Comparative Analysis of the Chemical Composition, Antimicrobial and Antioxidant Activity of Essential Oils of Spices Used in the Food Industry in Brazil

There are many food-borne pathogens in the wild and they are considered the cause of serious public health problems in both developed and developing countries. The use of natural products, such as antimicrobial compounds, has been increasing, in an attempt to control bacteria present in foods, mainly pathogens resistant to conventional antibiotics. This chapter is intended to provide the antimicrobial and antioxidant activity of essential oils of Cinnamomum zeylanicum (cinnamon), Origanum vulgare (oregano), Zingiber officinale (ginger), Rosmarinus officinalis (rosemary), Citrus latifolia (tahiti lemon) and Curcuma longa (saffron) as well as to determinate its chemical composition. The oils had been extracted by hydrodistillation with a Clevenger type apparatus and the antimicrobial activity was performed against standard strains Escherichia coli , Pseudomonas aeruginosa and Staphylococcus aureus . The antioxidant activity was carried out using the ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] method. The essential oils presented a mixture of mono- and sesquiterpenes. The best minimum inhibitory concentration was determined to C. zeylanicum against S. aureus . O. vulgare antioxidant activity presented inhibition of 90.74% and EC 50 of 14 μ g mL − 1 . These results demonstrate that the essential oils analyzed presented efficient antibacterial activity and antioxidant action being able to satisfy the demand of use as control of microorganisms in the food.


Introduction
Brazil has an extensive diversity of species in its flora, and great tradition in the use of medicinal plants linked to the popular medicine [1]. Medicinal plants are characterized by common sense within communities as an alternative for Comparative Analysis of the Chemical Composition, Antimicrobial and Antioxidant Activity… DOI: http://dx.doi.org/10. 5772/intechopen.86576 Food-borne illness is a real problem in the present scenario as the consumerism of packaged food. Pathogens entering the packaged foods may survive longer. Therefore, antimicrobial agents either alone or in combination are added to the food or packaging materials to eliminate these agents [31].
Treatment in these cases leads to the indiscriminate use of antibiotics. These have provided a growing multidrug resistance of microorganisms, generating public health problems due to the residues in foods [2]. The antibiotics act as an important selective pressure for the emergence and persistence of resistant strains [32].
Exploiting the antimicrobial property, essential oils are considered as a "natural" remedy to this problem. Alternatives to the use of synthetic antimicrobial agents have been proposed in recent years, and some approaches include herbal products [28]. This promising determination of the action of these essential oils on microorganisms using Gram-positive and Gram-negative bacteria should be performed due to its low cost of acquisition, use and therapeutic action, such as the viability of medicinal potential and its use in the food industry, cosmetics and pharmaceuticals, whereas bacterial resistance is one of the most significant challenges to human health [33].
Thus, the objective of this chapter was to provide the antimicrobial and antioxidant activity of Cinnamomum zeylanicum (cinnamon), O. vulgare (oregano), Z. officinale (ginger), R. officinalis (rosemary), C. longa L. (saffron) and C. latifolia (tahiti lemon) essential oils as well as to determine its chemical composition. The essential oil was extracted by hydrodistillation using Clevenger system. A quantity of 300 g of dry plant material diluted in water at a ratio of 1:10 was boiled at 100°C for 3 h. The oil was dried with anhydrous sodium sulfate and kept in an amber bottle under refrigeration. For in vitro biological assay, the essential oils and reference drugs were dissolved in dimethylsulfoxide (DMSO) at 100 times the highest concentration of use, and subsequently diluted in an appropriate medium to a final concentration of DMSO less than 1%.

