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Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 969143, 9 pages
http://dx.doi.org/10.1155/2014/969143

Research Article
Eucalyptus Essential Oil as a Natural Food Preservative:
In Vivo and In Vitro Antiyeast Potential

Amit Kumar Tyagi,1,2 Danka Bukvicki,3 Davide Gottardi,2 Giulia Tabanelli,4

Chiara Montanari,4 Anushree Malik,1 and Maria Elisabetta Guerzoni2
1
Applied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology
Delhi, New Delhi 110 016, India
2
Dipartimento di Scienze degli Alimenti, Universita` degli Studi di Bologna, Sede di Cesena, Piazza G. Goidanich 60,
47023 Cesena, Italy
3
Faculty of Biology, University of Belgrade, Institute of Botany and Botanical Garden “Jevremovac”, 11 000 Belgrade, Serbia
4
Interdipartimental Center for Industrial Research-CIRI-AGRIFOOD, Alma Mater Studiorum,
University of Bologna, Piazza Goidanich 60, 4752 Cesena, Italy

Correspondence should be addressed to Amit Kumar Tyagi; [email protected]

Received 23 February 2014; Revised 23 June 2014; Accepted 21 July 2014; Published 7 August

2014 Academic Editor: Kimon Andreas Karatzas

Copyright © 2014 Amit Kumar Tyagi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

In this study, the application of eucalyptus essential oil/vapour as beverages preservative is reported. The chemical composition
of eucalyptus oil was determined by gas chromatography-mass spectrometry (GC-MS) and solid phase microextraction GC-MS
(SPME/GC-MS) analyses. GC-MS revealed that the major constituents were 1,8-cineole (80.5%), limonene (6.5%), 𝛼-pinene
(5%), and 𝛾-terpinene (2.9%) while SPME/GC-MS showed a relative reduction of 1,8-cineole (63.9%) and an increase of
limonene (13.8%),
𝛼-pinene (8.87%), and 𝛾-terpinene (3.98%). Antimicrobial potential of essential oil was initially determined in vitro against 8
different food spoilage yeasts by disc diffusion, disc volatilization, and microdilution method. The activity of eucalyptus
vapours
was significantly higher than the eucalyptus oil. Minimum inhibitory concentration (MIC) and minimum fungicidal
concentration (MFC) varied from 0.56 to 4.50 mg/mL and from 1.13 to 9 mg/mL, respectively. Subsequently, the combined
efficacy of essential oil and thermal treatment were used to evaluate the preservation of a mixed fruit juice in a time-dependent
manner. These results suggest eucalyptus oil as a potent inhibitor of food spoilage yeasts not only in vitro but also in a real food
system. Currently, this is the first report that uses eucalyptus essential oil for fruit juice preservation against food spoiling yeast.

1. Introduction significantly within microbial species and within microbial

Eucalyptus is an evergreen, tall tree, or shrub, belonging strains. The strong antimicrobial activity may be directly
associated with their major compounds in the oil (such as
to Myrtaceae family. Although it is native to Australia and
1,8-cineole and 𝛼-pinene) or with the synergy between the
Tasmania, nowadays it has extensively spread to other
major and minor constituents [3]. Previous results reported
coun- tries [1]. The genus Eucalyptus contains about 700
that Gram positive bacteria are more susceptible than Gram
species; among them, more than 300 contain volatile oils in
negative bacteria; moreover, activity against fungi and
their leaves. Essential oils of various eucalyptus species are
yeasts (Candida albicans and Saccharomyces cerevisiae)
used in the pharmaceutical, toiletries, cosmetics, and food
has also been detected [4]. According to one of the reports,
industries [1]. These broad applications are due to the
Eucalyp- tus odorata had the strongest activity against
antiseptic, antihy- perglycemic, anti-inflammatory, bacteria and yeasts while E. bicostata had the best antiviral
flavouring, and antioxidant properties of the molecules activity [3]. Although several studies about eucalyptus oils
present in the oil [2]. The antimi- crobial activity of have been published [5–7], only few of them evaluated
eucalyptus oils has been found to vary their activity
2 BioMed Research International

