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Vol. 63 No. 4 2017
From Botanical to Medical Research
DOI: 10.1515/hepo-2017-0021
Chemical compounds and antimicrobial activity
of petitgrain (Citrus aurantium L. var. amara)
essential oil
MAŁGORZATA GNIEWOSZ*1, KAROLINA KRAŚNIEWSKA1,
OLGA KOSAKOWSKA2, KATARZYNA POBIEGA1, IWONA WOLSKA1
1Department of Biotechnology, Microbiology and Food Evaluation
Warsaw University of Life Sciences – SGGW
Nowoursynowska 159c
02-776 Warsaw, Poland
2Department of Vegetable and Medicinal Plants
Warsaw University of Life Sciences – SGGW
Nowoursynowska 159c
02-776 Warsaw, Poland
* corresponding author: e-mail: malgorzata_gniewosz@sggw.pl
Summary
Introduction: Due to its low cost and easy availability on the market, the petitgrain oil is commonly used
in food, cosmetics, and aromatherapy.
Objective: The examination of chemical composition and antibacterial activity of commercial petitgrain oil.
Methods: Identification of chemical components of the petitgrain oil was performed by gas chromatography
(GC). The minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations
(MBC/MFC) were determined using macrodilution method for the reference strains of bacteria and fungi.
Results: Twenty components were identified. The petitgrain oil contained mostly oxygenated monoter-
pene hydrocarbons (98.01%), and the main components included linalyl acetate (48.06%) and linalool
(26.88%). The MIC/MBC of the petitgrain oil for bacteria was in the range of 0.63–5.0/1.25–5.0 mg/ml
and for fungi in the range of 1.25–40/5.0–80 mg/ml.
Conclusion: The petitgrain oil had higher antibacterial activity than antifungal activity. Bacillus subtilis
among the tested bacteria and Aspergillus niger and Penicillium expansum among the fungi were found to
be highly inhibited by the petitgrain oil.
Key words: petitgrain, essential oil, chemical composition, antimicrobial activity
Received: 2017-03-21
Accepted: 2017-08-30
SHORT COMMUNICATION
Herba Pol 2017; 63(4): 18-25
19
Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil
Vol. 63 No. 4 2017
INTRODUCTION
Citrus aurantium L. var. amara of the Rutaceae
family, commonly known as bitter (“sour”) orange, is
regularly cultivated in the Mediterranean area and in
Central and South America [1]. Composition of es-
sential oils from bitter orange is not the same and it
depends, to a large extent, on the geographic origin
and parts of the plant (zest, fruit, and owers) [2].
Essential oil from fruit zest is obtained through the
cold-pressed extraction method and it is referred to
as “cold-pressed essential oil”. e most expensive
in production is “Neroli oil”, which is obtained from
petals using steam distillation or hydrodistillation
method [1]. Petitgrain bigarade oil is obtained by
steam distillation [3] or hydrodistillation of the leaves
and twigs from pruning of the trees at dierent times
in the year [4]. Among essential oils obtained from
bitter orange, petitgrain is the lowest-priced oil [5].
e largest producer of the petitgrain oil is Paraguay,
followed by Egypt, Spain, France, and Italy [6]. e
total production of this essential oil is estimated at
260 tons per annum [7]. Petitgrain oil is also recom-
mended for aromatherapy, particularly for the antide-
pressant treatment, since it relieves anxiety, agitation,
stress, and challenging behaviors [8]. Due to its low
cost, and easy availability in the market, this essential
oil is commonly used in the production of marma-
lades and in avoring of some types of beers [9].
e study was aimed at the examination of the
chemical composition and antimicrobial activity
of commercial petitgrain oil against four bacterial
strains, including the pathogens transferred via food
and four fungi strains found in food.
MATERIAL AND METHODS
Essential oil
In this research, petitgrain essential oil (from Cit-
rus aurantium L. var. amara) was used. e oil was
purchased from the Pollena Aroma Company, Po-
land (commercial producer of plant essential oils and
aromatic substances), from three dierent batches.
