported for oregano EOs included thymol, carvacrol, qcymene, and c-terpinene (1, 2, 14). Compounds detected in the thyme EO tested herein agree with those reported by Consentino et al. (9), Baydar et al. (5), and Imelouane et al. (27), identifying linalool, thymol, carvacrol, a-pinene, and q-cymene as major components. It is well known that the chemical composition determines the antimicrobial activity of EOs and that their major components are primarily responsible for their activity; so, if there is an increased presence of these components, the antimicrobial activity will be an enhanced (26). Antimicrobial activity depends not only on the chemical composition but also on the lipophilic properties, the potency of functional groups, and aqueous solubility; thus, a mixture of compounds with different biochemical properties may increase EO efficacy.
MICs of EOs tested individually. To determine the effectiveness of EOs against the tested bacteria, selected concentrations (0.02, 0.05, 0.08, 0.10, 0.12, 0.15, 0.20, 0.30, 0.40, 0.50, and 0.60% [vol/vol]) of the EOs were tested
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TABLE 3. Effect of combinations of thyme and mustard EOs on studied bacteria according to the proportions of their MIC tested and corresponding fractional inhibitory concentration index (FICIndex)a % of MIC Thyme
0
25
50
75
100
a
Mustard
L. monocytogenes
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
G G G G NG G G G G NG G G NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
S. aureus
1.00
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
1.00
FICIndex
0.75
0.75
Salmonella Enteritidis
G G G G NG G G NG NG NG G NG NG NG NG G NG NG NG NG NG NG NG NG NG
FICIndex
0.75
0.75
G, growth; NG, no growth.
using the broth dilution method. The MIC of each EO is presented in Table 5. For these MICs, no colonies were observed after 24 h of further incubation when aliquots were spread plated on TSA. EOs exhibit inhibitory effects against both gram-positive and gram-negative bacteria. Results showed that antimicrobial activity varied among the studied EOs; concentrations from 0.05 to 0.10% (vol/vol) of mustard EO were required to inhibit the growth of L. monocytogenes, S. aureus, or Salmonella Enteritidis, whereas concentrations from 0.15 to 0.40% (vol/vol) of Mexican oregano EO and from 0.10 to 0.12% (vol/vol) of thyme EO were necessary to inhibit the growth of tested bacteria (Table 5). S. aureus was the most susceptible bacterial species, followed by L. monocytogenes, and Salmonella Enteritidis was the most resistant among the studied microorganisms. It has been reported that gram-positive bacteria are more susceptible to EOs than gram-negative bacteria, and this tolerance of gram-negative bacteria to EOs has been related to the presence of a hydrophilic outer membrane that blocks the penetration of hydrophobic EOs into target cell membranes (16, 29, 33). Based on the MICs, mustard EO was the most effective against the studied bacteria; its antimicrobial activity is largely due to the presence of AITC, the main active compound in mustard EO. The antimicrobial mode of action
of AITC is related to enzyme inhibition and protein alteration by oxidative cleavage of disulfide bonds (26). This compound has been shown to have strong antimicrobial activity in liquid media as well as when applied in its vapor form (31, 34, 42). According to different reports, MICs of mustard EOs varied from 0.02 to 0.2% (vol/vol) (28, 38, 42, 44). Some reports indicate that mustard EO affects the concentration of intracellular components such as ATP in bacteria and affects the pH, suggesting that the cytoplasmic membrane is involved in the antimicrobial action of mustard EO (46). In general, it is difficult to compare published results for selected EOs because some reports do not provide the chemical composition of the EOs, or different methods were used to investigate their antimicrobial activity (24, 25). In this study, the MICs obtained for the tested EOs agree with those reported in other studies. For example, Dorman and Deans (13), Elgayyar et al. (15), Nostro et al. (39), Nevas et al. (37), Becerril et al. (6), and Nedorostova et al. (36) reported MICs from 0.06 to 0.50% (vol/vol) for different species of oregano EO against some foodborne pathogenic bacteria. Likewise, different values of thyme EOs have been reported. For example, Friedman et al. (18), Burt (8), and Imelouane et al. (27) reported MICs ranging from 0.02 to 0.60% (vol/vol) for different pathogenic bacteria. In both oregano and thyme, the antimicrobial
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TABLE 4. Main compounds of Mexican oregano, mustard, and thyme essential oils (EOs) determined by gas chromatography– mass spectrometry
Compound
Retention index
Mustard EO (%)
Mexican oregano EO (%)
Thyme EO (%)
Diallyl sulfide a-Pinene Camphene b-Pinene Myrcene AITC a-Terpinene q-Cymene c-Terpinene Linalool Isoborneol Borneol a-Terpineol Pulegone Thymol Diallyl trisulfide Carvacrol Caryophyllene Caryophyllene oxide
847 933 944 974 989 1081 1015 1022 1053 1095 1154 1165 1187 1232 1290 1297 1299 1411 1583
0.1 — — — — 98.4 — — — — — — — — — 0.2 — — —
— 3.9 4.1 4.7 — — 0.3 35.5 — 3.1 — — 11.0 0.1 — — 26.9 7.5 2.3
— 7.6 3.5 1.0 2.2 — — 19.8 9.9 13.6 6.7 3.8 5.2 — 12.1 — 7.1 6.2 0.4
activities are mainly attributed to the presence of carvacrol and thymol; previous reports indicate that these compounds have the ability to alter bacterial membranes, increasing their permeability. Furthermore, this increased permeability promotes pH gradients and causes leakage of inorganic ions (30). The antimicrobial activity of the studied EOs against gram-negative and gram-positive bacteria suggests that they may be useful as antimicrobials in selected food formulations. However, the aroma of the studied EOs is quite strong; thus, their usefulness may be limited to foods where oregano, mustard, or thyme flavor would be desirable.
