The antimicrobial activity of plant essential oils is applied by mankind for various purposes since ancient times. They are used in food preparation, forming the sensory profile, and increasing the shelf life of the resulting foods. They facilitate the absorption of proteins, fats, and carbohydrates. The antimicrobial and antioxidant properties of plants and the aromatic products derived from them are due to the different chemical substances in their composition – essential oils and glyceride oils, alkaloids, flavonoids, tannins, glycosides, and other compounds , . Citrus aurantium L (Rutaceae), commonly known as sour orange, bitter, bigarade, or Seville orange, is generally consumed as marmalade in Mediterranean countries and is used as a flavoring agent . In Haiti, it was found to be used as a medicine to treat colds, fevers, hepatic disorders, gall bladder problems, rheumatism, epilepsy, emotional shock, bruising internally and externally, skin blemishes and digestive problems .
Recently, consumers’ health concerns in relation to food ingredients have led to an increase in the request of foods processed without the addition of synthetic chemical preservatives. The use of essential oils may provide a “natural” alternative to the chemical preservation of foods. Novel or emerging nonthermal food processing technologies including natural antimicrobials are gaining increasing importance , .
It was reported that volatile compounds from Citrus zest exhibit antifungal activity correlated to its monoterpene and sesquiterpene content. Anxiolytic and sedative effects were also reported for both Citrus aurantium L extract and essential oil. Moreover, flavonoids from citrus were reported as effective cytostatic anticancer agents , . The purpose of the present study was to determine the chemical composition, the antioxidant, and the antimicrobial activity of Citrus aurantium L zest essential oil against some pathogens.
2 Materials and methods
2.1 Essential oil extraction and chemical substances
Essential oil from Citrus aurantium L zest was used for the conduction of the experiments. The essential oil content was determined by steam distillation of the British Pharmacopoeia . Steam distillation, the method used for essential oil extraction, takes advantage of the volatility of a compound to evaporate when heated with steam and the hydrophobicity of the compound to separate into an oil phase during condensation.
Fresh zest of Citrus aurantium L was used for the extraction of essential oil. The zest of Citrus aurantium L was threshed into small pieces and was thoroughly washed with distilled water at least two times. The excess water was drained out and the zest was dried for 2 days at 25 °C. The extraction was carried out during 4 h from the first drop of distillate until the amount of essential oils stabilized. The oil was collected in a glass tube and was kept at 4 °C till further use.
Synthetic monoterpene – limonene (≈99%) was purchased from Sigma, Munich, Germany.
2.2 Gas chromatography/mass spectrometry (GC/MS) analysis
The composition of the oil was determined by GC-MS .
The GC-MS analyses were performed on a 7890A gas chromatograph (Agilent Technologies) coupled to a 5975C quadrupole mass spectrometer (Agilent Technologies) (Agilent, Santa Clara, CA, USA). The analytes were separated on a HP-5MS capillary column (30 mm×0.25 mm with a phase thickness of 0.25 μm). The split/splitless injector temperature was set at 250 °C and the temperature program was 60 °C for 3 min, 6 °C min−1 ramp rate to 250 °C and held constant for 3 min. The carrier gas was helium (99.999%) at a 1 mL min−1 flow rate. In the solid phase microextraction analysis, splitless injection (3 min) was used at 250 °C. The mass spectrometer was operated in the electron-impact mode (EI) at 70 eV.
The identified components were arranged according to the retention time and their quantity is given in percentages.
The obtained mass spectra were analyzed using 2.64 Automated Mass Spectral Deconvolution and Identification System (AMDIS) (National Institute of Standardization and Technology, NIST, Gaithersburg, MD, USA). The separated polar and nonpolar compounds were identified by comparison of their GC-MS spectra and Kovach retention index (RI) with referent compounds in NIST 08 database (NIST Mass Spectral Database, PC-Version 5.0, 2008). The RIs of compounds were recorded with standard n-hydrocarbon calibration mixture (C10–C40, Fluka) using the 2.64 AMDIS software.
2.3 DPPH radical scavenging assay
The ability of Citrus aurantium L zest essential oil to scavenge free radicals was assayed with the use of a synthetic free radical scavenger compound 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma-Aldrich, St. Louis, MO, USA) according to the method employed as shown in Ref. . Briefly, essentials oils were serially diluted [0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL (w/v)] in methanol. A solution of DPPH [0.004% (w/v)] was prepared in the same solvent. Then 300 μL of each dilution were mixed with 2700 μL of DPPH solution. The reaction was performed at 37 °C in darkness and the absorption at 517 nm was recorded after exactly 15 min against methanol. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard. Each test was performed in triplicate.
The antioxidant activity was calculated as follows:
AA: antioxidant activity, Abs: absorbance.
