*Eucalyptus globulus* **Essential Oil as a Natural Food Preservative: Antioxidant, Antibacterial and Antifungal Properties** *In Vitro* **and in a Real Food Matrix (Orangina Fruit Juice)**

**Mohamed Nadjib Boukhatem 1,2,\*, Asma Boumaiza 2, Hanady G. Nada 1,3, Mehdi Rajabi <sup>1</sup> and Shaker A. Mousa 1,\***


Received: 5 May 2020; Accepted: 19 May 2020; Published: 12 August 2020

**Abstract:** The potential application of *Eucalyptus globulus* essential oil (EGEO) as a natural beverage preservative is described in this research. The chemical composition of EGEO was determined using gas chromatography analyses and revealed that the major constituent is 1,8-cineole (94.03% ± 0.23%). The *in vitro* antioxidant property of EGEO was assessed using different tests. Percentage inhibitions of EGEO were dose-dependent. In addition, EGEO had a better metal ion chelating effect with an IC50 value of 8.43 ± 0.03 mg/mL, compared to ascorbic acid (140.99 ± 3.13 mg/mL). The *in vitro* antimicrobial effect of EGEO was assessed against 17 food spoilage microorganisms. The diameter of the inhibitory zone (DIZ) ranged from 15 to 85 mm for Gram-positive bacteria and from 10 to 49 mm for yeast strains. *Candida albicans*, *C. parapsilosis* and *Saccharomyces cerevisiae* were the most sensitive fungal species to the EGEO vapor with DIZ varying from 59 to 85 mm. The anti-yeast effectiveness of EGEO alone and in association with heat processing was estimated in a real juice matrix (Orangina fruit juices) in a time-dependent manner. The combination of EGEO-heat treatment (70 ◦C for 2 min) at different concentrations (0.8 to 4 μL/mL) was effective at reducing *S. cerevisiae* growth in the fruit juice of Orangina, compared to juice preserved with synthetic preservatives. Current findings suggest EGEO as an effective and potent inhibitor of food spoilage fungi in a real Orangina juice, and might be a potential natural source of preservative for the food industry.

**Keywords:** natural food preservative; *Eucalyptus globulus* essential oil; eucalyptol; antioxidant effect; vapor phase; Orangina fruit juice

#### **1. Introduction**

Food spoilage by fungi, yeasts and bacteria is a major problem in food production, and it considerably impacts the price and availability of the food [1]. The use of synthetic and chemical antibacterial and antifungal compounds and food preservatives is considered as one of the ancient methods for reducing foodborne pathogens and contamination. The addition of synthetic antioxidant and antimicrobial food preservatives is an active way for storage to slow down food alteration and oxidation [2]. Nevertheless, due to increasing confirmation of the dangerous properties of synthetic food additives, there is constant pressure from consumers to decrease the quantity of these chemicals in food [3] and deliver minimally processed foodstuffs without compromising food preservation, safety and quality [4].

Thus, other sources of nontoxic, bioactive and suitable natural food preservatives need to be discovered and investigated, such as plant secondary metabolites, phytochemicals and volatile oils or essential oils (EOs). A new approach to prevent and avoid the proliferation of microorganisms or protect food from oxidation is the use of EOs as antifungal, antibacterial and antioxidant preservatives. The potential application of EOs as functional components in drinks and beverages is gaining force because of growing anxiety about possibly dangerous and toxic synthetic additives [5,6]. Within the context of the extensive variety of the aforementioned foodstuffs, a collective need is the accessibility of phytochemical extracts and EOs with an agreeable flavor or scent associated with a conserving effect and that can avoid lipid alteration, oxidation and contamination by food spoilage microorganisms and pathogens [7–9].

Therefore, there has been growing attention to the discovery and investigation of safe, effective and natural antioxidant bioactive molecules because they can defend the human body from free radicals and delay the development of several chronic or acute illnesses [10]. The antimicrobial molecules found in aromatic and medicinal plants are of interest because multidrug resistant bacteria are now becoming a global community health alarm, particularly in terms of foodborne infections and nosocomial contaminations [11–13]. Several studies have reported antiseptic, anti-inflammatory, wound-healing, analgesic, antioxidant and free radical-scavenging activities [14] from aromatic and medicinal plants, herbs, spices and EOs and, in most cases, a direct food-related application has been verified [15].

The *Eucalyptus* genus is a tall shrub belonging to the family of Myrtaceae. Although some papers about *Eucalyptus globulus* essential oils (EGEOs) have been done [16–20], only a limited of them estimated *Eucalyptus* oil's effect against pathogens and food spoilage species [7]. In spite of the well-reported *in vitro* antibacterial and antifungal effects, food manufacturing has used *Eucalyptus* EOs principally as flavoring agents. Consequently, the application of EOs and phytochemical extracts as natural food additives has been restricted [16].