Essential oils chemical profile
Chemical characterization of the essential oils was performed by gas chromatography coupled to mass spectrometry (GC-MS). The essential oils under study were dissolved in 1 mg/mL ethyl acetate and analyzed on Shimadzu QP 5000 gas chromatograph with ZB-5 ms capillary column (5% phenyl arylene 95% dimethylpolysiloxane) coupled at 70 eV (40-500 Da) electronic impact detector HP 5MS with a transfer temperature of 280°C. The chromatographic conditions were injection of 0.3 μL of ethyl acetate; helium carrier gas (99.99%); injector temperature: 280°C; split mode (1:10); then an initial temperature of 40°C and a final temperature of 300°C; initial time of 5 min and final time of 7.58min. The results obtained for C. zeylanicum leaves are shown in Table 1.
A total of 15 compounds were identified and their main constituents such as cinnamic aldehyde (46.30%), α-copaene (16.35%) and trans-β-caryophyllene (8.26%) were identified and quantified. Various researchers have identified and quantified different chemical compounds of C. zeylanicum essential oil. A study identified nine Essential Oils -Oils of Nature compounds [34] with (E)-cinnamaldehyde as its major component [35,36]. The essential oils of this plant generally have cinnamaldehyde [37], which corroborates the results obtained in this study. We already presented similar results to chemical composition of C. zeylanicum essential oil [38].
The chemical profile obtained for the aerial parts such as R. officinalis and O. vulgare is shown in Table 2. Its total composition presented 17 components with the major constituents being camphor (37.00%), 1,8-cineol (11.32%) and α-terpineol (7.12%). In O. vulgare essential oil, 20 compounds were found, represented by the main constituents cis-ρ-menth-2-en-1-ol (33.88%), linalyl acetate (13.90%) and p-cymene (8.29%). Probst also identified camphor as the major component of R. officinalis essential oil [39], being possible to observe similarity with the essential oil composition of this study, while other study obtained 1,8-cineol in a higher percentage, but camphor was present in the second place among the major components [40].
With respect to O. vulgare essential oil, while there is a description of similar chemical composition to our study [38,41], another study found three different chemotypes in 25 samples of this essential oil: linalool/linalyl acetate chemotypes with predominant linalyl acetate; the second major chemotypes rich in carvacrol and c-terpinene; and a third rich chemotype in thymol [42].
In the essential oil of Z. officinale, 18 components were identified, constituting α-zingiberene (27.14%), geranial (14.06%) and nerolidol (13.51%) in greater percentage. Diemer studing essential oil of Z. officinale quantified the chemical profile in 12 constituents and concluded that α-zingiberene was the predominant The chemical composition obtained from C. latifolia leaves is presented in Table 4. The essential oil obtained presented from 17 components with the main constituents being limonene dioxide (25.92%), cis-carveol (11.59%) and ρ-cymene (10.86%). Similarly, it was found in the researches carried out in C. latifolia tanaka, identifying 17 compounds and limonene as the major compound with 46.3% [48] and 58.43% [49].

Antimicrobial activity
Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 12600) and Pseudomonas aeruginosa (ATCC 27853) strains were cultured in brain heart infusion  The inoculum (100 μL) of each bacterium was seeded in Mueller-Hinton agar, with filter paper impregnated with 50 μL of essential oil placed on the surface. The plates were incubated at 35°C and after 24 h, the inhibition halo was measured with a millimeter ruler [51]. The minimum inhibitory concentration (MIC) was determined using the broth dilution methodology [52] and performed in triplicates with the same bacterium used in solid media diffusion techniques. Initially, an aliquot of the essential oil prepared in DMSO was transferred to a test tube containing BHI broth. Serial dilutions were then performed resulting in concentrations of 5-2000 μg/mL. Microbial suspensions containing 1.5 × 10 8 CFU/mL of the bacteria were added at each concentration and incubated at 35°C for 24 h. Tubes without bacteria were reserved for control of broth sterility and bacterial growth. After the incubation period, the minimum essential oil inhibitory concentration was defined as the lowest concentration which visibly inhibited bacterial growth observed by the absence of visible turbidity. To confirm growth inhibition, the BHI broth was subjected to the inoculum microbial seeding test on the surface of the plate-count agar.