against pathogenic and food spoilage microorganisms [4,

injection. Five millilitres of 10 ppm solution of the
8]. Despite the well-reported antimicrobial activity in vitro,
eucalyptus oil was placed in 10 mL vials and the vials were
the food industry has applied eucalyptus essential oils
sealed by PTFE/silicone septa. 1 𝜇L of the samples was
mainly as flavouring agents. Therefore, the use of essential injected directly into the column with a split ratio of 1 : 100.
oils as preser- vatives in food has been limited. Because the Component separation was achieved following the method
required con- centration against microorganisms is affected described above.
by the interac- tions of the oil compounds with the food For the SPME analysis, a divinylbenzene-poly(dimeth-
matrix components, higher concentrations are needed to ylsiloxane) coated stable flex fiber (65 𝜇m) and a manual
achieve sufficient activ- ity. This negatively impacts the SPME holder (Supelco Inc., Bellefonte, PA, USA) were
organoleptic properties of the final product [9]. To used in this study after preconditioning according to the
overcome this problem, a promising alternative is the use of manu- facturer’s instruction manual. Samples were put into
a combination of mild temperature treatment with essential sealed vials for 10 min at room temperature. The SPME
oils [10]. A mild thermal treatment, in fact, enhances the fiber was exposed to each sample for 10 min by manually
antimicrobial efficacy of the essential oil influencing the penetrating the septum, and, finally, the fiber was inserted
vapour pressure of the molecules [11]. into the injec- tion port of the GC for 10 min sample
In the present study, after a chemical characterization desorption.
by GC-MS, in vitro effect of eucalyptus oil against 8 The identification of the molecules was based on
different food spoilage yeast species was studied through the compar- ison of mass spectra of compounds both with those
disc diffu- sion method, the disc volatilisation method, and contained in available databases (NIST version 2005) and
MIC/MFC. Moreover, to evaluate the antiyeast activity in with those of pure standards (Sigma-Aldrich, Milan, Italy)
vivo, we employed a real food system based on the analyzed under the same conditions.
preservation of a mixed fruit juice inoculated with S.
cerevisiae and stored at room temperature for 8 days.
2.3. Antiyeast Activity of Eucalyptus Oil and Vapour
Further, in order to improve the efficiency of the essential
oil, the combined effect of oil and thermal treatment was 2.3.1. Disc Diffusion Method. The agar disc diffusion
also evaluated in the same real system. method was employed for the determination of
antimicrobial activ- ities of the essential oils [12] (NCCLS,
2. Materials and Methods 1997). Briefly, a suspension of the tested microorganism
(100 𝜇L of 1 × 106 CFU/mL) was confirmed by viable
2.1. Chemicals and Strains. The essential oil was procured counts and spread on the YPD agar media plates. These
from Erbamea, “olio essenziale naturale,” Italy, and stored plates were allowed to dry. Filter paper discs, 6 mm in
in an airtight sealed glass bottle at 4∘C till further use. diameter (Schleicher & Schuell, Germany), were soaked
Growth media and Tween 80 were purchased from Oxoid with 10 𝜇L of the oil and placed on the inoculated plates
Ltd., Basingstoke, Hampshire, UK, and Merck Schuchardt, and, after storing at 4∘C for 2 h, were incubated at 28∘C for
Germany, respectively. Different yeast strains 48 h. Volume of essential oils tested was varied (10, 20, or
(Saccharomyces cerevisiae SPA, Zygosaccharomyces bailii 30 𝜇L) by using appropriate number of sterile discs. The
45, Aureobasidium pullulans L6F, Candida diversa T6D, diameters of the inhibition zones were measured in
Pichia fermentans T2A1, Pichia kluyveri T1A, Pichia millimetres.
anomala, and Hansenula poly- morpha CBS 4732) were
obtained from the strain collection of the Dipartimento di 2.3.2. Disc Volatilisation Method. Standard experimental
Scienze degli Alimenti, University of Bologna, Italy, and setup as described by Lopez et al. [13] was used. Briefly, a
used to evaluate the effect of essential oil. The yeast strains 100 𝜇L portion of each suspension containing
were grown in yeast peptone dextrose (YPD) medium at 28∘C approximately 106 CFU/mL was spread over the surface of
for 24 h in an orbital shaking incubator (Universal Table YPD agar plate and allowed to dry. A paper disc (diameter
Shaker 709, Milan, Italy) at 120 rpm. 6 mm; Schleicher & Schuell, Germany) was laid on the
inside surface of the upper lid and 10 𝜇L eucalyptus oil was
2.2. Gas Chromatography-Mass Spectrometry (GC-MS) and soaked on each disc. The plates inoculated with
Solid Phase Microextraction-Gas Chromatography-Mass Spec- microorganisms were immediately inverted on top of the lid
trometry (SPME/GC-MS) Analyses of Eucalyptus Essential and sealed with parafilm to prevent leakage of eucalyptus
Oil. GC-MS analyses were carried out on an Agilent 7890 oil vapour. Plates were incubated at 28∘C for 48 h and the
gas chromatograph (Agilent Technologies, Palo Alto, CA) diameter of the resulting inhibition zone in the yeast lawn
coupled to an Agilent 5975 mass selective detector was measured.
operating in electron impact mode (ionization voltage, 70
eV). A CP- Wax 52 CB capillary column (50 m length, 0.32 2.3.3. Determination of Minimum Inhibitory Concentra-
mm inner diameter, and 1.2 𝜇m film diameter) was used. tion (MIC) and Minimum Fungicidal Concentration
The temper- ature program started from 50 ∘C and then was (MFC). Broth microdilution assays were performed as
programmed at 3∘C/min to 240∘C which was maintained for recommended by NCCLS [12]. All tests were performed in
1 min. Injector, interface, and ion source temperatures were YPD agar sup- plemented with Tween 80 (final
250∘C, 250∘C, and 230∘C, respectively. Injections were concentration of 0.5% v/v). Yeast strains were cultured
performed in split mode and helium (1 mL/min) was used overnight at 28∘C in YPD broth. Test strains were
as carrier gas. The mass selective detector was operated in suspended in YPD to give a final density of 1 × 106
the scan mode between 20 and 400 m/z. Data acquisition CFU/mL. Geometric dilutions ranging from 0.036 mg/mL
started 4 min after to 72 mg/mL of the eucalyptus oil were
BioMed Research International 3