Effect of EO combinations. To explore the possibility of reducing undesirable sensory impacts of the studied EOs on foods, we examined the antimicrobial effect of mixtures of these EOs. The antimicrobial activities of the EO combinations were evaluated using the broth dilution method (32). According to a checkerboard design, the concentrations of the EOs were calculated based on the individual MICs to provide different proportions. Tables 1 through 3 show the percentages of MICs tested as well as the corresponding FICIndex values of EO combinations where their effect on bacterial response was no growth. For the combinations where no growth was observed, no colonies were observed after 24 h of further incubation when aliquots were spread plated on TSA. The FICIndex results indicated synergistic effects against L. monocytogenes and Salmonella Enteritidis by combining different proportions of mustard and Mexican oregano EOs (FICIndex ¼ 0.75), thereby inhibiting these microorganisms (Table 1). Also, a synergistic effect was obtained by
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TABLE 5. Individual MIC of tested EOs against studied bacteria MIC (% [vol/vol]) Bacteria
Mexican oregano
Mustard
Thyme
L. monocytogenes S. aureus Salmonella Enteritidis
0.20 0.15 0.40
0.05 0.05 0.10
0.10 0.10 0.12
combining mustard and thyme EOs against S. aureus and Salmonella Enteritidis (Table 3). When thyme EO was combined with Mexican oregano EO (Table 2), an additive effect was usually obtained, except for Salmonella Enteritidis, for which a synergistic effect was obtained (FICIndex ¼ 0.75). Bassol´e et al. (4) reported a synergistic effect against L. monocytogenes by combining carvacrol with linalool. de Azeredo et al. (10) reported a synergistic effect against L. monocytogenes by combining oregano (Origanum vulgare) EO with rosemary (Rosmarinus officinalis) EO. According to Techathuvanan et al. (44), an additive effect was obtained against L. monocytogenes by combining white mustard and olive EOs. Some authors have reported additive effects against S. aureus by combining thymol with carvacrol (30) and cinnamaldehyde with eugenol (35); however, Gallucci et al. (20) reported antagonistic effects in some combinations of carvacrol with eugenol and carvacrol with myrcene and synergistic effects when combining menthol with geraniol. Techathuvanan et al. (44) also obtained additive effects against S. aureus by combining white mustard and olive EOs. Lv et al. (33) observed synergistic effects against S. aureus by combining oregano EO with basil or bergamot EOs. Zhou et al. (48) found synergistic effects against Salmonella Typhimurium when combining thymol with carvacrol, cinnamaldehyde with carvacrol, and cinnamaldehyde with thymol. Techathuvanan et al. (44) obtained synergistic effects against Salmonella Enteritidis by combining white mustard and olive EOs. Because higher concentrations of EOs are generally required when applied individually, a challenge for practical application is to develop combinations of the EOs to maintain product safety and shelf life, while minimizing undesirable flavor and sensory changes associated with the addition of high concentrations of EOs (23). The synergistic effect of Mexican oregano and mustard EOs may be useful to control L. monocytogenes and Salmonella Enteritidis, and combining thyme and mustard EOs against S. aureus in foods may diminish their sensory impact by reducing the amount of required EOs. The tested EOs demonstrated antimicrobial effectiveness against Salmonella Enteritidis, S. aureus, or L. monocytogenes under in vitro conditions; therefore, they have great potential to be used in foods compatible with their flavor. Although the EOs individually effectively inhibited tested microorganisms, several of their combinations enhanced the antimicrobial activity; thus, these
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combinations could reduce costs and have lower impact on sensory attributes, but still maintain microbial safety and quality. Additional studies are needed to assess the effectiveness of these combinations and their effects on sensory quality in selected foods.
ACKNOWLEDGMENTS We acknowledge financial support (Project 180748) from the National Council for Science and Technology of Mexico (CONACyT) and Universidad de las Am´ericas Puebla (UDLAP). F. Reyes-Jurado greatly acknowledges financial support for her Ph.D. studies in Food Science from CONACyT and UDLAP.
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