LWT - Food Science and Technology 69 (2016) 522e528

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Role of Curcuma longa L. essential oil in controlling the growth and

zearalenone production of Fusarium graminearum
Kalagatur Naveen Kumar a, Mudili Venkataramana b, *, Joseph Anthuvan Allen b,
Siddaiah Chandranayaka c, Harishchandra Sreepathi Murali a, Harsh Vardhan Batra a
a
b
c

Food Microbiology Division, Defence Food Research Laboratory, Mysore, 570011, Karnataka, India
DRDO-BU-Centre for Life Sciences, Coimbatore, 641046, Tamil Nadu, India
Department of Biotechnology, Mysore University, Mysore, 570005, Karnataka, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 September 2015
Received in revised form
21 December 2015
Accepted 3 February 2016
Available online 11 February 2016

The present study was aimed to determine the inhibitory effects of Curcuma longa L. essential oil (CLEO)
against the growth and zearalenone (ZEA) production of Fusarium graminearum. Chemical proling of
CLEO was carried out by GC-FID analysis and major compound was ar-turmerone (53.10%). Antifungal
activity of CLEO was assessed by using micro-well dilution method, and minimum inhibitory (MIC) and
minimum fungicidal concentrations (MFC) were determined as 2450 and 3300 mg/mL, respectively.
Interestingly, ROS played a major role in antifungal activity as evidenced by reactive oxygen species
estimation from CLEO treated fungal cultures. Further, Scanning electron microscopy observations were
carried at MIC and MFC concentrations. Results indicated that, CLEO signicantly affected the
morphology of mycelia and spore structures compared to untreated control culture. Effects of CLEO on
fungal biomass as well as ZEA production were assessed by dry-weight and UHPLC analysis, respectively.
Results indicated that, fungal biomass and zearalenone production was completely inhibited at 3500 and
3000 mg/mL, respectively. The results of the study conclude that, CLEO may nd a potential application in
the food industry to control the fungal infestation and ZEA contamination.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Curcuma longa L. essential oil
Fusarium graminearum
Zearalenone
Scanning electron microscope
Reactive oxygen species

1. Introduction
Fungal infestation and mycotoxin contamination are the biggest
threat to food and feed industry globally (Pitt & Hocking, 2009). It
was estimated that ~25% of agricultural products are contaminated
with fungi and their toxic metabolites (McKellar & Lu, 2003).
Fungal infestation leads to the deterioration in nutritional quality,
altered color, texture and rancidity thus to spoilage of food
(Dhingra, Mizubuti, Napoleao, & Jham, 2001; Morgavi, Wiseman, &
Riley, 2007 and Magan, Aldred, Mylona, & Lambert, 2010). In
addition to these, presence mycotoxins in food and feed matrices
were possessing great threat to humans and animal husbandry.
Recently, Zearalenone (ZEA) also known as F-2 toxin and nonsteroidal
estrogenic
mycotoxin
produced
by
Fusarium

* Corresponding author. Toxicology and Immunology Division, DRDO-BU-Centre

for Life Sciences, Bharathiar University Campus, Coimbatore, 641046, Tamil Nadu,
India.
E-mail addresses: ramana.micro@gmail.com (M. Venkataramana), drmuralihs@
gmail.com (H.S. Murali).
http://dx.doi.org/10.1016/j.lwt.2016.02.005
0023-6438/ 2016 Elsevier Ltd. All rights reserved.

graminearum, Fusarium culmorum, Fusarium equiseti, and Fusarium

crookwellense possess great attention towards researchers
(Zinedine, Soriano, Molto, & Manes, 2007). Several toxic properties
of ZEA were documented, which includes, immunosuppression,
neurotoxic,
hepatotoxic
and
carcinogenic
in
nature
(Venkataramana et al., 2014; Zinedine et al., 2007). Moreover, ZEA
is the known mycotoxin accountable for abortions, expansion of
gonads and sometimes causes hyperestrogenism in females
(Zinedine et al., 2007). International Agency for Research on Cancer
(IARC) proven the carcinogenic property of ZEA in laboratory animals and categorized as a Group 3 carcinogen (IARC, 1999).
Assessing the risk factors of ZEA to humans and animals several
countries implemented maximum allowed limits for ZEA in food
and feed matrices. The European Union (EU) established a
maximum allowed limit for ZEA was 100 mg/kg in unprocessed
cereals omitting maize (European Commission 2007). The joint
FAO/WHO Expert Committee on Food Additives (JECFA) also
implemented Provisional Maximum Tolerable Daily Intake (PMTDI)
as 0.5 mg/kg body weight (JECFA, 2014).
In context of the present study, F. graminearum is well known