prepared in a 96-well microtiter plate, including one growth

TABLE 1: Chemical constituents of eucalyptus essential oil obtained
control (YPD broth + Tween 80) and one sterility control through GC-MS.
(YPD broth + Tween 80 + test oil). Plates were incubated
at 30∘C for 48 h. The yeast cell growth was indicated by the RT (min) Compound Percentage
presence of a white pellet on the well bottom. The MIC 15.776 𝛼-Pinene 5.02
values were determined as the lowest concentration of oil 17.843 𝛽-Pinene 0.54
preventing visible growth of microorganisms. MFC was 18.267 𝛽-Myrcene 0.77
defined as the lowest concentration at which no growth was
19.092 𝛼-Phellandrene 0.53
observed after subculturing into fresh media.
19.685 Terpinolene 0.10
20.128 Limonene 6.45
2.4. Mixed Fruit Juice Preservation by 20.646 1,8-Cineole 80.44
Eucalyptus Oil and Thermal Treatment 21.75 𝛾-Terpinene 2.90
2.4.1. Preparation of Fruit Juice Mixture Inoculated with S. 23.237 4-Carene 0.34
cerevisiae SPA. Apples (Golden Delicious) and oranges at 23.616 Linalool 0.16
commercial maturity were purchased from a local market 25.889 Pinocarveol 0.17
(Ipercoop, Cesena). After being washed, apples were cut 27.69 4-Terpineol 0.55
into about 35 × 25 × 5 mm slices and then immersed in 28.296 𝛼-Terpineol 1.72
0.2% ascorbic acid solution (to avoid undesirable Monoterpene hydrocarbons 16.67
enzymatic browning during the processing), drained Oxygenated monoterpenes 83.04
quickly, and made into juices using a blender. After being Total of identified compound 99.71
washed, oranges were peeled off and made into juices. Both
RT: retention time (min), relative area percentage. Results are based on GC-
juices were mixed in 1 : 1 ratio. The suspension of yeast MS. MS acquisition started after 4 min.
strain (S. cerevisiae SPA) was mixed with fruit juice
mixture to result in final concen- tration of 103CFU/mL and
the inoculated juice mixtures were transferred into 10 mL differences among treatments (𝑃 ≤ 0.05) was analysed
sterilized glass vials. using one-way ANOVA (SPSS, 10.0 version). For all
experiments, three replicates were used and the data
2.4.2. Effect of Thermal Treatment. The effect of thermal presented here represents the mean of these replicates with
treatment was studied by exposing the mixed juice samples standard error or deviation. Moreover, as regards yeast load
at 70∘C for 30, 60, and 90 s. Subsequently, the treated vials counts during juice storage, a principal component analysis
were stored at room temperature up to 8 days and samples (PCA) was carried out with Statistica 6.1 (StatSoft Italy srl,
were drawn on 0, 2nd, 4th, and 8th day. Vigonza, Italy), using the different concentrations of
eucalyptus essential oil and duration of the thermal
2.4.3. Effect of Eucalyptus Oil. 1.0% ethyl alcohol solution treatments as variables.
of eucalyptus oil was mixed in the inoculated fruit juice
mixture at MIC level (4.5 mg/mL), half of MIC level (2.25 3. Results and Discussion
mg/mL), and one-fourth of MIC level (1.125 mg/mL). Fruit
juice sample inoculated with S. cerevisiae alone was 3.1.Chemical Composition of Eucalyptus Oil. Thirteen
considered as positive control. Subsequently, the treated com- pounds were identified by GC-MS for the total of
vials were stored at room temperature up to 8 days and 99.7% (Table 1). The major constituents of the oil were
samples were drawn on 0, 2nd, 4th, and 8th day. 1,8-cineole (80.4%), followed by limonene (6.5%), 𝛼-
pinene (5%), and
2.4.4. Effect of Eucalyptus Oil and Thermal Treatment: 𝛾-terpinene (2.9%). On the contrary, seventeen molecules
Com- bined Effect. A set of inoculated fruit juice mixtures were detected by SPME/GC-MS for the total of 99.9%
vials added with three different concentrations of (Table 2). The major constituents were the same as the
eucalyptus oil were exposed to thermal treatment (70 ∘C) for reported
30, 60, and 90 s. Each condition was treated in triplicate. ones in Table 1 but with different relative composition: 1,8-
Subsequently, the treated vials were stored at room cineole (63.9%), limonene (13.8%), 𝛼-pinene (8.9%), and
temperature up to 8 days and samples were drawn on 0, 𝛾-terpinene (3.9%). The differences between the chemical
2nd, 4th, and 8th day. All treated samples were serially contents of oil and vapour and their reasons were also
diluted and plated on PDA. The plates were incubated for evaluated in our previous reports [4]. As reported in the
72 h at 28∘C and CFU counts were made. The efficacy of literature, essential oil of eucalyptus was characterized by
the thermal treatment alone and the combination with very high concentration of 1,8-cineole. Damjanovic´-Vratnica
different doses of eucalyptus oil were quantified in time- et al. [14] determined an 85.8% 1,8-cineole in eucalyptus
dependent manner by the variation in log CFU/mL of the essential oil from Montenegro and reported its significant
inoculated yeast strains. activity against different bacteria and yeasts. Moreover,
Elaissi et al. [3] showed strong antibacterial, antifungal, and
antiviral effect of eight eucalyptus oils from Tunisia.
2.5. Statistical Analyses. All the experiments were done in
triplicate and repeatability was established. Significance of 3.2. Antiyeast Activity of Eucalyptus Oil. In this work, the
antiyeast activity of eucalyptus oil was evaluated with 8
 4 BioMed Research International

