Compounds listed in order of elution from a non-polar column. **§** RI (retention index) calculated relative to C6–C17 n-alkanes.

#### *3.2. Antioxidant E*ff*ect of the EGEO*

The antioxidant effect was determined by DPPH radical scavenging and metal ion chelating activity (Table 2). The capacity of the EGEO to scavenge DPPH radical and its reducing ability was assessed on the basis of its dose giving 50% inhibition (IC50). In addition, its ability to chelate Fe2<sup>+</sup> metal ions was evaluated and calculated using different doses. The radical scavenging activities of positive controls (BHA and ascorbic acid) were more active than those obtained from EGEO. However, EGEO had a superior metal ion chelating activity with an IC50 value of 8.43 ± 0.03 mg/mL, followed by BHA (104.73 ± 7.30 mg/mL) and gallic acid (136.97 ± 9.09 mg/mL). Current results show that EGEO has the best chelating activity.

**Table 2.** Antioxidant property of *Eucalyptus globulus* volatile oil *in vitro*.


IC50 = medium inhibitory concentration; values are given as mean ± SD (n = 3). Means within the same column followed by the same capital letters are significantly not different (*p* > 0.05) from one another according to the ANOVA test followed by Tukey's comparison tests.

#### *3.3. In vitro Antibacterial and Antifungal E*ff*ects*

#### 3.3.1. Disc Diffusion Assay

The microbial inhibitory effect of EGEO was assessed against different food spoilage microorganisms: six yeast, six bacteria and five fungal strains. The results of the antibacterial tests of EGEO are presented in Table 3. The EGEO displayed a dose-dependent inhibition effect. EGEO was found to be active against all the Gram-positive bacteria species. The EGEO inhibited the growth of *Staphylococcus aureus*, *Bacillus cereus* and *Escherichia coli* with DIZ ranging from 11 to 18 mm at the lower volume of EGEO (20 μL/mL) and from 19 to 85 mm at the higher amount (60 μL/mL). The DIZ increased with the increasing quantity (20, 40 and 60 μL) of EGEO in each paper disc. However, no inhibitory action was found in the case of *Pseudomonas aeruginosa*. For the fungal strains, the DIZ varied from 10 to 49 mm (Table 4). The highest DIZs were shown by *Candida albicans* ATCC (40 mm) and *Saccharomyces cerevisiae* (49 mm) at a greater volume of EGEO (60 μL). The sensitivity of these bacteria to chloramphenicol and EGEO can be explicated by the fact that they have a similar mechanism of action on Gram-negative strains.

The highest susceptible yeast was *Saccharomyces cerevisiae* (49 mm), followed by *Candida albicans* ATCC (40 mm), *Trichosporon* sp. (39 mm) and *Candida parapsilosis* (20 mm) (Table 4). Further, the DIZ due to the *Eucalyptus* volatile oil was bigger for yeast and Gram-positive strains than for mycelial species and Gram-negative bacteria.

#### 3.3.2. Disc Volatilization Method

The *in vitro* antifungal and antibacterial effects of EGEO were assessed according to the absence or the presence of inhibition zones. The DIZ resulting from the exposure to EGEO vapors is shown in Tables 3 and 4. The DIZ due to the same quantity of EGEO was larger for yeast than for mycelial species (Figure 1A,B).


**Table 3.** Susceptibility of bacterial strains to *Eucalyptus globulus* essential oil and to classic antibiotics (positive controls).

<sup>a</sup> Diameter of inhibition zone (mm) comprising a disc diameter of 9 mm; <sup>b</sup> Amoxicillin-clavulanic acid (AMC, 20/10 μg), erythromycin (E, 15 μg) and chloramphenicol (C, 30 μg) were tested as a positive control for microbial species. EGEO: *Eucalyptus globulus* essential oil. (-) No activity.


**Table 4.** Susceptibility of fungal strains to *Eucalyptus globulus* essential oil and antiseptic solution.

<sup>a</sup> Diameter of inhibition zone (mm) including a disc diameter of 9 mm; <sup>b</sup> Antiseptic solution (Hoxamidine 0.1%) used as a positive control for fungal strains. EGEO: *Eucalyptus globulus* essential oil; (-) No inhibitory effect; ATCC: American Type Culture Collection.

