Chemical characterization of the Allium sativum and Origanum vulgare essential oils and their inhibition effect on the growth of some food pathogens

This study sought to evaluate the chemical composition of the Allium sativum and Origanum vulgare essential oils and their effect on the growth inhibition of microorganisms, such as P. aeruginosa, S. Choleraesuis, A. flavus, A. niger and P. simplicissimum, important food contaminants. The main constituents of the oregano essential oil were 4-terpineol (27.03%), γ-terpinene (20.04%), and β-cymene (6.34%), and the main constituents of the garlic essential oil were diallyl trisulfide (38, 81%), diallyl disulfide (25.23%), and methyl allyl trisulfide (12.52%). Inhibition zones were formed in in vitro tests on the bacteria S. Choleraesuis and P. aeruginosa, except for A. sativum against P. aeruginosa. The inhibition of mycelial growth caused by the oregano essential oil occurred with the concentrations of 0.10, 0.03 and 0.05 mg mL-1 for the A. flavus, A. niger and P. simplicissimum fungi, respectively. The CMI for the garlic oil began at the 0.03 mg mL-1 concentration for all species of fungi. The oils presented an inhibitory effect against the microorganisms studied and constitute an alternative for microbiological control in food.


INTRODUCTION
It is essential that measures be taken during food production to ensure that the safety and stability of the final products be ensured throughout the life of the product.The preservation processes have become more complex over the years because the continuous appearance of new foods in the market requires a longer shelf life and because there exists a high potential for contamination by pathogenic and/or deteriorating microorganisms.Thus, the plant elements that possess antimicrobial properties have received emphasis for possible use in food conservation (Kizil & Sogut, 2003).Research on plants has created wide interest because of the great diversity of the natural products, which is much greater than that of synthetic products, and because of their possible antimicrobial activities.
The investigation of the antimicrobial activity of plant products has been increasing.Various antimicrobial compounds have been extracted from plants, and the essential oils have been the principal objects of study (Siqui et al., 2000).In view of their prominent antimicrobial properties, they are viable for use in food preservation.Thus, these metabolites have a potential for use in the food industry, where risk of contamination is great.According to Sridhar et al. (2003), essential oils are known to contain a natural blend of monoterpenes and sesquiterpenes, with a variety of functional groups that lead to antifungal and antibacteial properties.
Fungi are one of the major natural degraders of organic matter.They have the ability to grow on various substrates and can cause huge economic losses.According to Pasin et al. (2002), some fungi have the ability to produce toxic substances, mycotoxins, which can cause severe poisoning to humans when present in food.
In view of this scenario, there is a need to create new alternatives for microbiological control of food that could be applied in combination with existing methods.Accordingly, the aromatic species Allium sativum and Origanum vulgare, commonly known as garlic and oregano, either fresh or dried, present an interesting source of antimicrobial agents whose properties are of great value to industries in general.
The present study sought to evaluate the chemical composition of the essential oils from Allium sativum and Origanum vulgare and their inhibitory activities against the growth of the microorganisms Pseudomonas aeruginosa, Salmonella Choleraesuis, Aspergillus flavus, Aspergillus niger and Penicillium simplicissimum.

MATERIAL AND METHOD
The plant materials were purchased at the Central Market in Belo Horizonte, MG, Brazil, in March 2008.The Allium sativum ("Soeto" brand "lot 460130807) used for extractions was in the form of dehydrated flakes and Origanum vulgare ("Pirata Especiarias" brand, lot F5L B3EO) in its dried form.

Obtaining the essential oils
The extraction of the essential oils from the plant material was performed at the Laboratory of Organic Chemistry, Department of Chemistry, Federal University of Lavras (UFLA) by steam distillation using a modified Clevenger apparatus.The material was packed in six-liter flasks, and the oil was removed by steam distillation over a twohour period.The distillate was collected and was centrifuged at 321.8g for five minutes.The yield was expressed as volume of oil per unit weight of fresh leaves (% v/w) on a dry weight basis (% v/w BLU) (Pimentel et al., 2006).