Quality of the essential oil was ascertained to be
higher than 98% pure. e oils were stored in tightly
closed dark vials at 4°C until further tests and analysis.
Identication of chemical components
Gas chromatography analysis was performed us-
ing a Hewlett Packard 6890 gas chromatograph
equipped with aame ionization detector (FID) and
capillary, polar column HP 20M (25 m × 032 mm,
0.3 µm lm thickness). e analysis was performed
following the temperature program: oven tempera-
ture isotherm at 60°C for 2 min, then from 60°C to
220°C at a rate of 4°C/min and held at 220°C for
5 min. Injector and detector temperatures were set
at 220°C and 260°C, respectively. e carrier gas
(He) ow was 1.1 ml/min. e split ratio was 1:70.
Atotal of 0.1 µl of pure essential oil was manually
injected. Component identication was performed
by comparison of their retention times with those of
pure authentic samples and by their linear retention
indices (RI) relative to the series of n-hydrocarbons
(C7–C30), under the same conditions. Retention in-
dices of compounds were also compared with those
reported in the literature. e percentage composi-
tion of the oils was computed by the normalization
method from the GC peak areas, without the use of
correction factors. All the analyzes were performed
in triplicate.
Microorganisms and inoculum preparation
e reference strains Bacillus subtilis ATCC 6633,
Staphylococcus aureus ATCC 25923, Escherichia
coli ATCC 25922, Salmonella ser. Enteritidis ATCC
13076, Saccharomyces cerevisiae ATCC 9763, Can-
dida krusei ATCC 14243, Aspergillus niger ATCC
9142, and Penicillium expansum ATCC 7861 were
obtained from the culture collection (Division of
Food Biotechnology and Microbiology, Warsaw
University of Life Sciences, Warsaw, Poland). e
strains were stored in 20% glycerol at -80°C in
afreezer.
e frozen subcultures of bacteria were trans-
ferred to tubes containing 5 ml of sterile nutrient
broth (BTL). Aer incubation the test cultures of the
bacteria strains were separately inoculated on slants
of nutrient agar (BTL) and incubated at 37°C for
24 hours. Bacterial inocula were prepared in asterile
saline (0.85% NaCl) (w/v) solution with the quantity
corresponding to 0.5 McFarland (~1×108 cfu/ml)
and diluted to ~1×107 cfu/ml.
e yeasts were separately inoculated on Sab-
ouraud Agar slopes (SA, Merck) and were incubated
at 28°C for 48 hours. Yeast inocula were prepared
in asterile saline solution. e density of the yeast
suspension were measured with a hemocytometer
and diluted as necessary with sterile saline to obtain
the inoculum density of ~1×106 cfu/ml. e molds
were separately inoculated on Potato Dextrose Agar
20
Gniewosz, K. Kraśniewska, O. Kosakowska, K. Pobiega, I. Wolska
slopes (PDA, BTL) and incubated at 28°C for 7 days.
Slopes were ooded with 1 ml of phosphate-buered
saline (PBS) containing 0.05% Tween 80 and gently
probed to dislodge the conidia [10]. Aer the set-
tling of larger particles, suspensions were adjusted
by nephelometry and diluted as necessary to obtain
the inoculum density of ~1×106 cfu/ml.
Determination of MIC and MBC/MFC
e antimicrobial activity of the petitgrain oil was
assayed with dilution broth method according to
the EUCAST guidelines [11]. Double series of petit-
grain oil dilutions ranging from 0.078 to 80 mg/ml
(v/v) were prepared for the respective bacteria in
Mueller-Hinton Broth (MHB, Merck) and for fungi
in Sabouraud Broth (SB, Merck) [12] supplemented
with 0.002% (v/v) of Tween 80 (Sigma-Aldrich).
