2.1. Materials

Fruits of Citrus of four species: bitter orange (Citrus aurantium) cultivar Larange, lemon (Citrus limon) cultivar Beldi, orange maltaise (Citrus sinensis) cultivar Lsen asfour, mandarin (Citrus reticulate)cultivar Elarbi, were evaluated in this study. Samples were collected at three harvesting periods: stage 1 (green colour, immature), stage 2 (yellow colour; semi-mature), and stage 3 (orange colour: mature), in 2009, from Menzel Bouzelfa in the North East of Tunisia (latitude 36°42′13′.17′′; longitude 10°29′46.93′′). The fruits peels including flavedo (epicarp) and albedo (mesocarp) layers were peeled off carefully and discarded.

2.2. GC and GC-MS Analysis

Essential oils were analyzed by gas chromatography (GC) using a Hewlett-Packard 6890 apparatus (Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A HP-Innowax capillary column (polyethylene glycol: 30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent Technologies, Hewlett-Packard, CA, USA) was used; the flow of the carrier gas (N₂) was 1.6 mL/min and the split ratio 60 : 1. Analyses were performed using the following temperature program: oven temps isotherm at 35°C for 10 min, from 35 to 205°C at the rate of 3°C/min, and isotherm at 205°C over 10 min. Injector and detector temperature were held, respectively, at 250 and 300°C.

2.3. Gas Chromatography-Mass Spectrometry

GC-MS analyses of essential oil volatile components were carried out on a gas chromatograph HP 5890 (II) coupled to a HP 5972 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with electron impact ionization (70 eV). A HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies, Hewlett-Packard, CA, USA) was used. The column temperature was programmed to rise from 50°C to 240°C at a rate of 5°C/min. The carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was 60 : 1. Scan time and mass range were 1 s and 40–300 m/z, respectively.

2.4. Compounds Identification

Identification of essential oil volatile compounds was based on the calculation of their retention indices (RI) relative to (C₈–C₂₂)n-alkanes with those of authentic compounds available in our laboratory. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC-MS data systems and other published mass spectra [16].

2.5. Screening of Antibacterial Activity

Antibacterial activity was analyzed by the disc diffusion method [17] against three human pathogenic bacteria including Gram-positiveStaphylococcus aureus (ATCC 25923), and Gram-negative bacteriaEscherichia coli(ATCC 35218) andPseudomonas aeruginosa (ATCC 27853). All bacteria were grown on Mueller Hinton plate at 30°C for 18–24 h previous inoculation onto the nutrient agar. A loop of bacteria from the agar slant stock was cultivated in nutrient broth overnight and spread with a sterile cotton swap onto Petri dishes containing 10 mL of API suspension medium and adjusted to the 0.5 McFarland turbidity standards with a Densimat (BioMerieux). Sterile filter paper disks (6 mm in diameter) impregnated with 10 μL of essential oil were placed on the cultured plates. After 1-2 h at 4°C, the treated Petri dishes were incubated at 25 or 37°C for 18–24 h. The antimicrobial activity was evaluated by measuring the diameter of the growth inhibition zone around the discs. Each experiment was carried out in triplicate, and the mean diameter of the inhibition zone was recorded.

2.6. Statistical Analyses

All data are reported as means ± standard deviation of three samples. Statistical analysis was performed with STATISTICA. Differences were tested for significance by using the ANOVA procedure, using a significance level ofP < 0.05.

3.1. Yields of Essential Oils

The essential oils yields of four citrus peels during fruit maturation are shown inTable 1. Species and harvest time had significant effect on essential oil yield. Independently to ripening stage, mandarin exhibited the highest yield (2.70%) followed by lemon (1.30%) and orange (0.74%) while bitter orange showed the smallest value of 0.46%.

On the other hand, essential oil yields varied during ripening to reach maximum values during the middle stage of maturity (stage 2) for mandarin and orange while the highest lemon yield was found at the beginning of fruit maturation and decreased after that. Bitter orange showed different pattern behaviour evolution from other species since the yield doubled during ripening from 0.23 at stage 1 to 0.46% at stage 3. Vekiari et al. [14] reported a seasonal variation of the yield of lemon peel essential oil extracted from Zambetakis variety cultivated in the island of Crete with the highest value reached at the middle of the season.