Chemical characterization
The qualitative analysis of the essential oils was performed at the Department of Chemistry, Federal University of Sergipe (UFS), Aracaju, SE, Brazil, by gas chromatography (GC/MS).A Shimadzu model GC-17 gas chromatograph equipped with a model QP5050A mass-selective detector was employed.The equipment was operated under the following experimental conditions: fused-silica capillary column (3.0 m x 0.25 mm) with a BD-SMS bound stationary phase (Folsom, CA, USA), the temperature of the ion source was 280 o C; the column temperature was programmed with an initial temperature of 50°C for 2 min, an increase of 4ºC min -1 to 200ºC, then 10ºC min -1 to 300ºC, finalizing at 300°C for 10 minutes.The carrier gas was helium (1 mL min -1 ); the initial column pressure was 100.2 kPa, the split ratio was 1:83; and the injected volume was 0.5 mL (1% solution in dichloromethane).The configuration of the mass spectrometer (MS) included an impact energy of 70 EV; decomposition rate = 1000; and decomposition interval of 0.50.Fragments from m/z 40 to 550 Da were recorded.A series of hydrocarbon standards (C 9 H 20 to C 26 H 54 ) was injected under the same conditions as the sample.The quantitative analysis was performed at the Laboratory of Organic Chemistry, Federal University of Lavras, using a Shimadzu model GC-17 gas chromatograph equipped with a flame ionization detector (FID) under the following operating conditions: DB5 capillary column containing 35% phenylmethylpolysiloxane, initial temperature of 45°C, programmed to 240ºC, injector temperature, 200°C, FID detector temperature, 240°C, nitrogen carrier gas (2.2 mL min -1 ), split ratio, 1:10; injected volume, 1 mL (1% solution in dichloromethane); column pressure of 115 kPa.The quantity of each constituent was obtained by normalization of areas (%).

Antibacterial Activity
The microbiological tests for determining the susceptibility of microorganisms to the essential oils were performed at the Laboratory of Food Microbiology, Department of Food Science, Federal University of Lavras.Strains of Pseudomonas aeruginosa ATCC 15442 and Salmonella Choleraesuis ATCC 6539 were employed to evaluate the in vitro inhibitory effect of the essential oils of A. sativum (garlic) and O. vulgare (oregano).The organisms were maintained at 4 o C in Eppendorff tubes containing freezing medium.The counting of the number of colony-forming units was standardized by employing the McFarland scale (0.5%) with an inoculum concentration of 10 6 CFU mL -1 .For the activation of the cultures, they were subcultured in brain heart infusion broth (BHI, Oxoid) and incubated for 24 hours at 37°C.Aliquots were transferred to TSB (Triptic Soy Broth) and incubated again at the same temperature.The increase in the number of microbial cells was monitored every half hour using a spectrophotometer (UV Shimatzu -160 1PC) at 625 nm until the reading reached or exceeded the reading of the 0.5% McFarland solution (NCCLS, 2003).The method employed was that of diffusion in an agar cavity using the Mueller Hinton Agar culture media.This medium was inoculated with cultures (P.aeruginosa and S. Choleraesuis) and deposited on the same agar layer where the slots were prepared with the aid of glass beads.These slots were filled with the essential oils diluted in dimethylsulfoxide (DMSO) at concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90 and 1.95 µL mL -1 .The plates were incubated in a BOD at 37°C for 48 hours (Ogunwande et al., 2005), and the diameters of the halos were measured.A randomized block design with bacteria (P.aeruginosa and S. Choleraesuis) as the block factor was employed for statistical analysis, where the factorial design was 2 x 9 x 2 [two essential oils (garlic and oregano), nine concentrations (500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90 and 1.95 µL mL -1 ), totaling 18 treatments with three replicates].The SISVAR program was used for statistical analysis (Ferreira, 2000).The analysis of variance of the treatments utilized the Scott-Knott test at the 5% significance level.