Despite the great efficacy of EOs and their phytochemical compounds against food-related and spoilage pathogens with *in vitro* methods, a similar result in a food matrix is only accomplished with a greater dose of EOs [3]. This statement suggests a sensorial and organoleptic influence from changing the ordinary flavor of the food and beverages by surpassing suitable taste thresholds [7]. Therefore, to reduce the dose of EO in a real juice matrix, studies on the associated effect of EOs with traditional conservation methods such as heat processing are required. In the current research, the chemical composition of EGEO was analyzed with gas chromatography-mass spectrometry (GC-MS). Then, the *in vitro* antioxidant effect was carried out using DPPH radical scavenging and metal ion chelating activity. The inhibitory effects of Algerian EGEO against several food spoilage bacterial and fungal strains were assessed *in vitro* (disc diffusion and disc volatilization tests) and in a real food matrix (inhibition of *Saccharomyces cerevisiae* strain for the preservation of Orangina fruit juices) and stored at laboratory temperature for 6 days. Further, for reducing the dose of EGEO in the Orangina fruit juice, the associated effect of EGEO with moderate heat treatment was evaluated.

#### **2. Materials and Methods**

#### *2.1. Material*

#### 2.1.1. Distillation of *Eucalyptus globulus* Essential Oil

*Eucalyptus globulus* EO was purchased from "Ziphee-Bio" company of essential oils (Lakhdaria, Bouira, Algeria). EGEO was extracted from the aerial part with alembic steam distillation (SD). SD is a method used to obtain EOs from *Eucalyptus globulus* by passing steam generated in a pot still through the plant material. A quantity of fresh plant (leaves and small branches of the tree) was loaded in the still and stacked in layers to allow the appropriate delivery of the steam. When the steam

passed through the *Eucalyptus globulus*, tiny pockets that hold the EOs opened to release the volatile compounds. This is referred to as the distillate. The distillate will contain a mix of hydrosol (aromatic water) and EO which return to their liquid form in the condenser and are separated using a Florentine separator. EGEO was stored in air-tight sealed glass bottles at 4 ◦C until further use.

#### 2.1.2. Food Spoilage Microorganisms

Different food-spoilage bacterial strains (*Escherichia coli*, *Enterobacter sakazakii, Pseudomonas aeruginosa, Klebsiella ornithinolytica, Bacillus cereus* and *Staphylococcus aureus*), fungal strains (*Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus* and *Aspergillus brasiliensis*) and yeasts (*Saccharomyces cerevisiae, Candida parapsilosis, Candida albicans* and *Trichosporon* sp.) were collected and identified from different food matrices (water, milk, juices and honey) in the Laboratory of Food Microbiology (Laboratoire d'Hygiène, Blida, Algeria) and used to evaluate the microbial inhibitory effect of EGEO.

The identification of microorganisms was carried out using morphologic and biochemical characterization tests. After cell identification through Gram staining and microscopic observation was done, the traditional biochemical tests (using API 20E) were carried out to assess the tested bacteria classification following the Gram-negative bacterial identification method [21]. Different biochemical assays were used such as oxidase, TSI medium, gelatin hydrolysis, sugar assimilation, amino acid degradation, hydrogen sulfide production, citrate and Voges-Proskauer.

The fungal species were identified based on their morphological arrangements such as pigmentation, diameter of the mycelia, and microscopic determination of formation of the germ tube, spores and chlamydoconidias. Yeast strains were identified using the AuxacolorTM kit which is an identification method based on sugar digestion [22]. The growth of yeasts is assessed by the color change of a pH indicator. The AuxacolorTM system contains 16 wells in a plastic microplate. All assays with the AuxacolorTM method were carried out following the manufacturer's guidelines. The AuxacolorTM system was stored at 4 ◦C and was carried to laboratory temperature before use. The fungal and bacterial species were identified with standard microbiology assays and stored in mueller-hinton agar (MHA) and sabouraud dextrose agar (SDA) for bacteria and fungi, respectively.

#### 2.1.3. Chemicals and Reagents

Dimethyl sulfoxide (DMSO), gallic acid, butylated hydroxyanisole (BHA), L-ascorbic acid (vitamin C.), ethanol, tween 80, FerroZine™ iron reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and iron (II) chloride (FeCl2) were obtained from Sigma Aldrich (St. Louis, MO, USA). Isosaline (0.9% NaCl), MHA and SDA medium were purchased from the Ideal-Labo company (Blida, Algeria). Filter paper discs (9 mm in diameter) were provided by Schleicher and Schull GmbH (Dassel, Germany). Antibiotic discs of amoxicillin-clavulanic acid (AMC, 20/10 μg), erythromycin (E, 15 μg), chloramphenicol (C, 30 μg) (Bio-Rad Laboratories, France) and antiseptic solution of Isomedine® 0.1% (Hexamidine dermal solution, Isopharma, Algiers, Algeria) were used to assess the sensitivity of isolated microorganism species.