2.4 Determination of the antimicrobial activity against pathogenic microorganisms
Staphylococcus aureus NBIMCC 3703, Salmonella sp. (clinical isolate), Pseudomonas aeruginosa NBIMCC 1390, Bacillus subtilis NBIMCC 1208, and Escherichia coli NBIMCC 3702. All strains are deposited in the culture collection of the Food Research and Development Institute, Plovdiv, Bulgaria.
Preparation of the suspensions of the test pathogenic microorganisms:
The test pathogenic microorganisms were cultured on Luria Bertani Medium with glucose (LBG) agar medium (LB Broth, Miller-Novagen, Merck, Germany) at 37±11 °C for 24–48 h. Using sterile loop biomass of the developed test pathogenic microorganisms was suspended in sterile saline solution in order to obtain suspensions of the test pathogenic microorganisms.
The antimicrobial activity was studied by the disc-diffusion method:
Agar disc-diffusion testing which was developed in 1940  is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing. Nowadays, many accepted and approved standards are published by the Clinical and Laboratory Standards Institute for bacteria and yeasts testing. In this well-known procedure, agar plates are inoculated with a standardized inoculum of the test microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test compound at a desired concentration, are placed on the agar surface. The Petri dishes are incubated under suitable conditions. Generally, antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism and then the diameters of inhibition growth zones are measured.
Sterile melted LBG agar medium (LB Broth, Miller-Novagen, Merck, Germany) was poured in Petri dishes and after the hardening of the agar, the dishes were spread plated with suspensions of the test pathogenic microorganisms. Decimal dilutions of the essential oil in saline solution containing 1% (v/v) Tween 80 were prepared. The experiments were conducted with dilutions 1×, 10×, and 100× in order to determine the minimum inhibitory concentration (MIC). The used paper discs were 6 mm in diameter. Six μL of the corresponding dilution were pipetted on the corresponding paper discs. Paper discs soaked in distilled water were used as blanks. The results were recorded as diameters of the clear zones around the paper discs, in millimeters, after 24–48 h of incubation of the Petri dishes at an optimal temperature for the growth of the corresponding test-microorganism −37 °C . The MIC was defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth .
The experiments were performed in quadruplicate. The mean values and the standard deviations were calculated using MS Office Excel 2010. The MICs, in ppm, were calculated based on the obtained results.
3 Results and discussion
3.1 Chemical composition
Essential oils are complex mixtures of volatile compounds derived from a large number of plants. Their content in plants is a very small part (less than 5% of the dry matter content of the plant) and consists primarily of hydrocarbonterpenes . Citrus oils are characterized by complex mixtures containing mostly terpenes as well as oxygen-containing compounds. For most citrus fruits, oils consist almost exclusively in the form of monoterpenes, sesquiterpenes, and other aliphatic hydrocarbons. Limonene is a clear, colorless liquid hydrocarbon classified as a cyclic monoterpene, and was the major component in oil of citrus fruit peels and it is used as a functional index of ripeness in GC-MS analyses.
In previous reports , , limonene was observed as the main constituent (77.49%) in the zest essential oil of sweet orange, followed by myrcene (6.27%), α-farnesene (3.64%), and γ-terpinene (3.34%). The main compounds in zest essential oil from Uganda and Rwanda were limonene (87.9 and 92.5%), myrcene (2.4 and 2.0%), α-pinene (0.5 and 2.4%), and linalool (1.2 and 0.9%) . In every oil, limonene, α-pinene, sabinene, and α-terpinene were the major compounds . The chemical composition of the essential oil from Citrus aurantium L zest is presented in Table 1. Forty eight compounds, 3 of them being over 1% and the other 45 >1% were identified in the Citrus aurantium L zest essential oil. The monoterpene hydrocarbons prevailed. The monoterpene hydrocarbons were represented mainly by limonene (85.22%), α-pinene (1.29%), and β-myrcene (4.30%) (Table 1). These results were similar, with some numerical differences, to the works of others authors, limonene was the major compound with a percentage of 96% , , . These variations probably occur due to factors related to the oil extraction method, genetic characteristics of the species, and environmental conditions in which they were grown.
3.2 Total antioxidant capacity based on the DPPH radical-scavenging assay
The free radical-scavenging activity of fruit, vegetable, and medicinal plant extracts and essential oils were extensively studied. Essential oils rich in monoterpenes are recognized as food preservatives and monoterpenic essential oils are natural antioxidants that are active against certain cancer , , , . Indeed, a number of monoterpenes have antitumoral activity that can prevent the formation or progress of cancer and cause tumor regression. Limonene and perillyl alcohol have a well-established protective activity against many types of cancer .