TABLE 2: Chemical constituents of eucalyptus essential oil obtained

50
through SPME/GC-MS.

RT (min) Compound Percentage 40

Zone of inhibition (mm)

8.069 𝛼-Pinene 8.87
30
11.315 𝛽 -Pinene 0.82
13.422 𝛽 -Myrcene 1.07
13.677 𝛼-Phellandrene 1.05 20
13.908 3-Carene 0.19
14.323 Terpinolene 0.19 10
15.221 Limonene 13.84
15.877 1,8-Cineole 63.96 0

S. cerevisiae
17.255 𝛾-Terpinen 3.98

C. diversa
Z. bailii

P. kluyveri

P. anomala

H. polymorpha
A. pullulans
18.423 o-Cymene 4.13
18.948 4-Carene 0.15
22.443 trans-5-Methyl-2-isopropyl-2-hexen-1-al 0.05
25.958 p-Cymene 0.14 10 𝜇L Yeast strains
30.132 Linalool 0.10 20
𝜇L 30 𝜇L
32.868 4-Terpineol 0.26
35.08 Pinocarveol 0.08 FIGURE 1: Antiyeast potential of eucalyptus oil evaluated with
36.609 𝛼-Terpineol 0.43 disc diffusion method. Zone of inhibition due to the different
Monoterpene hydrocarbons 34.46 concen- trations (10, 20, and 30 𝜇L) of eucalyptus oil against S.
Oxygenated monoterpenes 64.83 cerevisiae SPA, Z. bailii 45, A. pullulans L6F, C. diversa T6D, P.
Total of identified compound 99.29 kluyveri T1A, P. anomala, and H. polymorpha CBS 4732 was
measured. Column height represents the mean of triplicate results
RT: retention time (min), relative area percentage. Results are based on
and error bar represents the standard deviation.
GC- MS.