Our results concerning the mature stage are in accordance with those of Hosni et al. [15] who demonstrated that Tunisian mandarin peel was the richest on essential oil compared to orange and bitter orange. However, these authors reported higher values (varying from 1.24 to 4.62%). Such differences could be due to the effect of extraction procedure and environmental conditions. In fact, these authors used dried and ground material from citrus cultivated in Mograne region which is known to belong to the semiarid region, while in our experiment we used a fresh material collected from Menzel Bouzelfa which belongs to the humid region. Extractions parameters are known to greatly influence the essential oil yield [18,19]; moreover, water supply during ripening was reported to influence considerably the essential oil content with an enhancement of the yield under moderate water shortage conditions [20,21].

On the other hand, the yields obtained in our study were higher than those reported in the literature; Ahmad et al. [22] reported yields varying from 0.30 to 1.21% for the four citrus varieties from Pakistan. Moreover, lower yields were reported for the mandarins from France (yield ranging from 0.05% to 0.60%) by Lota et al. [8] and the mandarin from Colombia (yield of 0.79%) by Blanco Tirado et al. [23].

3.2. Essential Oil Composition

Analysis of citrus peel essential oils composition showed 39 identified compounds presenting fluctuations during ripening (Table 2).

3.3. Antibacterial Activity

The antibacterial activity of citrus essential oils was tested against human pathogenic bacteria. The results presented inTable 4 showed great differences in the activity between citrus species and during ripening stages. The oils were effective against Gram (+) and Gram (−) bacteria, with a major activity againstS. aureus andE. coli. Bitter orange, lemon, and orange were effective againstP. aeruginosa only at maturity while mandarin oil remained inactive against this strain. In accordance with our finding, Espina et al. [12] reported no activity of the peel oil extracted from mature mandarin fruit againstP. aeruginosa. On the other hand, lemon and mandarin essential oils extracted from immature fruit exhibited the highest antibacterial activity againstE. coliwhich was comparable of positive control activity. In the case ofS. aureus, the oils were mostly active at mature stage with mandarin oil showing the highest antibacterial activity.

The antimicrobial activity of essential oils is believed to be associated with phytochemical components especially monoterpenes [35] which diffuse into and damage cell membrane structures. However, the differences on the oils activity found in our experiment between ripening stages may be related to the modifications of the oils composition during fruit maturation. For the four citrus species, the variations of the activity were not correlated to that of the major compound level: limonene. This is in agreement with literature data where limonene is a weak antibacterial compound [36]. Moreover, the inhibitory activity of an essential oil is known to result from a complex interaction between its different constituents, which may produce additive, synergistic, or antagonistic effects, even for those present at low concentrations. Thus, at immature stage, antibacterial compounds including camphor [37] andα-thujone [38] in bitter orange,α-terpineol [39] and nerol in lemon [40], and borneol [37] and caryophyllene oxide [41] in mandarin may be involved in the found activities of the corresponding oils.

At semimature stage, the higher antibacterial capacity of orange oil compared to the citrus oils could be linked to the presence of camphor at appreciable level (4.81%). Moreover, borneol could be involved in the found activity since this monoterpene alcohol was reported to exhibit moderate antibacterial activity againstS. aureus andE. colibut to remain inactive againstP. aeruginosa[42]. Our results demonstrated also an important increase of 1,8 cineole in bitter orange and mandarin oils. Literature data dealing with the activity of 1,8 cineole are contrasting; Randrianarivelo et al. [43] reported that this ether is effective against Gram(+) and Gram(−) bacteria includingE. coli andS. Aureus; however, Hussain et al. [44] found that 1,8 cineole remained inactive againstP. aeruginosa andE. coli. The low activity of the bitter orange and mandarin oils at the semimature stage in spite of a high level of 1,8 cineole suggests that this compound is inactive.

The activity of the four oils extracted from mature fruits could mainly be due to the presence of the phenolic compound carvacrol. This later is a well-known antibacterial compound acting at low concentration [38]. Moreover, mandarin was distinguished from other citrus species by the presence of farnesol which was reported to be highly effective againstS. aureus [45] while the activity of orange oil could be ascribed to the enhancement of humulene percentage, which was reported to be moderately active [46].

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