The disc diffusion method evaluated the antibacterial activity of C. zeylanicum, O. vulgare, Z. officinale, R. officinalis, C. longa and C. latifolia essential oils to form inhibition halos against the growth of Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria strains. The diameters of the inhibition halos developed by the essential oils are shown in Table 5. The halos ranged from 7.67 to 15.33 mm. The largest inhibition halo against Gram-negative E. coli bacteria was quantified at 21 mm by C. latifolia essential oil. The best bactericidal activity for S. aureus was quantified at 15.66 mm by C. longa, while the Gram-positive P. aeruginosa was strongly inhibited by C. longa essential oil of, quantifying a halo of 12 mm.
The minimum inhibitory concentration (MIC) in μg mL −1 , the lowest visible concentration that prevents visible microbial growth in the culture medium by the action of the natural product, is being reported in Table 5.
The bactericidal activity of C. zeylanicum essential oil was demonstrated by larger halos against the Gram-positive bacteria. Similar results were found in C. zeylanicum essential oil [34,38,53] and the authors reported cinnamaldehyde as responsible for the antimicrobial action. However, lower results were also described when evaluating the antimicrobial activity of this oil against Salmonella typhimurium and E. coli, being its better inhibition against S. aureus [54].
The MIC's of C. zeylanicum essential oil were quantified using S. aureus strain and obtained a similar inhibitory concentration to that in this research, however E. coli and Salmonella typhi concentrations were far superior to that described in our study [55]. In another research conducted by Trajano et al.
[4], concentrations are relatively lower than those observed in this study.
To the antimicrobial potential of R. officinalis essential oil, Cordeiro [56] also obtained inhibition halos for S. aureus, as well as Ribeiro [57] for E. coli, both using this same essential oil as antimicrobial. The antimicrobial activity of this oil for the strains tested in broth dilution showed MIC's similar to the experiment performed by Silva et al. [58]. However, values for such concentrations have also been reported in smaller units by Thanh et al. [59].
On the other hand, O. vulgare essential oil has demonstrated efficiencies for both Gram-positive and Gram-negative strains which can be observed by Stefanakis et al. [60] and Sankar et al. [61] who reported antimicrobial activity of this essential oil similar to this research against the same bacteria tested.
Soković et al. [62] obtained MIC's for O. vulgare essential oil smaller than those described in the results of this study for S. aureus, E. coli, Salmonella enteritidis Essential Oils -Oils of Nature and Salmonella typhimurium. Sarikurkcu et al. [38,63], also performed an assay to determine the MIC against S. aureus and E. coli, obtaining results similar to those observed.
For C. longa essential oil, the largest halos were quantified for Gram-positive bacteria, similar to results found by Gupta et al. [64], Teles et al. [38] and Mishra et al. [65] who reported the formation of halos against the same bacteria in this study submitted to antimicrobial activity assays. Singh et al. [66] also observed satisfactory MIC values for the control of the microorganisms tested in this study.
In relation to the bactericidal effect of the Z. officinale essential oil, the values obtained in our disc diffusion test are superior to those obtained in the study by Singh et al. [67], where the authors did not obtain inhibition halos for E. coli and S. aureus, and the same were reported by Grégio et al. [68]. However, MIC values were similar to those found by Sasidharan and Menon [69].

Antioxidant activity
The antioxidant activity by the ABTS method [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] was adapted according to the methodology suggested by [70]. The ABTS˙+ radical was prepared by the reaction of 5.0 mL of a 3840 μg mL −1 of ABTS solution with 88 μL of the 37,840 μg mL −1 potassium persulfate solution. The mixture was left in the dark for 16 h. After formation of the radical, the mixture was diluted in ethanol (approximately 1:30 v/v) and absorbance was obtained at 734 nm. From the extracts and essential oils concentrations (5-150 μg mL −1 ), the reaction mixture was prepared with the ABTS radical cation. In a dark environment, a 30 μL aliquot of each extract and essential oil concentration was transferred into 23 test tubes containing 3.0 mL of the ABTS radical cation and homogenized on a tube shaker. After 6 min, absorbance of the reaction mixture was obtained in a spectrophotometer at 734 nm. The analyzes were performed in triplicate and the capture of the free radical was expressed as percent inhibition (% I) of the ABTS radical cation.  The ABTS method allowed the calculation of the 50% effective concentration of the essential oils, which express the minimum concentration required to reduce the initial concentration of ABTS by 50%, and these are expressed in Table 6. The lowest concentration and consequently the best antioxidant activity was observed to oregano, with an EC 50 quantified in 14 μg mL −1 and consequently also the highest percentage of ABTS inhibition.