different food spoilage yeasts: S. cerevisiae, Z. bailii, A. a reduced activity was detected when using 1,8-cineole
pullulans, C. diversa, P. fermentans, P. kluyveri, P. alone. This shows that possible synergistic effect of minor
anomala, and H. polymorpha. and major components determines the final antimicrobial
activity of the essential oils [22]. Based on the chemical
composition, it can be concluded that the antimicrobial
3.2.1.Disc Diffusion Method. The antiyeast activity of activity of the oil is apparently attributed to its high content
euca- lyptus essential oil was assessed by the presence or the of oxygenated monoterpenes.
absence of inhibition zones. Three different concentrations
of the oil (10, 20, and 30 𝜇L) were tested. The highest 3.2.2. Disc Volatilisation Method. The zone of inhibition
susceptible yeast was H. polymorpha (10, 18, and 32 mm), in yeast strains due to eucalyptus essential oil vapours is
followed by A. pullulans (10, 16, and 30 mm), C. diversa (10, shown in Figure 2. Zone of inhibition due to essential oil
15, and 23 mm), vapour increased in a dose-dependent manner similar to
Z. bailii (10, 14, and 22 mm), P. kluyveri (12, 16, and 20 mm), disc diffusion method. The inhibition zones observed using
and S. cerevisiae (9, 12, and 17 mm) (Figure 1). P. 30 𝜇L of eucalyptus oil vapours were P. kluyveri (22 mm) <
anomala presented the lowest inhibition zone (9, 12, and 14 S. cerevisiae (36 mm) < Z. bailii (38 mm) = P. anomala (38
mm). The results demonstrated that eucalyptus oil was mm)
effective against all the considered strains. Previous data < C. diversa (39 mm) < A. pullulans (42 mm) < H.
already reported that eucalyptus oil possesses antimicrobial polymorpha
activity against differ- ent microorganisms [14–18]. For (44 mm). H. polymorpha was the most susceptible yeast to
example, Eucalyptus staige- riana oil showed strong eucalyptus oil vapours since 14, 26, and 44 mm inhibition
activity (with 14.3–18.2 mm zones of inhibition) against zones were generated using 10, 20, and 30 𝜇L eucalyptus
several microorganisms (Escherichia coli, Staphylococcus oil vapours, respectively (Figure 2). As compared to the oil,
aureus, and Alcaligenes faecalis) and no activity against the eucalyptus vapours resulted in a significantly larger
yeast C. albicans [19]. Eucalyptus cinerea oil exhibited zone of inhibition (𝑃 ≤ 0.05) in all the strains tested. This
significant activity against S. pyogenes (26 mm) and better result could be attributed to the variation in relative
P. aeruginosa (17 mm). The zone of inhibition for S. composition of the oil and vapours, as already shown in
aureus was 13 mm. Only isolated and purified 1,8-cineole Table 2.
(eucalyp- tol) presented no antimicrobial activity against S.
aureus and 3.2.3. MIC and MFC of Eucalyptus Oil. MIC and MFC of
C. albicans [20]. Also, Vilela et al. [21] tested the eucalyptus essential oil were determined against food
antimicrobial activity of both the eucalyptus essential oil spoiling yeasts (Table 3). The MIC values varied from 0.56
and 1,8-cineole against two Aspergillus species. They mg/mL to
reported a complete fungal growth inhibition when using 4.5 mg/mL. MIC value for S. cerevisiae and A. pullulans
the essential oil, while was higher (i.e., 4.5 mg/mL) than that for Z. bailii, C.
BioMed Research International 5
diversa,
6 BioMed Research International

50
bacteria and human pathogens, varied between 0.3 and
40 3.13 mg/mL, which can be attributed to the different
Zone of inhibition (mm)

amount of active molecules observed in the tested

30 eucalyptus oils. In fact, according to Sokovic´ et al. [23] and
Inouye et al. [24], not only the major compounds (1,8-
20 cineole) but also the minor ones (such as 𝛾-terpinene, 𝛼-
pinene, 𝛽-pinene, myrcene, and linalool) play a significant
10
role in the antimicrobial activity.
0
S. cerevisiae