Anegative control (medium and inoculum) was ad-
ditionally prepared for each experimental series. Fi-
nally, each test tube contained 2 ml of medium with
the appropriate concentration of petitgrain oil and
0.1 ml of inoculum.
e 100 µl of inoculum containing approxi-
mately 1 × 107 cfu/ml of bacteria were transferred
in MBH so that the nal concentration in each
tube was ~ 5.0 × 105 cfu/ml and 100 µl of the in-
oculum containing approximately 1×106 cfu/ml of
fungi were added in SB, so that the nal inoculum
concentrations were in the range of 0.4 × 104 –
5.0 × 104 cfu/ml [10]. Apositive control (contain-
ing inoculum without petitgrain oil) was includ-
ed in each series. Aer incubation at appropriate
temperature and time (bacteria at 37°C for 24 h,
yeast at 28°C for 48 hours, and molds at 28°C for
72 hours), the MIC was checked. e MIC is the
lowest concentration of petitgrain oil at which the
bacteria and fungi failed to grow, no visible chang-
es were detected in the broth medium.
In order to evaluate the minimum bacteri-
cidal/fungicidal concentration (MBC/MFC) of
petitgrain oil, 100 µl of each culture from tubes
in which microbial growth was not observed was
spread on Mueller-Hinton Agar (MHA, Merck)
for bacteria and on Sabouraud Agar (SA, Merck)
plates for fungi. Plates were incubated at appropri-
ate temperature and time (bacteria at 37°C for 24 h,
yeast at 28°C for 48 hours, and molds at 28°C for
72 hours).e complete absence of growth of bac-
terial or fungal colonies on the agar surface at the
lowest concentration of petitgrain oil was dened
as the minimum bactericidal concentration (MBC)
or minimum fungicidal concentration (MFC). e
evaluation of MIC and MBC/MFC was carried out
in triplicate.
Ethical approval: e conducted research is not re-
lated to either human or animal use.
RESULTS AND DISCUSSION
Characterization of essential oil
e chemical composition of petitgrain oil analyzed
using the GC/FID is presented in table 1. Twenty
chemical compounds were determined in the ana-
lyzed essential oil. e percentage content of its
components was 98.90%. e highest percentage of
content characterized was of monoterpenoid com-
pounds and oxygenated monoterpene hydrocar-
bons, which formed 98.01% of the oil. e majority
of the identied compounds belong to the oxygen-
ated monoterpene hydrocarbons: linalyl acetate
(48.06%), linalool (26.88%), α-terpineol (5.74%),
geranyl acetate (3.92%), geraniol (3.05%), and gera-
nial (2.44%).
Based on chemical analysis of petitgrain oil pre-
sented in this study and several other previous ex-
aminations, it can be concluded that oxygenated
monoterpenes are present at higher concentrations
than monoterpene hydrocarbons in the oil. eir
content constitutes over 90% of total composition of
petitgrain oil. e major components of petitgrain oil
are linalyl acetate, linalool, α-terpineol, and geranyl
acetate [7, 13]. Petitgrain oils originating from vari-
ous areas of the world dier in the content of main
components which may be due to dierent plant gen-
otype, climate, and soil conditions [13, 14]. Linalool
was the predominant among the main components
of the Tunisian oil (62.57−22.35%), with lower con-
tent of linalyl acetate (25.38–5.64%) and α-terpineol
(3.0–15.69%) [15]. e petitgrain oil originating from
Greece contained 88.09% oxygenated monoterpenes,
mostly linalool (58.21%). Apart from these, the Greek
petitgrain oil contained neryl acetate and trans-β-
ocimene, which were not determined in the com-
position of commercial petitgrain oil. On the other
hand, Sicilian petitgrain oil was characterized with
higher values of linalyl acetate (0.3–73.1%) and lower
linalool content (8.7–16.7%) [16]. Petitgrain oil from
Turkey has ahigh content of oxygenated compounds
(89.6%) with linalyl acetate (50.1%) and linalool
(24.8%) being the main components [3]. e chemi-
cal composition of petitgrain oil is further inuenced
21
Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil
Vol. 63 No. 4 2017
by the procedure and the time of extraction from the
plant raw material [15]. Ellouze and Abderrabba [15]
determined that during hydrodistillation of essential
oil from C. aurantium leaves for 180 min, adecline in
monoterpene content was observed (89.93–47.32%
in 15 min and 165 min, respectively), while the ses-
quiterpene content increased. Ellouze et al. [4] have
found the eect of seasonal variations of leaves col-
lection on chemical composition of essential oils.
e essential oils obtained from the fresh leaves of
C. autrantium L. ssp. aurantium, which were gathered
during January noted 14 components only, while the
July one presented 35 among the 46 identied com-
ponents.