The antioxidant effect of C. zeylanicum essential oil differs from that observed by [71] using the ABTS technique, where these authors verified a lower EC 50 . This difference is explained by the authors due to variation in the chemical composition of the essential oils studied, which depends on factors such as the geographic location and the time of collection of the plant.
The result for R. officinalis essential oil exhibited by [57] shows a fairly high effective concentration comparing that obtained in this article. Also [72], when evaluating the antioxidant activity of this essential oil from five different crop fields, found a lower EC 50 than this study. According to [73], the main responsible for the free radical stabilization capacity of this species is 1,8-cineol. In this way, it is possible to relate the lower antioxidant potential of the rosemary essential oil obtained in this research with the low content of 1,8-cineol.
On the other hand [72], while evaluating the antioxidant capacity of rosemary essential oil using DPPH, it had a relatively higher concentration than this study Salmonella thyphimurium. Also highlighting the difference of methods used, since its concentration, it presented higher concentrations at levels of approximately 700 more units.
Regarding the antioxidant potential of oregano essential oil, the author [74] obtained a higher value of efficient concentration than presented in our study, which highlights the data obtained satisfactory in this research. However [75], while still evaluating the antioxidant activity of O. vulgare essential oil, using the ABTS radical discoloration technique, a lower EC 50 is observed than that observed in this study. According to these authors, the antioxidant potential of this oil is related to the presence of phenolic compounds, but it can also be attributed to a possible synergy between the various constituents.
When checking the antioxidant activity of C. longa [76], it is found that concentrations are much higher than those quantified for the essential oil using the DPPH assay, whereas, when using the ABTS assay, we exposed satisfactorily lower concentrations.
When studying the anti-inflammatory and anti-inflammatory activity of ginger essential oil [77], a much higher EC 50 value is obtained which was presented in this study. These results are lower than that obtained in this study, and the authors attribute to this fact that the low concentration of phenolic compounds is mainly responsible for the antioxidant activity. A relatively lower EC 50 value was found for Z. officinale essential oil by the β-carotene/linoleic acid system [78]. The author attributes the antioxidant activity of the oil to the geranium and neral aldehydes, which were found in their essential oil at concentrations much higher than in this work.

Conclusions
These studies have shown that the essential oils of C. zeylanicum (cinnamon), O. vulgare (oregano), Z. officinale (ginger), R. officinalis (rosemary), C. longa L. (saffron) and C. latifolia (Tahiti lemon), in the chemical composition, presented a mixture of mono-and sesquiterpenes, with the major constituents being cinnamic aldehyde (46.30%), cis-p-menth-2-en-1-ol (33.8%), α-zingiberene (27.14%), camphor (37%), turmerone (55.43%) and limonene dioxide (25.92%), respectively. Results of disc diffusion showed the essential oil of C. longa as the oil with the highest bactericidal action independent of the bacteria strain; however, the lowest bactericidal concentration observed was the lowest concentration of C. zeylanicum essential oil (83.33 μg mL -1 ) in S. aureus strains. The antioxidant activity of O. vulgare presented the percentage of inhibition of the 90.74% radical by 50 μg mL −1 having the EC 50 of 14 μg mL −1 . These results indicate that bioactive molecules present in the essential oils of the species tested presented antimicrobial activity and antioxidant action. These characteristics contribute to the control of microorganisms and help increase the shelf life of foods.