The antioxidant properties of citrus essential oils were described by several authors. Choi et al. investigated the radical scavenging activities of 34 kinds of citrus essential oils by DPPH assay, four essential oils from Citrus aurantium L zest showed scavenging effects in the range from 17.7% to 34.1% . These results are different from us, in our study. While in our study, the essential oil from Citrus aurantium L showed the highest antioxidant activities valued 88.1% in DPPH assay. These differences would be explained by the different chemical compositions of the same citrus species in different regions.
Figure 1 shows the free radical-scavenging potential of different concentrations of Citrus aurantium L zest essential oil and limonene – synthetic monoterpene, as determined by the DPPH assay. As shown in Figure 1, Citrus aurantium L zest essential oil had a good antioxidant activity in line with previously results obtained on Citrus spp. When the reaction was stable, even lower concentrations of the essential oil scavenged the radicals. Taking into account that limonene is the major compound of essential oil our results confirm the study of other authors , who described the antioxidant activity of this compound. The essential oil showed considerable free radical-scavenging ability, whereas limonene was less active. The greater free radical-scavenging ability of the Citrus aurantium L zest essential oil compared to the limonene may have resulted either from the synergistic action of a mixture of terpenes, or from the action of nonvolatile compounds, other than monoterpenes, present in the essential oil.
The biological function of the chemical components of the Citrus aurantium L zest essential oil is not limited to their antioxidant and antimicrobial activity. Some of them also have antitumor (linalool, borneol), anti-inflammatory (sabinene, pinene), and analgesic function (citral) , , , , , .
3.3 Antimicrobial activity
The antimicrobial activity of essential oils is strictly connected to their chemical composition. At present, the mechanism of action of terpenes is not fully understood but it is speculated to involve a damage of the plasma membrane stability with subsequent membrane disruption by the lipophilic compounds. Limonene and α-pinene, which were found to be abundant in the present study, were reported to possess antimicrobial activity . α-Pinene (monoterpene hydrocarbon) had slight activity against pathogenic microorganisms . It was concluded that limonene had an antibacterial effect weaker than the antifungal activity. The antimicrobial activity of a citrus essential oil is enhanced by the presence of bioactive alcohols, such as carveol and linalool, a monoterpene alcohol, known as a potent antimicrobial and antifungal compound. However, for a deeper comprehension of this subject, the reader is referred to other authors . The results from the determination of the antimicrobial effect of the Citrus aurantium L zest essential oil are presented in Table 2. The essential oil exhibited antimicrobial activity against the investigated test microorganisms. Gram-positive bacteria were more sensitive to the activity of the essential oil, the measured zones of inhibition were 12.5 mm, and the minimum inhibitory concentration was 60 ppm. The tested Gram-negative bacteria showed zones of inhibition between 9 and 10 mm, with a minimum inhibitory concentration of more than 600 ppm. The observed difference in the sensitivity of the different test-microorganisms to the examined essential oil was due to the difference in the cell wall structure and composition of the two groups of bacteria. The presence of an outer membrane in Gram-negative bacteria hinders the diffusion of the essential oil through the membrane to the cytoplasm of the cell, making them more resistant to the action of the oil. S. aureus NBIMCC 3703 and B. subtilis NBIMCC 1208 exhibited a high sensitivity at low essential oil concentration; these findings are in agreement with other authors , , , who found that citrus essential oils accounted for a potent antimicrobial activity on these species, although the antimicrobial activity was strain-dependent. The results obtained for the different resistance of Gram-positive and Gram-negative bacteria to inhibitors of microbial growth were consistent with literature data , . The greater antimicrobial activity of the essential oil compared to the limonene – synthetic monoterpene may have resulted either from the synergistic action of the mixture of terpenes, or from the action of nonvolatile compounds, other than monoterpenes, present in the Citrus aurantium L zest essential oil.
The Citrus aurantium L zest essential oil can be used for food flavoring in the production of sausages, vegetable, meat, and fish canned food, chutneys, mayonnaise, ketchup, salad dressings, processed cheese, beverages, candies, and more. It also has potential application in perfumery and cosmetics, in the manufacture of perfumes, cosmetics, soaps, and shampoos. The essential oil is also widely used in medicine because of multilateral physiological effect.
The chemical composition of the essential oil from Citrus aurantium L zest was determined and its antimicrobial and antioxidant activities were examined. The essential oil exhibited pronounced antimicrobial activity against Gram-positive bacteria as compared to Gram-negative bacteria. This was due to the difference in the structure and composition of the cell wall of bacteria belonging to both groups and the essential oil chemical composition. The demonstrated antioxidant and antimicrobial activities of the Citrus aurantium L zest essential oil, allow its application as natural preservative, helping increase food safety.
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Published Online: 2019-01-26