**Figure 1.** *In vitro* inhibitory activity of *Eucalyptus globulus* essential oil against bacterial and fungal species: Disc diffusion (**A**) versus vapor diffusion (**B**) methods using three different quantities of EO (20, 40 and 60 μL/disc). Red arrows are the diameter of the inhibitory zone (DIZ).

As detected in the previous analyses using EGEO in the liquid phase, the DIZ due to EGEO vapors augmented with an increasing amount of the EGEO and followed a similar tendency with respect to the different microbial species. Nevertheless, in comparison with the liquid phase, the EGEO vapors resulted in considerably superior DIZ in all the yeast strains tested, except *Candida albicans* ATCC. In addition, a total inhibitory effect (85 mm) of EGEO vapors was found in the case of *Staphylococcus aureus* and *Enterobacter sakazakii* bacterial strains. However, no inhibitory action was found for *Aspergillus* species, *Pseudomonas aeruginosa* and *Klebsiella ornithinolytica*. *Candida albicans* (Ca2) and *Candida parapsilosis* were the most sensitive yeasts to EGEO vapors because total inhibition zones were generated using 20, 40 and 60 μL of EO per disc.

#### *3.4. Orangina Fruit Juice Preservation*

#### 3.4.1. Effect of Varying Dose of EGEO

As EGEO was active to inhibit different food-borne spoilage bacteria and fungi in *in vitro* methods, its effect in a fruit juice matrix (Orangina juice) was also studied (Figure 2). The reduction in viability of *Saccharomyces cerevisiae* due to EGEO use in a concentration-dependent way (0.8, 2 and 4 μL/mL) and a time-dependent manner (i.e., 0, 1, 2, and 6 days) was evaluated. Complete growth inhibition was observed in Orangina fruit juice only when a high concentration of EGEO (4 μL/mL) was used. However, the doses of 2 and 4 μL/mL did not show an important decrease in the final amount of yeasts (2.8 log CFU/mL and 2 log CFU/mL, respectively) in comparison to untreated Orangina juice (2.3 log CFU/mL).

**Figure 2.** Effect of different doses of EGEO (0.8, 2 and 4 μL/mL) on the viability of *Saccharomyces cerevisiae* cells in Orangina beverage during storage. The fungal growth was followed up to 6 days after the application. HT: Heat treatment at 70 ◦C for 2 min; PG: Positive control or juice with synthetic antimicrobial additives (sodium benzoate and potassium sorbate); EGEO: *Eucalyptus globulus* essential oil; CFU: Colony-forming unit. \* significant difference (*p* < 0.05) according to ANOVA one-way analysis followed by Tukey's *post hoc* multiple comparison tests.

3.4.2. Combined Effect of EGEO and Moderate Heat Processing

The log decrease in CFU count of the *Saccharomyces cerevisiae* due to the associated action of EGEO at different doses along with medium heat treatment at 70 ◦C for 2 min of Orangina juices was calculated for a specific time interval (0, 1, 2 and 6 days). In juices treated using an association of medium heat processing and all EGEO doses, total fungal inhibition of *Saccharomyces cerevisiae* was recorded on first sampling after 2 days (Figure 3). Therefore, the association of medium heat processing with EGEO reduced the EGEO concentration requirement considerably. Even in the Orangina juices treated with a lower quantity of EGEO, the association of medium heat processing at 70 ◦C for 2 min improved the log reduction by 3.5 log CFU/mL in comparison to EGEO-treated juices. Therefore, the application of moderate heat processing with EGEO can offer improved Orangina juice preservative.

**Figure 3.** Effect of EGEO at different doses (0.08, 0.2 and 0.4 μL/mL) in association with moderate heat processing (70 ◦C for 2 min) on the viability of *Saccharomyces cerevisiae* cells in Orangina beverage during storage. The fungal growth was followed up to 6 days after the heat processing. HT: Heat treatment at 70 ◦C for 2 min; PG: Positive control or juice with synthetic antimicrobial additives (sodium benzoate and potassium sorbate); EGEO: *Eucalyptus globulus* essential oil; CFU: Colony-forming unit. \* significant difference (*p* < 0.05) according to ANOVA one-way analysis followed by Tukey's post hoc multiple comparison tests.