K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

ZEA producing fungi because of its potential to adapt and grow in

different climatic conditions with diverse cereal grains (Bernhoft,
Torp, Clasen, Les, & Kristoffersen, 2012). Due to the well-known
toxic ability and pathogenic nature, there is a need of control
measures for the successive management of F. graminearum and
toxins from cereal grains intended for consumption. Unfortunately
the use of synthetic fungicides is toxic to the environment, when
excessively used and accumulated in the environment (Reimann &
Deising, 2000). Thus, there is need to propose safe, cost effective
and easy to use strategies to control the growth and mycotoxin
production by F. graminearum from cereals.
Due to its potential antioxidant and antimicrobial features,
essential oils of aromatic plants attained much interest in development for bio-control herbal agents. FDA as well as Environment
Protection Agency (EPA) declared the use of essential oils for safe
application in food industry (Burt, 2004 and da Cruz Cabral, Pinto, &
Patriarca, 2013). Curcuma longa L. widely used as spice for avor
and color in South Asian countries. The essential oil of C. longa L.
(CLEO) rhizomes are rich in several aromatic medicinally important
compounds and possess potential biological features such as antioxidant, antimicrobial, anti-inammatory, antihepatotoxic activities and etc (Gounder & Lingamallu, 2012; Sindhu, Chempakam,
Leela, & Bhai, 2011 and Singh et al., 2010). Owing to the medicinal properties of CLEO, it was used to control the growth of several
fungal species including, Aspergillus avus, Fusarium semitectum,
Curcuma gloeosporioides and Curcuma musae, Curcuma albicans and
A. niger (Dhingra, Jham, Barcelos, Mendona, & Ghiviriga, 2007,
Singh et al., 2011). In a recent study, Ferreira et al., (2013) reported that, the antifungal and aatoxin inhibitory activity of
C. longa essential oil on A. avus and similar ndings were also
made by Sindhu et al., (2011). However, to date, there is no such
reports were made the use of CLEO against the control of
F. graminearum and ZEA production.
Hence the present study was aimed to investigate the inhibitory
effects of CLEO on F. graminearum growth and ZEA production.
Chemical proling of CLEO was carried out using GC-FID analysis.
Effects of CLEO on growth of F. graminearum were studied using
micro-well dilution, scanning electron microscope and fungal
biomass determination methods. Effects on ZEA production were
analyzed using UHPLC analysis and also the role ROS production in
fungal control was investigated.

2. Materials and methods

523

2.3. Chemical prole of C. longa L. essential oil

2.3.1. GC-FID analysis
The chemical prole of CLEO was determined by GC-FID analysis
using a PerkinElmer Clarus 600 C (Waltham, USA) equipped with
ame-ionization detector (FID) and DB-5MS fused silica column
(30 m  0.25 mm; 0.25 mm lm thickness) included with TurboMass software application. Working conditions were as follows:
Helium was used as a carrier gas with a ow rate of 1 mL per min,
and temperature of the injector at 250
C and sensor at 280
C were
used. The column temperature was set at 40
C for 3 min and linearly increased by 4
C/min up to 280
C. Mass spectra were
operated in EI mode (70 eV) with scanning speed of 0.5 scans/sec in
a range of m/z 40e450. CLEO was diluted in acetone (10 mL/mL) and
1 mL solution was injected in a split-mode of 1:30. Proling of CLEO
was determined by comparing their retention indices and mass
spectra with respect to a homologous series of normal alkanes
(Adams, 2007) and mass spectra computer library of the NIST05.LIB/Wiley 8th Edition, respectively. Percentage composition of
chemical compounds was computed from GC peak areas without
devoid of FID response factor.
2.4. Antifungal activity of C. longa L. essential oil
2.4.1. Fungi and culture conditions
Antifungal activity of CLEO was carried out using F. graminearum
(MTCC 1893) culture. Fungi was grown on SDA at 28
C for one
week and spores were collected using peptone water with 0.001%
Tween 80. The spore number was counted using haemocytometer
and adjusted to 1  106 spores per mL.
2.4.2. Micro-well dilution method
The Minimum inhibitory (MIC) and fungicidal concentrations
(MFC) of CLEO were determined by micro-well dilution method in
accordance with CLSI (2008). A volume of 10 mL spore suspension
(1  106 spores per mL) was added to 96-well microtiter plate and
mixed with various concentrations of CLEO (500e3500 mg/mL) and
nal volume was adjusted to 100 mL with SDB and incubated at
28
C for 3 days. The wells without CLEO, and with nystatin and
amphotericin B were as control and reference standards, respectively. MIC value was determined as the minimum concentration of
CLEO at which no visible growth observed. After 3 days of incubation, 10 mL of sample from each well was inoculated into SDA
plate and incubated for 3 days at 28
C. MFC value was determined
as the minimum concentration at which no detectable growth was
observed.