3.3. Mixed Fruit Juice Preservation by Eucalyptus Oil

and Thermal Treatment
Z. bailii

C. diversa

P. kluyveri

P. anomala

H. polymorpha
A. pullulans

3.3.1.Effect of Thermal Treatment. A thermal treatment for

30 and 60 s at 70∘C did not have effect on the growth of
10 𝜇L S. cerevisiae in the mixed fruit juice samples (Figure 3(a)).
20 Yeast strains
Indeed, only a 0.49 log CFU/mL reduction was observed in
𝜇L 30 samples, subjected to a thermal treatment for 90 sec, after
𝜇L eight days at room temperature. Hence, this kind of
treatment was almost ineffective for preserving the juice.
A similar
FIGURE 2: Antiyeast potential of eucalyptus oil vapour evaluated from Montenegro against 17 microorganisms, including
with disc volatilisation method. Zone of inhibition due to the food poisoning and spoilage
different concentrations (10, 20, and 30 𝜇L) of eucalyptus oil
against
S. cerevisiae SPA, Z. bailii 45, A. pullulans L6F, C. diversa T6D,
P. kluyveri T1A, P. anomala, and H. polymorpha CBS 4732 was
measured. Column height represents the mean of triplicate results
and error bar represents the standard deviation.

TABLE 3: MIC/MFC of eucalyptus oil for different yeast strains.

S. number Name of the strain MIC MFC

(mg/mL) (mg/mL)
1 Saccharomyces cerevisiae SPA 4.5 9
2 Zygosaccharomyces bailii 45 2.25 4.5
3 Aureobasidium pullulans L6F 4.5 9
4 Candida diversa T6D 2.25 4.5
5 Pichia fermentans T2A1 2.25 4.5
6 Pichia kluyveri T1A 0.56 2.25
7 Pichia anomala 1.13 2.25
8 Hansenula polymorpha CBS 4732 2.25 4.5
MIC: minimum inhibitory concentration by microbroth dilution method;
MFC: minimum fungicidal concentration by streak plate method (𝑛 = 3;
𝑃 ≤ 0.05).

P. fermentans, and H. polymorpha (i.e., 2.5 mg/mL). P.

anomala and P. kluyveri showed the lowest MIC values,
1.13 mg/mL and 0.56 mg/mL, respectively. MFC varied
from
1.13 mg/mL to 9 mg/mL and showed a similar pattern to
MIC; that is, S. cerevisiae and A. pullulans (9 mg/mL) had
higher values than Z. bailii, C. diversa, P. fermentans, H.
polymorpha (4.5 mg/mL), P. anomala. (2.25 mg/mL), and
P. kluyveri (1.13 mg/mL). Silva et al. [20] reported the
minimal inhibitory concentrations of the eucalyptus
essential against different bacteria: Streptococcus pyogenes
(MIC: 0.39 mg/mL), Staphylococcus aureus (MIC: 0.78
mg/mL), Pseudomonas aeruginosa (MIC: 1.56 mg/mL),
and Candida albicans (MIC: 0.78 mg/mL). Damjanovic´-
Vratnica et al. [14] reported that the MICs of eucalyptus oil
BioMed Research International 7
pattern was also observed in our previous studies [25, 26].

3.3.2. Effect of Varying Concentrations of Eucalyptus Oil.

Since eucalyptus oil was able to kill several food spoilage
yeasts in in vitro tests, its activity in a real food system
(mixed fruit juice) has also been studied. The reduction
inviability of S. cerevisiae due to eucalyptus oil treatment
in dose-dependent manner (MIC, 1/2 MIC, and 1/4 MIC
level) and time-dependent manner (i.e., 0, 2, 4, and 8
days) was evaluated. As shown in Figure 3(b), a complete
growth inhibition was observed in the mixed fruit juice
when MIC levels of essential oil were used. However, 1/2
MIC and 1/4 MIC level samples showed a significant
reduction in the final number of cells (3.2 log CFU/mL
and 6.2 log CFU/mL, resp.) compared to untreated sample
(7.2 log CFU/mL). These data represented that the yeast
growth has been inhibited in a dose-dependent manner
even in food matrix. As previously reported [27], different
essential oils showed an excellent activity against food
spoilage yeasts (Saccha- romyces cerevisiae, Candida
rugosa, Debaryomyces hansenii, Kluyveromyces marxianus,
Rhodotorula glutinis, Rhodotorula minuta, Trichosporon
cutaneum, Yarrowia lipolytica, and Zygosaccharomyces
rouxii). For example, cardamom oil acted as a good
antimicrobial agent in real system such as pine apple fruit
juice [28], sweet orange juice [29], and apple juice [30]. In
the present study, it is the first attempt to evaluate the
antiyeast potential of eucalyptus oil in fruit juice mixture.
In some cases, the natural compounds performed even
better than the chemical preservatives [27].