#### **4. Discussion**

As published in previous reports, the EO of the *Eucalyptus* genus was described by a great quantity of 1,8-cineole (eucalyptol). Current data are in accordance with those published by Elaissi et al. [18] and Goldbeck et al. [26], who found that the principal chemical element of EGEO was eucalyptol. The chemical composition of a diversity of other *Eucalyptus* species have been reported [7,19,27] and are also in accordance with our data.

Different percentages of eucalyptol in *Eucalyptus globulus* leaf EO have been shown: 53.7% in Tunisia, 14.5% in Germany, 33.6% to 66.7% in India and 71% in Brazil. The majority of EO extracted from *Eucalyptus* trees contains at least 10% to as high as 97% eucalyptol (1,8-cineole). Ecological position (Table 5), climatic conditions and extraction methods have been invoked as reasons for the chemical composition disparities and variations [18,20].


**Table 5.** Comparison of the chemical composition of *Eucalyptus* essential oil from different countries.

A few investigations have been completed to study the antioxidant activity of different *Eucalyptus* EO such as *E. globulus* [28,30,34–37]. Our IC50 values are not in agreement with those published for the DPPH radical scavenging method [34]. The scavenging activity on the DPPH radical reported as IC50 value was 57 μg/mL for Tunisian EGEO, but this result is inferior as compared to the BHT value obtained with the same test (11.5 μg/mL) [34]. *E. tereticornis* EO presented powerful DPPH, OH and O2<sup>−</sup> radical scavenging activity [35]. In opposition, *E. camaldulensis* and *E. radiata* EOs have been reported to display a medium DPPH scavenging effect [38]. This variance in antioxidant power may be related to the variation in chemical composition, distillation methods and environmental aspects, age of the trees, storage conditions and geo-climatic situations [12].

In addition, Singh et al. [35] tested the *Eucalyptus* EO and its three main oxygenated terpenes (isopulegol, β-citronellol and citronellal) for antioxidant and scavenging effect. The authors reported that the EO extracted from *Eucalyptus* displayed medium to powerful antioxidant effect in terms of metal chelating (877.3% ± 9.27% inhibition), DPPH radical (IC50 = 425.4 ± 6.79 mg/mL) and lipid peroxidation inhibition. This research revealed that *Eucalyptus* EO leaves contain oxygenated monoterpenes rich EO presenting antioxidant action.

The chelating property on metal ions is one of the important mechanisms of antioxidant activity. *Eucalyptus* EO showed a better chelating effect (8.43 ± 0.03 mg/mL) on ferrous ions in comparison with standards (BHA = 104.73 ± 7.30 mg/mL and ascorbic acid = 140.99 ± 3.13 mg/mL). Analysis of metal ion chelating activities revealed that all EOs extracted from *Eucalyptus* species were capable of

chelating iron (II) in a dose-dependent way [9]. Another study reported that the antioxidant action of phytochemicals in lemon eucalyptus oil may be due to their redox activities of the phenolic compounds and oxygenated terpenes and that make them good reducing, scavenging and chelating EO [8]. One of the probable modes of action of the antioxidative effect is the chelation of transition metals.

There are very limited published studies about the antioxidant property of eucalyptol as the principal chemical compound of the EGEO [36]. Consequently, antioxidant property detected for the volatile oil could be linked to the remaining compounds [35]. El-Ghorab et al. [37] revealed a medium antioxidant property by preventing the oxidative alteration of linoleic acid (20%) for 12 days for the EO of *Eucalyptus camaldulensis*. Instead, EOs extracted from aerial parts of *Eucalyptus camaldulensis*, growing wild in different regions of Sardinia Island (Italy), have exhibited an *in vitro* antioxidant potential that varied between 0.5 and 5.8 mM [38]. The mode of action involved in the lipid peroxidation and inhibition of DPPH assay is not analogous, thus, the results of the current experiment are not similar to those formerly published by El-Ghorab et al. [37] and Barra et al. [38] because the positive controls and units used in both assays for the determination of the IC50 values are not identical.