2.1. Chemicals and materials

Rhizomes of C. longa L. were obtained from the Ooty, Tamil
Nadu, India. Sabouraud dextrose agar (SDA) and broth (SDB) were
obtained from HiMedia (Mumbai, India). Zearalenone and other
chemicals were purchased from SigmaeAldrich (Bangalore, India).

2.2. Extraction essential oil from rhizomes of C. longa

Rhizomes of C. longa L. were dried under shade at room temperature for four weeks and ground into powder (500 g) and
essential oil was extracted using Clevenger type apparatus followed
by European Pharmacopeia (1997). Extracted oil (CLEO) was dried
over sodium sulfate anhydrous to remove moisture and stored in
amber glass vial at 4
C. The percentage of the essential oil yield was
calculated as the weight of the essential oil divided by the weight of
the dry rhizomes.

2.4.3. Scanning electron microscopic observation

The effect of CLEO on fungi was analyzed by SEM method
following the technique of Yamamoto-Ribeiro et al., (2013) with
minor modications. A mycelia was collected from a seven day old
culture and transferred onto SDA slide prepared with MIC and MFC
concentration of CLEO and incubated for 7 days at 28
C. Following
the incubation period, the mycelia was xed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 6.5) and used for
SEM analysis. The mycelial sample was xed on dual carbon adhesive tape and dehydrated out in CO2 and sputter coated with
gold, and observed under SEM (Quanta 200, FEI, USA) at 20.0 kV in
environmental mode.
2.5. Effects of CLEO on fungal biomass and ZEA production
2.5.1. Liquid broth culture
The nal aim of the present study was to know the inhibitory activity
of CLEO on ZEA production in F. graminearum. Various

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K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

concentrations of CLEO and 10 mL of spore suspension (1  106 spores

per mL) were inoculated into 100 mL SDB in 250 mL of Erlenmeyer
ask and the ask with spore suspension and without CLEO was
referred as a control, and asks were incubated under shaking
condition (140e160 rpm) for 14 days at 28
C. Following incubation,
the mycelia was collected in a pre-weighed Whatman no.1 lter
paper and dried at 60
C for 24 h and fungal biomass was determined
(Denver instruments, India). ZEA was extracted from liquid broth

n
~ ez-Vea et al., (2011) with minor modications. The
followed by Iba
broth was blended with equal volume of acetonitrile (1:1, v/v) at
145 rpm and 15 mL was passed through ZEA specic immunoafnity
column (Vicam, Milford, USA), pre-conditioned with 10 mL of
phosphate-buffered saline pH 7.4 (PBS) for 5 min. Following, the
column was washed with 5 mL of PBS and distilled water, and air
dried. The ZEA was eluted with 5 mL of acetonitrile by maintaining
contact between antibodies for 5 min. The collected fraction was
dried completely at 60
C and redissolved in 1 mL of acetonitrile and
used for the UHPLC determination of ZEA using Nexara UHPLC system (Shimadzu, Kyoto, Japan) attached with C18 column and uorescence detector. The analysis was carried out in reverse-phase at
excitation and emission wavelength of 334 nm and 450 nm,
respectively. Acetonitrile and water (50:50 v/v) was mobile phase
with a ow rate of 1 mL/min, and injection volume was 25 mL for both
standard ZEA and test sample. The calibration curve for standard ZEA
(100 nge500 mg/mL) was constructed with peak area versus concentration and the concentration of ZEA in test samples were
determined against the calibration curve.