3.3.3. Combined Effect of Eucalyptus Oil and Thermal

Treat- ment. The combined effect of eucalyptus oil (at
MIC, 1/2 MIC, and 1/4 MIC level) with thermal treatment
(at 70∘C for 30, 60, and 90 sec) on S. cerevisiae growth
was determined in a time-dependent manner (i.e., 0, 2, 4,
and 8 days) at room temperature (Figure 4). MIC and 1/2
MIC levels of eucalyptus oil combined with thermal
treatment showed complete growth inhibition of S.
cerevisiae after two days. In fact, the same growth
recovery was also found in samples treated with only
eucalyptus oil at 1/2 MIC dose up to
8 BioMed Research International

8
8

6
6
Viability (log CFU)

Viability (log CFU)

4
4

2 2

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Sampling time (days) Sampling time (days)

0
0.5 1 E0 E1
1.5 E2 E3
(a) (b)

FIGURE 3: Variation in viability of S. cerevisiae SPA in fruit juice mixtures during storage after (a) thermal treatment at 70∘C for 0.5, 1, and
1.5 min; (b) eucalyptus oil treatment at different concentrations (𝐸1 = MIC, 𝐸2 = 1/2 MIC, 𝐸3 = 1/4 MIC level, and 𝐸0 = control). The
data represents the mean of triplicate results and error bar represents standard deviation.

eight days of storage. However, the 30 and 60 sec thermal

compounds present in the eucalyptus oil when they have to
treatments combined with the oil produced a final reduction
diffuse in the agar compared to the diffusion in vapour
of 4.5 and 5.16 log CFU/mL, respectively (Figure 4), when
phase [35]. In our study, the oil dose requirement was
compared with 3.98 log CFU/mL measured in those treated
significantly reduced with the combination of the two
with essential oil alone (Figure 3(b)). Finally, the samples
treatments. This combination can be used as a better
with 1/4 MIC level of the oil were not affected by 30 sec
preservative with minimal impact on the organoleptic
thermal treatment, if compared with Figure 3(b).
properties of the bev- erage. Even our previous studies
Nevertheless, the 60 and 90 sec of thermal treatments
using a combination of oils (mentha and lemongrass) and
reduced the growth by 0.89 to 1.90 log CFU/mL,
thermal treatment have been published [25, 26]; this is the
respectively, as compared to the control. It has been
first report that uses eucalyptus essential oil for fruit juice
reported that the use of thermal treatment affects the
preservation against S. cerevisiae.
volatile compounds by increasing their vapour pressure,
which in turn improves the possibility to solubilize the
3.3.4.Principal Component Analysis. In order to confirm
yeast cell membrane. Though, the use of only one
the interactive effects between the different variables
treatment cannot guarantee the microbial stability of the
(concentra- tions of eucalyptus essential oil and thermal
beverages without affecting the final organoleptic
treatments) on the yeast growth, a principal component
properties of the product [31]. The combination of thermal
analysis (PCA) was carried out. Figure 5 reports the PCA
treatment with essential oils offers a very useful synergy
loadings plot on the first two factors of the samples. As
whereby increase in temperatures during storage could
expected, factor 1 (essential oil concentration) accounted for
enhance the vapour phase concentration of volatiles,
the great part of variability (about 94%) while factor 2
thereby enhancing the antimicrobial activity for better food
(thermal treatment) had a limited effect. In particular, four
preservation [25]. In some of our previous reports [32–34],
clusters can be identified. In the first, the control juice and
it was also observed that antimicrobial activity of
the heat treated juices (without eucalyptus essential oil)
essential oils was higher in vapour phase than in liquid
were grouped. In the second, the juices added with 1/4 MIC
phase, which was observed by different microscopic
thermal treated or not were grouped together. This cluster
techniques: scanning electron micro- scope, transmission
was characterized by a lower difference compared with
electron microscope, and atomic force microscope.
cluster 1 in relation to factor 1. This means that the addition
Basically, the differences in inhibition of yeast strain
of this amount of oil had scarce activity on the
obtained from essential oil (liquid phase, direct contact with
effectiveness of heat treatment on yeast viability during
the culture media) and the vapour can be attributed to the
storage. At last, clusters 3 and 4 were characterized by
differences in diffusion coefficients of the antimicrobial
pronounced differences with respect to the
BioMed Research International 9

8
8

6
6
Viability (log CFU)

Viability (log CFU)

4
4

2
2

0
0 2 4 6 8 10 0
0 2 4 6 8 10
Sampling time (days)
Sampling time (days)
(a)
(b)
8

6
Viability (log CFU)

0
0 2 4 6 8 10
Sampling time (days)

E0 E2
E1 E3
(c)