Numerous published reports have assessed and confirmed the *in vitro* antibacterial, anti-yeast and antifungal potential of EGEO against a varied collection of pathogens [39,40]. For example, *Eucalyptus citriodora* EO has been demonstrated to have a wide range of anti-yeast action. Moreover, *Eucalyptus urophylla* and *Eucalyptus camaldulensis* EOs are also recognized for their microbial inhibitory effect [38]. Though much research has been dedicated to the antifungal and anti-yeast effects of EGEO [40], only a limited number of studies have estimated their bio-activity against foodborne bacteria and fungi [7]. Damjanovi´c-Vratnica et al. [41] reported 85.8% of eucalyptol in EGEO from Montenegro and demonstrated its important and significant effect in the inhibition of different yeast and bacteria growth. Furthermore, another study [18] revealed powerful antiviral, antiseptic and antimicrobial activities of eight EGEOs from Tunisia.

The microbial inhibitory action of EGEO has been shown to diverge considerably within microorganisms and species. The higher antiseptic potential could be directly linked to their main chemical constituents detected in the EGEO (such as eucalyptol and α-pinene) or with the interaction among the minor and major components [18]. Previously published studies revealed that Gram-positive strains are more vulnerable than Gram-negative bacteria; the inhibitory effect against yeasts (*C. albicans* and *S. cerevisiae*) and fungi (*Aspergillus*, *Mucor*, and *Penicillium* species) has also been investigated [7]. According to one of the reports, *Eucalyptus odorata* volatile oil presented the strongest *in vitro* inhibitory effect against micro-organisms and *Eucalyptus bicostata* EOs possess the greatest antiviral action [18].

The EGEO vapor could be extremely active against food spoilage microorganisms in minor doses in comparison with the liquid diffusion, thus producing less influence on the organoleptic and sensorial food properties [42]. Hence, estimating the *in vitro* antifungal and antibacterial actions of EGEO vapor might open up a promising dimension with many possible uses, especially in the food industry.

Current data obtained by both vapor diffusion and agar disc diffusion tests were not similar. The efficiency of the EGEO had previously been described but their use in the vapors phase requires an advanced approach [20]. This could be related to the variance in the chemical composition profile of the vapors and liquid oil because the oil must be enriched in terms of its volatile compounds [7]. A supplementary clarification that has been proposed for the vapor phase being more active is that the hydrophobic compounds in the aqueous phase associate to create micelles and thus restrain that connection of the EO to the microorganism, while the vapor phase permits free attachment [43]. Despite published papers on the *in vitro* antibacterial and antifungal properties of EGEO, few published articles exist on the bioactivity of EGEO vapors. The research done by Goni et al. [44] showed that the *in vitro* antibacterial activity of a combination of cinnamon and clove volatile oils presented a greater inhibitory action with less active doses in the vapor phase in comparison to the liquid phase.

Current results revealed that Algerian EGEO was effective against all yeast and Gram-positive bacteria strains. Previous data already showed that EGEO has *in vitro* inhibitory activity against several microorganisms [7,18]. For example, EGEO displayed powerful action (with 14.3 to 18.2 mm DIZ) against different bacterial species (*S. aureus*, *E. coli*, and *A. faecalis*) and no inhibitory effect was reported against yeast strain (*C. albicans*) [45]. Vilela et al. [42] tested the *in vitro* antifungal property of both the EGEO and its main compound (eucalyptol) against two *Aspergillus* strains. They obtained complete inhibition when using the EGEO, while less antifungal activity was obtained when testing eucalyptol alone. This proves that the potential synergistic influence of minor and main chemical compounds defines the final *in vitro* inhibitory potential of the volatile oils [3]. Based on the chemical profile of EGEO, it can be suggested that the *in vitro* antiseptic potential is seemingly due to its great concentration of oxygenated monoterpenes (94.09%).

Tserennadmid et al. [46] assessed the antifungal activity of different volatile EOs such as lemon, clary sage, marjoram and juniper in fruit juices. The minimum inhibitory concentration (MIC) of these EOs in fruit juices was considerably greater than *in vitro* MIC values. As great doses of several EOs are necessary to accomplish a suitable fungal inhibitory activity, undesirable levels of unsuitable tastes and smells may exist [47]. To more decrease the necessary EGEO dose for monitoring the *S. cerevisiae* load in Orangina juices, the interaction between EO and heat processing was evaluated.

To avoid the development of spoilage bacteria and yeasts in foods, numerous protection methods, such as thermal processing, the additions of acids or salts, and drying have been investigated in food production [48–52]. In this background, several volatile molecules and EOs containing oxygenated monoterpenes (linalool, geraniol, citronellol, citral, limonene and pinene), combined with moderate thermal processing, were tested to decrease the growth of *S. cerevisiae* in beverages [48].