Antimicrobial activity of petitgrain oil
e evaluation of antimicrobial activity of petitgrain
oil was conducted in compliance with the standard-
ized techniques, determining the minimum inhibi-
tory concentrations (MIC) that inhibits the growth
of microbes and minimum bactericidal and fungi-
cidal concentrations (MBC/MFC). e assessment
of the eciency of petitgrain oil was conducted for
the reference test strains of bacteria and fungi.
e MIC and MBC/MFC of petitgrain oil are
presented in table 2. e highest inhibitory activ-
ity of petitgrain oil was determined for bacteria,
including Gram-positive bacteria. e MIC values
Table 1.
Gas chromatographic composition (% peak area) of petitgrain (Citrus aurantium L. var. amara) essential oil
Compound RIaRIbRI b range Mean ±SDc
α-Pinene 1028 1025 1008–1039 0.16±0.02
β-Pinene 1113 1110 1085–1130 1.14±0.12
Sabinene 1124 1120 1098–1140 0.27±0.37
β-Myrcene 1166 1161 1140–1175 1.62±0.15
Limonene 1203 1198 1178–1219 1.40±0.12
1,8 Cineole 1209 1211 1186–1231 0.04±0.03
γ-Terpinene 1248 1245 1222–1266 1.29±0.10
p-Cymene 1273 1270 1246–1291 0.06±0.01
α-Terpinolene 1278 1282 1261–1300 0.23±0.02
Linalool 1540 1543 1507–1564 26.88±0.88
Linalyl acetate 1557 1554 1532–1570 48.06±1.05
β-Caryophyllene 1593 1588 1570–1685 0.96±0.04
Isoborneol 1657 1659 1635–1675 0.09±0.01
α-Terpineol 1681 1694 1659–1724 5.74±0.11
Borneol 1687 1699 1653–1728 0.35±0.01
Geranial 1722 1725 1680–1750 2.44±0.12
Geranyl acetate 1731 1751 1728–1772 3.92±0.23
Nerol 1795 1794 1752–1832 1.36±0.31
Geraniol 1826 1839 1795–1865 3.05±0.04
Caryophyllene oxide 1955 1986 1936–2023 0.08±0.02
Identied components [%] 99.14
Grouped components
Monoterpene hydrocarbons 6.17
Oxygenated monoterpene
hydrocarbons
91.93
Sesquiterpene hydrocarbons 0.96
Oxygenated sesquiterpene
hydrocarbons
0.08
Notes: RIa – etention index relative on HP-20M capillary column; RIb – average retention indices on polar column reported by Babushok et al. [13],
RIb range – range of retention indices on polar column reported by Babushok et al. [13]; SDc – standard deviation, n=3
22
Gniewosz, K. Kraśniewska, O. Kosakowska, K. Pobiega, I. Wolska
of petitgrain oil for B. subtilis and S. aureus bacte-
ria remained in the range of 0.63–1.25 mg/ml, while
the MBC values were 1.25–5.0 mg/ml. e Gram-
negative bacteria strains of E. coli and S. ser. Enter-
itidis were less susceptible to the eect of petitgrain
oil. e MIC and MBC values remained in the range
between 2.5 and 5.0 mg/ml.
e weakest antimicrobial activity of petitgrain
oil was observed against fungi. e MIC of petit-
grain oil for the inhibition of P. expansum and A. ni-
ger mold was 1.25 mg/ml. In the case of S. cerevisiae
yeast, the MIC and MFC values were 2.5 mg/ml and
5.0 mg/ml, respectively. e weakest fungicidal ef-
fect of petitgrain oil was observed for C. krusei.