FIGURE 4: Combined effect of essential oil and thermal treatment. Variation in viability of S. cerevisiae SPA in fruit juice mixtures was
estimated. Different concentrations of eucalyptus oil ( 𝐸1 = MIC, 𝐸2 = 1/2 MIC, 𝐸3 = 1/4 MIC level, and 𝐸0 = control) combined with
different thermal treatments at 70∘C for (a) 30, (b) 60, and (c) 90 sec were tested. The growth of the yeast was followed up to 8 days after
the treatment. The data represents the mean of triplicate results and error bar represents standard deviation.

sample without essential oil. Cluster 3 was formed by

system). The chemical identification of the different mole-
juices with 1/2 MIC, not treated and thermal treated for 30
and 60 seconds. The last cluster grouped all the samples cules characterizing the eucalyptus oil evidenced the
having better antiyeast results (juices with MIC level and presence of oxygenated monoterpenes responsible for the
the sample with 1/2 MIC thermal treated for 90 seconds). antimi- crobial activity. The use of the combination of
The composition of the latter cluster highlights the eucalyptus essential oil with thermal treatment successfully
equivalence of the antiyeast effectiveness of this last sample inhibited the development of yeast (S. cerevisiae SPA) in
with the juices containing a double concentration of oil. fresh fruit juices. The results provide an excellent record of
This fact can allow for obtaining the same antiyeast effect eucalyptus oil as antimicrobial agent and suggest its
using a concentration of essential oil with a lower impact on potential application for beverages preservation. Additional
organoleptic profile of juices. studies should be conducted to confirm the potentiality of
eucalyptus essential oil in order to use it as a preservative in
4. Conclusion other food models.

The results of this work demonstrated that eucalyptus Conflict of Interests

essen- tial oil could be used as a potential antimicrobial
compound against food spoilage yeasts (in vitro and in The authors declare that there is no conflict of interests
a real food regarding the publication of this paper.
1 BioMed Research International
0
0.6
pathogenic microorganisms: a review,” Issues in Biological
Cluster 2: juices 13 Sci- ences and Pharmaceutical Research, vol. 2, no. 1, pp. 1–7,
0.4 added with 1/4 MIC
15
heat treated (11, 8 2014.
13, and 15) or not (9) 11
0.2 Cluster 3: juices
10
[7] I. Somda, V. Leth, and P. Se´re´me´, “Antifungal effect of
Factor 2: 4.20%

9 added with 1/2

Cymbo- pogon citratus, Eucalyptus camaldulensis and
MIC not treated (8)
0.0 Azadirachta indica oil extracts on sorghum feed-borne fungi,”
or treated for 30 (10)
and 60 (12) seconds 12 Asian Journal of Plant Sciences, vol. 6, no. 8, pp. 1182–1189,
−0.2 2007.
12 [8] P. Sartorelli, A. D. Marquioreto, A. Amaral-Baroli, M. E. L.
−0.4 3
4 Cluster 4: juices Lima, and P. R. H. Moreno, “Chemical composition and anti-
added with MIC 6 7 16 microbial activity of the essential oils from two species of
treated or not (5, 6, 7,
−0.6 5 14
Cluster 1: control juice (1),
heat treated juices without oil
and 16) and juice
added with 1/2 MIC
Euca- lyptus,” Phytotherapy Research, vol. 21, no. 3, pp. 231–
−3 addition (2,−2
3, and 4) −1 0 treated for 901 2 3 233, 2007.
seconds (14)
Factor 1: 94.77% [9] M. Hyldgaard, T. Mygind, and R. L. Meyer, “Essential oils in
food preservation: mode of action, synergies, and interactions
FIGURE 5: PCA loadings plot on the two first factors of control with food matrix components,” Frontiers in Microbiology, vol.
and treated juice. The clusters generated were as follows. Cluster 3, no. 12, pp. 1–24, 2012.
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and 4). Cluster 2: juices added with 1/4 MIC heat treated (11, 13, [10] F. Gardini, R. Lanciotti, and M. E. Guerzoni, “Effect of trans-
and 15) or not (9). Cluster 3: juices added with 1/2 MIC not 2-hexenal on the growth of Aspergillus flavus in relation to
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The present work was supported by the research exchange
fellowship to AKT (EMECW13c) and from the Ministry of [12] National Committee for Clinical Laboratory Standards
(NCCLS), “Publication M27-A: reference method for Broth
Education, Science and Technological Development of Serbia
dilution antifungal susceptibility testing of yeasts,” in
(Project no. 173029). Approved Standard, vol. 17, no. 9, pp. 1–28, NCCLS, Wayne, Pa,
USA, 1997.
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