#### *2.2. Methods*

#### 2.2.1. Chemical Composition of *Eucalyptus globulus* Essential Oil by GC-MS Analysis

Analysis of *Eucalyptus globulus* volatile oil was done using GC-MS. Analyses were carried out on an HP 6890 gas chromatograph (Agilent Technologies, Wilmington, DE, USA) fitted with an HP-5MS fused silica column (30 m × 0.25 mm, 0.25 μm film thickness), interfaced with an HP mass selective detector 5790A (Agilent Technologies) operated by HP Enhanced ChemStation software. The oven temperature program was 45–280 ◦C (2 ◦C/min). The injector temperature was 250 ◦C; the carrier gas was helium, adjusted to a linear velocity of 30 cm/s; the splitting ratio was 1:20 and the detector temperature was 250 ◦C. Separate peaks were recognized by comparison of their Retention Index (RI) to RI of authentic models, and by comparing their mass spectra with the NIST 2007 (National Institute

of Standards and Technology, Gaithersburg, MD, USA) and Wiley 6.0 library (New York, NY, USA) mass spectral database and the literature [23].

#### 2.2.2. *In Vitro* Antioxidant Activity

#### DPPH Radical Scavenging Assay

The radical scavenging effect was evaluated with a spectrophotometric assay based on the reduction of an ethanol solution of DPPH [24]. Briefly, a series of dilutions of EGEO was made. Then, 1.5 mL of each concentration was mixed with 1.5 mL of a 30 μg/L ethanoic DPPH solution that was incubated at laboratory temperature in a dark storeroom for 40 min. Afterward, the absorbance at 520 nm (maximum absorbance of DPPH) was noted spectrophotometrically. Separately, a negative control was made comprising all reagents except the EGEO. The free radical scavenging effect of each solution was then measured and calculated as percentage inhibition in accordance to the following equation:

$$\% \text{ inhibition} = 100 - \left(\frac{\text{DPPHs}}{\text{DPPHb}}\right) \ast 100 \text{ }$$

where DPPHs corresponds to the absorbance of DPPH with the sample and DPPHb to the absorbance of DPPH without sample (blank).

The antioxidant ability was recorded as the IC50 (medium inhibitory concentration). The IC50 is defined as the quantity of antioxidant required to reduce the primary DPPH quantity by 50%. The results are expressed as the mean ± standard deviation (SD) of three tests. Ascorbic acid and BHA were used as positive standards.

#### Metal Chelating Activity

Briefly, different concentrations of 1 mL of EGEO were dissolved in DMSO and added to a solution of 0.05 mL of 2 mM FeCl2 in H2O. The reaction was started by adding 0.02 mL of 5 mM ferrozine. The mixture was vigorously shaken and incubated at room temperature for 15 min. The absorbance of the EGEO sample was then estimated and determined at 565 nm using a spectrophotometer [24]. The metal chelating activity was expressed according to the following formula:

$$\% \text{ inhibition} = 1 - \left(\frac{\text{Abs Control} - \text{AbsSample}}{\text{Abs Control}}\right) \ast 100$$

Gallic acid, ascorbic acid and BHA were used as positive standards. The amount of inhibition by the test samples was expressed as the percentage of concentration required to do 50% inhibition (IC50).

#### 2.2.3. *In Vitro* Antimicrobial Effect of EGEO

#### Disc Diffusion Method

Microbial media used for culture and growth of bacterial and fungal strains were MHA and SDA, respectively. Inoculum of each bacterial and fungal species to be tested was prepared with fresh cultures by suspending the strain in isosaline (0.9% NaCl). In the first step, the antimicrobial potential of EGEO was explored with the disc diffusion assay [7]. Antibiotic discs of amoxicillin-clavulanic acid, erythromycin, chloramphenicol and antiseptic solution of hexamidine were used to control the antibacterial of isolated micro-organisms strains according to the NCCLS guidelines [25]. Discs without EGEO were considered as a negative control. Filter paper discs (diameter 9 mm) were saturated with three different quantities (20, 40 and 60 μL per disc) of *Eucalyptus* EO and sited in the inoculated microbial media (MH for bacteria and SDA for fungi). After keeping at laboratory temperature for 40 min, the Petri dishes were incubated at 37 ◦C/24 h for bacterial strains and at 25 ◦C/72 h for fungal strains. The microbial inhibitory effect was calculated by measuring the diameter of the growth-inhibition zone (DIZ) in mm (including filter diameter of 9 mm) for the tested species and compared to the antibiotic standards.