In the last decade, numerous reports have revealed the incidence of additional activities when associating EOs with moderate heat and high hydrostatic pressure actions [49,50,53,54]. Synergistic effects between EOs with high hydrostatic pressure or moderate heat have been linked to the existence of lethal damages in the external membrane of surviving microorganisms, which may help the entrance of oil's compounds into the cells.

The research of Cherrat et al. [50] revealed the superior effect of EOs extracted from *Myrtus communis* and *Laurus nobilis* in association with medium thermal processing and high hydrostatic pressure to accomplish a greater level of bacterial inhibition of food spoilage microorganisms, and as result, decrease undesirable effects on sensorial food properties. Belletti et al. [48] have proved that the addition of citral and *Citrus* EO to soft juices in association with moderate heat processing can avoid yeast spoilage in beverages. In addition, the bacterial growth of *Listeria innocua* in orange juice was inactivated by the addition of vanillin in relation to the entity of heat processing [51].

As the required EO concentration against foodborne and spoilage bacteria and yeast is affected by the interactions of the EO chemical constituents with the food medium and ingredients, higher quantities are necessary to achieve appropriate food preservative action. This undesirably influences the organoleptic and sensorial properties of the foodstuff [7,52]. To resolve this problem, an interesting alternative is the application of an association of moderate heat treatment with EOs, which improves the antibacterial and antifungal effects of the EO influencing the vapor pressure of the compounds [55–57]. Consequently, the association of EOs with medium heat processing can be investigated for emerging food conservation technologies. Few studies have focused on the association of EOs (*Eucalyptus* and *Mentha*) with thermal processing at 55 ◦C [7,52]. This approach considerably decreases the EOs concentration necessity, offers a very valuable interaction, as the rise of heating increases the quantity of EO in the vapor phase, thus it improves its anti-yeast effect.

Belletti et al. [58] detected that neither the presence of the EO at their greater doses alone nor the heat treatment alone, was able to maintain or decrease the yeast and fungal stability of the drinks against *Saccharomyces cerevisiae*. In opposition, when applied in association, moderate heat processing (55 ◦C, 15 min) improved the activity of EO compounds (pinene, linalool and citral) and made an increase in their vapor pressure, which in turn amplified their chance to solubilize in the fungal cell membrane. The current research also revealed the improvement in the antifungal effect of EGEO on association with moderate heat action. The association of medium heat treatment with EGEO application has not been formerly published for avoiding Orangina juice contamination. Current findings demonstrated that EGEO can be used with moderate heat action for the food protections of Orangina juices.

#### **5. Conclusions**

Current findings revealed that EGEO could be used as a possible antifungal and antibacterial agent against foodborne and food spoilage microorganisms. The chemical composition of the different compounds characterizing the EGEO showed the predominance of oxygenated terpenes responsible for the microbial inhibitory effect against pathogens. The use of the EGEO in association with moderate heat processing effectively reduced the growth of spoilage yeast strain in Orangina fruit juices. Current data offer an outstanding record of EGEO as an antifungal agent and propose its possible use for beverage preservation. Supplementary research should be done to determine the effectiveness of EGEO in order to use it as a natural additive in different food matrices and/or improvement of food shelf life.

**Author Contributions:** Conceptualization, M.N.B.; methodology, M.N.B., A.B., H.G.N.; GC-MS analysis, M.N.B., M.R.; formal analysis, M.N.B., A.B., H.G.N.; resources, S.A.M.; writing—original draft preparation, M.N.B., S.A.M.; writing—review and editing, M.N.B., S.A.M.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank Kelly Keating (The Pharmaceutical Research Institute (PRI), Albany College of Pharmacy and Health Sciences, Rensselaer, NY, USA) for proofreading, constructive criticism and English editing of the manuscript. Also, we would like to thank the "Laboratoire d'Hygiène de Blida (Blida, Algeria)", especially Djamel Teffahi and Abdenacer Hmida for their help and making the facilities available for carrying out this research. M.N.B. sincerely thanks Noureldien Darwish and Thangirala Sudha from the PRI for their continuous support, advice and scientific interactions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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#### *Article*