e antimicrobial activity of essential oils is typi-
cally linked to their main components [17]. Petitgrain
oil is characterized with high content of oxygenated
monoterpenes, mostly linalyl acetate (48.06%) and
linalool (26.88%) (tab. 1). e mechanism of anti-
microbial activity of these compounds stems from
their lipophilic character, creating strong anity to
plasma membranes of microorganisms and strong
toxicity [18]. Linalyl acetate and limonene preferen-
tially divide from an aqueous phase and enter into
the plasma membrane structures. Accumulation of
linalyl acetate and limonene in the membrane dam-
ages the composition and structure of plasma mem-
brane, causing the increase of plasma membrane
uidity and leakage of intracellular molecules from
the cell. Blaskó et al. [18] conrmed that these dis-
turbances in plasma membrane determine the cell
death of Candida albicans. Similar eect is attributed
to 1,8-cyneol, which modies the characters of cel-
lular membranes by rupturing the hydrogen bonded
network at the membrane-water interface. Mono-
terpene alcohols (α-terpineol and 1,8-cineole) have
asimilar eect on microbial cells, i.e., they interact
with plasma membranes [12]. At relatively low con-
centrations, such interactions may lead to changes
like respiratory inhibition and altered permeability
[19], and at higher concentrations they may lead to
acomplete loss of homeostasis, plasma membrane
damage, and death. Monoterpene alcohols are con-
sidered to be ecient against microbes due to their
relatively high water solubility and the presence of
the alcohol moiety [19].
ere are published data from previous studies
regarding the antimicrobial activity of essential oils
produced from owers and fruits of Citrus auran-
tium L. var. amara, but no data is available on the
antimicrobial activity of petitgrain oil. Ellouze et
al. [4] registered that petitgrain oils were most ef-
fective against Gram-positive bacteria (S. aureus
and L. monocytogenes were more sensitive to the
essential oils), and weak against Gram-negative
strains. Essential oil from bitter orange owers ex-
amined by Hsouna et al. [17] inhibited the growth
of both Gram-positive and Gram-negative bacteria
in the concentration range from 0.312–2.5 mg/ml
and 0.625–2.5 mg/ml, respectively. e fungistatic
activity of this essential oil was even stronger. e
MIC for molds of the genus Aspergillus was in the
range of 0.078–1.25 mg/ml and for Fusarium, it
was 0.156–1.25 mg/ml. A comparison of MIC val-
ues of both the essential oils from Citrus aurantium
L. var. amara demonstrated that antimicrobial activ-
ity of the oil from owers was stronger than the oil
from leaves and twigs (petitgrain) of the plant. e
ower oil contained considerably higher amounts of
monoterpene hydrocarbons (36.2%), sesquiterpene
hydrocarbons (4.1%), and oxygenated sesquiterpene
hydrocarbons (26.2%), which were present in the
examined petitgrain oil at 6.17, 0.96, and 0.08%, re-
spectively. Strong fungicidal activity is also exhibited
Table 2.
e antimicrobial activity of petitgrain essential oil
Microorganism MIC MBC
mg/ml
B. subtilis ATCC 6633 0.63 1.25
S. aureus ATCC25923 1.25 5
Bacteria E. coli ATCC 25922 2.5 5
S. ser. Enteritidis ATCC 13076 5 5
S. cerevisiae ATCC 9763 2.5 5
A. niger ATCC 9142 1.25 40
Fungi P. expansum ATCC 7861 1.25 80
C. krusei ATCC 14243 40 >80
23
Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil
Vol. 63 No. 4 2017
by limonene and (E)-nerolidol, which were present
in the ower essential oil at concentrations 27 and
17.5%, respectively. In the petitgrain oil, limonene
was present only at aconcentration of 1.4%.
e unequal eect of the oil is linked to the dif-
ference in its composition of active compounds.
Monoterpenes might be responsible for the anti-
fungal eect of the petitgrain oil. Hammer et al.