#### Disc Volatilization Assay

The usual assay setup as designated by Tyagi et al. [7] was followed. Briefly, a 0.1 mL portion of each suspension was spread over the surface of MHA (bacteria) or SDA (fungi) plates. A filter disc was placed on the inside external of the upper lid and 10 μL EGEO was placed on each disc. The Petri dish inoculated with strains was directly overturned on top of the lid and wrapped with parafilm to avoid the escape of EGEO vapor. Petri dishes were incubated at 37 ◦C/24 h for bacterial strains and at 25 ◦C/72 h for fungal strains. The microbial inhibitory effect of *Eucalyptus* volatile oil was estimated by calculating the DIZ of bacterial and fungal growth above the disc. Blank discs were used as a negative control. The quantity of EGEO used was varied (20, 40 or 60 μL) by using a suitable number of sterile discs.

#### 2.2.4. Orangina Juice Preservation by *Eucalyptus globulus* Oil and Moderate Heat Processing

#### Preparation of Orangina Juice Inoculated with a Yeast Strain (*Saccharomyces cerevisiae*)

Orangina fruit juices were purchased from a local company (Djgaguen Company, Blida city, Algeria). The suspension of *Saccharomyces cerevisiae* was added to Orangina beverage, and the inoculated juices were moved into 250 mL sterilized glass flasks.

Orangina juice is a lightly carbonated beverage made from carbonated water, 12% citrus juice (10% from concentrated orange, 2% from an association of concentrated grapefruit, lemon, and mandarin juices), as well as 2% orange pulp. Orangina juice is sugared with high fructose corn syrup and natural orange flavors are added. Preservatives such as benzoate sodium (E211), potassium sorbate (E202) and citric acid (E330) are also added. All Orangina bottles were kept at 4 ◦C.

#### Influence of *Eucalyptus globulus* Essential Oil Alone

Tween 80 solution (0.5%) of EGEO was added in the inoculated Orangina at different doses (0.8, 2 and 4 μL/mL). The Orangina sample inoculated with *Saccharomyces cerevisiae* alone was considered as a positive standard. Afterward, the flasks were kept at laboratory temperature for up to 6 days and juices were drawn on days 0, 2, 4, and 6.

The microbiological method used for yeast counts was the standard plate count (SPC) agar method. This method is used by the food industry for estimating the microbial populations in most types of juices products and samples and for determining the quality and sources of contamination at successive stages of processing. All Orangina fruit juices were successively diluted and plated on SDA medium. The petri dishes were incubated for 48 h at 25 ◦C and CFU counts were estimated. The effect of different doses of EGEO treatment was measured in a time-dependent way by the difference in log CFU/mL of the inoculated *S. cerevisiae* [7].

#### Influence of *Eucalyptus globulus* Essential Oil and Moderate Heat Processing: Combined Action

Three different doses (0.8, 2 and 4 μL/mL) of EGEO were added and mixed to inoculated Orangina fruit juice vials and were exposed to a medium heat treatment (70 ◦C) for 2 min [7]. Then, the flasks were deposited at laboratory temperature for up to 6 days and juices were drawn on day 0, 2, 4, and 6. All Orangina fruit juices were successively diluted and plated on SDA medium. The Petri dishes were incubated for 48 h at 25 ◦C and CFU counts were estimated. The effects of different doses of EGEO in association with medium heat treatment were measured in a time-dependent way by the difference in log CFU/mL of the inoculated *S. cerevisiae*.

#### *2.3. Statistical Analysis*

The significance of variances was analyzed using the test of one-way ANOVA followed by Tukey's post hoc multiple comparison test. Differences with *p* < 0.05 between experimental groups were considered statistically significant. Statistical data analysis was performed using XLstat 2014 software, Addinsoft, Paris, France. For all tests, three replicates were done and the final results represent the mean of these repeats with standard deviation. The IC50 (median inhibitory concentration) was calculated from the dose-response curve obtained by plotting percentage inhibition versus concentrations.

#### **3. Results**

#### *3.1. Chemical Composition of Eucalyptus globulus Essential Oil*

Six compounds were identified with GC-MS (Table 1). The major constituent of the EGEO was 1,8-cineole or eucalyptol (94.03% ± 0.23%), followed by α-pinene (94.03% ± 0.23%) and γ-terpinene (94.03% ± 0.23%). The concentration of other compounds in the EGEO was less than 1%.


**Table 1.** Chemical profile of EO from *Eucalyptus globulus* analyzed with GC-MS.