Antifungal Activity
Evaluation of the antifungal activity of the garlic and oregano oils was performed at the Laboratory of Epidemiology and Management of the Department of Plant Pathology at the Federal University of Lavras.The pathogens were obtained from the mycology laboratory of the Department of Plant Pathology and registered with the following numbers: Aspergillus niger, CML N o 1815; Aspergillus flavus, CML N o 1816 and Penicillium simplicissimum, CML N o 1817.
The in vitro bioanalytical method was employed whereby the effect of the garlic and oregano oils on the growth of mycelial pathogens was evaluated.Petri dishes 9.0 cm in diameter were used.The oil concentrations were 0.03, 0.05, 0.07, 0.10, 0.12, 0.14, 0.16 and 0.18 g mL -1 .Two additional plates, one being the absolute control (containing only the pathogen), and one the relative control (containing only 0.25 mL of ether on a paper disk attached to the top of the Petri dish) were prepared.Both trials were conducted in triplicate.The PDA (potato-dextrose-agar) culture medium was used.After solidification of the culture medium in the Petri dishes, mycelia of fungus cultures were transferred from a pure culture and placed in the center of the plates.The respective solutions of essential oil were added, and the plates were incubated in a BOD chamber at approximately 25°C for seven days.
The measurements of pathogen growth were performed seven days after initiating the experiment, utilizing the means of the perpendicular diameters of the mycelial growth.The percentage of inhibition of growth was calculated for each dose and compared to that of the absolute control (Lindsey & Standen, 2004).Statistical analyses were accomplished using the SISVAR program (Ferreira, 2000).The treatments were arranged in a completely random manner, with three replicates, and the means were compared by the Scott-Knott test at a 5% significance level.3-vinyl-4(H)-1,2-dithiin and allyl methyl disulfide were encountered.These results differ from those obtained in the present study.The only similarity with the present study was the presence of diallyl disulfide, which was the second most abundant constituent in the oil samples analyzed.The inhibition halos for the S. Choleraesuis 6539 and P. aeruginosa ATCC 15442 bacteria, induced by the essential oils from O. vulgare and A. sativum, were measured and demonstrated an inhibitory effect for these oils, except for the case of A. sativum oil with P. aeruginosa.The results are described in Table 3.
It may be inferred that the essential oil from oregano has antibacterial activity against the species tested.The Minimum Inhibitory Concentration (MIC) for both S. Choleraesuis and P. aeruginosa was 15.62 mg mL -1 .Pereira et al. (2008) showed that the essential oil from O. vulgare was effective against E. coli and S. aureus at all the concentrations tested.
Unlike the findings of the present study, Ernandes & Garcia-Cruz (2007) found that oregano presented the lowest activity against the pathogens tested when they studied several essential oils.It is noteworthy, however, that, although the inhibitory effect was smaller, it was present at all the concentrations tested.Souza et al. (2006)   the antibacterial activity of the essential oil from O. vulgare against various Gram-positive and Gramnegative spoilage and/or pathogenic bacteria in food, including those bacteria investigated in the present study.The results demonstrated a strong antibacterial activity of the essential oil, as indicated by large growth-inhibition zones (30-37 mm).The MIC values oscillated between 20 and 40 μL mL -1 for most bacteria.The garlic oil did not inhibit the growth of P. aeruginosa.The MIC for S. Choleraesuis was 62.5 mg mL -1 .Packer & Luz (2007) found that samples of garlic oil showed no bacteriostatic or fungistatic activity against the microorganisms tested, including P. aeruginosa, E. coli and S. aureus.However, the results presented in Tables 4 and 5 demonstrate that the oil possesses fungitoxic activity.
Inhibition of mycelial growth of the A. flavus, A. niger and P. simplicissimum fungi caused by the essential oil from oregano occurred at concentrations of 0.10, 0.03 and 0.05 mg mL -1 , respectively, which are considered to represent the MIC.The oregano oil presented bacteriostatic and fungistatic activity against the microorganisms surveyed.
Recent studies of Carmo et al. ( 2008) evaluated the efficacy of the essential oil of O. vulgare against the growth of some species of Aspergillus (A. flavus, A. parasiticus, A. terreus, A. ochraceus, A. fumigatus and A. niger).They observed that it presented a strong activity against the species of the Aspergillus genus, inhibiting the mycelial growth of all the strains of fungi tested.MIC values ranged between 80 and 20 µL mL -1 , with a MIC 50 of 40 µL mL -1 .Moreover, the essential oil was able to inhibit the germination of fungus spores when tested at concentrations of 80 and 40 µL mL -1 .
The garlic oil exhibited inhibitory activity at the 0.03 mg mL -1 concentration.Thus, this oil inhibited mycelial growth at all the concentrations evaluated.Viegas et al. (2007) evaluated the in vitro toxicity of the essential oil of A. sativum against fungi of the A. flavus group isolated from peanut crops.They observed that the oil promoted inhibition zones ranging from 7.0 to 15.0 mm.However, these halos were greater than 12.0 mm for 34% of the isolates tested.These results illustrate a similarity between the two studies with respect to the inhibitory action of the oil against A. flavus.Souza et al. (2004) investigated the in vitro effect of the essential oil of garlic against the Rhizopus sp., Penicillium spp.Eurotium repens and Aspergillus niger fungi isolated from old bread.The oil was observed to inhibit the mycelial growth of all the fungi except Rhizopus sp., whose growth was inhibited only at the highest concentration tested.These results confirm those of previous studies that have demonstrated the antimicrobial and medicinal properties of these spices.

CONCLUSION
The oregano and garlic oils significantly inhibited the growth of the bacteria and fungi studied, 1 Means followed by the same letter do not differ significantly at 5% of probability by the Scott-Knott test.**Chloramphenicol (100 µg mL -1 )

TABLE 1 .
Principal components present in the essential oil of O. vulgare.
1 IKcal -Kovats indexes The chromatogram obtained for O. vulgare is presented in Figure1.

TABLE 2 .
Principal compounds identified in the essential oil from A. sativum.
FIGURE 2. Chromatogram of the essential oil from A. sativum.

TABLE 3 .
Mean diameters of the inhibition halos showing the effect of the essential oils from O. vulgare and A. sativum on the growth of S. Choleraesuis and P. aeruginosa submitted to different concentrations of the oils.

TABLE 4 .
Mean diameters of the inhibition halos showing the effect of the essential oil from O. vulgare on the growth of A. flavus, A. niger and P. simplicissimum submitted to different concentrations of the oil.

TABLE 5 .
Mean diameters of the inhibition halos showing the effect of essential oil from garlic on the growth of A. flavus, A. niger and P. simplicissimum submitted to different concentrations of the oil*.Means followed by the same letter do not differ significantly at 5% of probability by the Scott-Knott test. 1