[19] demonstrated monoterpene activity toward
yeasts and lamentous fungi. In the study, it was
determined that Candida albicans and C. tropi-
calis are characterized by high susceptibility to
monoterpene mixtures. High antifungal activity is
also exhibited by terpinen-4-ol, α-pinen, β-pinen,
1,8-cyneol, linalool, and 4-terpineol. is mixture
of terpenoid metabolites inhibits the growth of
dermatophytes, such as Trichophyton mentagro-
phytes, T. rubrum, and Microsporum gypseum, and
also the lamentous fungi Aspergillus niger and
A. avus [20].
CONCLUSION
Essential oils are natural plant products containing
mixture of components and thus having multiple an-
timicrobial properties. In the petitgrain essential oil,
the GC/FID analysis allowed to identied 20 com-
pounds. e major components of essential oil was
linalyl acetate (48.06%) and linalool (26.88%).
e results of the study may suggest eective an-
timicrobial activity of petitgrain essential oil. It was
found that tested oil eectively inhibits the growth
of Gram-positive bacteria. Less sensitive to the in-
hibitory activity were Gram-negative bacteria and
fungi. is nding also highlights the potential use
of the petitgrain essential oil as inhibitors of food
spoilage and pathogenic microorganisms.
ACKNOWLEDGEMENTS
e work was funded as statutory research by the
Department of Biotechnology, Microbiology and
Food Evaluation, Faculty of Food Science, Warsaw
University of Life Sciences-SGGW.
Conict of interest: Authors declare no conict of interest.
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Gniewosz, K. Kraśniewska, O. Kosakowska, K. Pobiega, I. Wolska
Skład chemiczny iaktywność przeciwdrobnoustrojowa olejku eterycznego
petitgrain (Citrus aurantium L. var. amara)
MAŁGORZATA GNIEWOSZ1*, KAROLINA KRAŚNIEWSKA1,
OLGA KOSAKOWSKA2, KATARZYNA POBIEGA1, IWONA WOLSKA1
1Katedra Biotechnologii, Mikrobiologii iOceny Żywności
Szkoła Główna Gospodarstwa Wiejskiego wWarszawie
ul. Nowoursynowska 159C
02-776 Warszawa
2Katedra Roślin Warzywnych iLeczniczych
Szkoła Główna Gospodarstwa Wiejskiego wWarszawie
ul. Nowoursynowska 159C
02-776 Warszawa
*autor, do którego należy kierować korespondencję: malgorzata_gniewosz@sggw.pl
Streszczenie
Wstęp: Olejek eteryczny petitgrain ze względu na niewielki koszt idostępność na rynku jest często stosow-
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Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil
Vol. 63 No. 4 2017
Cel: Zbadano skład chemiczny iaktywność przeciwdrobnoustrojową handlowego olejku petitgrain.
Metodyka: Identyfikację chemicznych składników olejku petitgrain wykonano przy użyciu chromatografii
gazowej (GC). Minimalne stężenia hamujące (mic) oraz minimalne stężenia bakteriobójcze/grzybobójcze
(MBC/MFC) zostały oznaczone metodą makrorozcieńczeń wobec referencyjnych szczepów bakterii igrzy-
bów.
Wyniki: Zidentyfikowano dwadzieścia kompotentów. Olejek petitgrain zawierał najwięcej utlenionych wę-
glowodorów monoterpenowych (98.01%), agłównymi składnikami były: octan linalilu (48,06%) ilinalool
(26,88%). MIC/MBC olejku petitgrain wobec bakterii były wgranicach 0,63–5,0/1,25−5,0 mg/ml, awobec
grzybów były wzakresie 1,25–40/5,0–80 mg/ml.
Wnioski: Olejek petitgrain miał większą aktywność przeciwbakteryjną niż przeciwgrzybiczną. Spośród ba-
danych bakterii Bacillus subtilis, aspośród grzybów Aspergillus niger iPenicillium expansum były najsilniej
hamowane przez olejek petitgrain.
Słowa kluczowe: petitgrain, olejek eteryczny, skład olejku eterycznego, aktywność przeciwdrobno-
ustrojowa