Table 1
Chemical prole of C. longa L. essential oil (CLEO) determined by GC-FID analysis.
S. No

Compounda

RIb

Quantity (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Total

a-Pinene

933
971
977
989
1003
1017
1021
1024
1027
1057
1099
1143
1166
1187
1288
1349
1372
1420
1455
1474
1480
1491
1506
1517
1525
1551
1623
1634
1639
1640
1654
1678
1673
1712
1762

1. 70
1.22
1.30
1.83
2.15
3. 28
1.0
3.15
4.39
2.70
1.17
0.32
0.59
1.08
0.46
0.06
0.85
1.80
0.28
4.81
0.10
0.19
0.31
0.08
0.03
0.91
6.15
6.42
53.10
0.30
0.15
0.19
0.16
0.28
0.05
97.58%

Sabinene
b-Pinene
Myrcene
a-Phellandrene
a-Terpinene
p-Cymene
Limonene
b-Phellandrene
g-Terpinene
Linalool
Camphor
Borneol
a-Terpineol
Bornyl acetate
a-Cubebene
a-Ylangene
b-Caryophyllene
a-Humulene
ar-Curcumene
g-Muurolene
b-Selinene
a-Muurolene
g-Cadinene
d-Cadinene
Elemol
a-Turmerone
beTurmerone
ar-Turmerone
Cubenol
a-Cadinol
Cadalane
Bulnesol
Cis-Thujopsenal
Cyclocolorenone

Compounds are listed in order of their elution.

Retention indices of compounds experimentally determined on DB-5MS column
based on n-alkanes (C-9 to C-24).
b

2.5.2. Maize culture

Maize grains were obtained from the regional agricultural
market, Mysore, India. Various concentrations of CLEO and 10 mL of
spore suspension (1  106 spores per mL) were inoculated into
100 g of pre-sterilized maize grains in 250 mL of Erlenmeyer ask,
and the ask with spore suspension and without CLEO was referred
as a control, and incubated at 28
C for 14 days. Following the incubation period, the grains were ground into ne powder and
dissolved in 500 mL of acetonitrile-water (60:40, v/v) and centrifuged at 6000 rpm for12 min. The supernatant was ltered through
Whatman no.1 lter paper and 15 mL was passed through ZEA
specic immunoafnity column, pre-conditioned with 10 mL of PBS
for 5 min. Following, the column was washed with 5 mL of PBS and
distilled water, and air dried. The ZEA was extracted with 5 mL of
acetonitrile by maintaining contact between antibodies for 5 min.
The ltrate was completely dried at 60
C and dissolved in 1 mL of
acetonitrile. A volume of 25 mL was injected into UHPLC and ZEA
concentration was determined from the standard calibration curve
of ZEA.

2.6. CLEO induced ROS production in F. graminearum

A volume of 10 mL spore suspension (1  106 spores per mL) was
added to various concentrations of CLEO (500e2000 mg/mL) in 96well microtiter plate, nal volume was adjusted to 100 mL with SDB
and was incubated for 3 days at 28
C. Followed by incubation,
wells were added with 5 mM dichloro-dihydro-uorescein diacetate (DCFH-DA) for 20 min. The absorbance was recorded using a
multimode plate reader (Biotek-H1M synergy, Germany) and
photo-micrographs were documented using EVOS uorescence
microscope (Life Technologies, USA) at excitation and emission
wavelength of 495 nm and 550 nm, respectively. The wells alone
with a spore suspension were referred as a control, and results were
expressed as percentage release of reactive oxygen species with
respect to the untreated control sample.

Reactive oxygen species %

Rt
 100
Rc

Where, R(t) and R(c) were absorbance of test sample and control.

Fig. 1. Minimum inhibitory (MIC) and minimum fungicidal concentration (MFC) of C.

longa essential oil (CLEO), nystatin and amphotericin B against F. graminearum. Data
were analyzed by one-way ANOVA in accordance to the Tukey's test (n 6) and p
value was signicant (<0.05).

K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

525

Fig. 2. Scanning electron microscopic observation of hyphae and spores of F. graminearum exposed with MIC and MFC concentration of C. longa essential oil (CLEO), (a) CLEO
untreated control hyphae, (b) hyphae exposed at a concentration of 1900 mg/mL (MIC), (c) hyphae exposed at a concentration of 3300 mg/mL (MFC), (d) CLEO untreated control
spores, (e) spores exposed at a concentration of 1900 mg/mL (MIC) and (f) spores exposed at a concentration of 3300 mg/mL (MFC) of CLEO.

2.7. Statistical analysis

All experiments were carried out independently for six times
and the data were analyzed using one-way ANOVA following the
Tukey's test. The graphical representation of the data was done
using GraphPad Prism 5.0 (GraphPad Software, Inc. USA).

compounds. In the present study no new compounds were identied, however the percentage composition of compounds varied
signicantly. This can be explained by, the chemical prole of
essential oils depends on genetics, nature of the raw material (dry
or fresh) and part of the plant, harvesting time, geographical conditions, luminosity and analytical method used for extraction of oil
(Gobbo-Neto & Lopes, 2007).

3. Results and discussion

3.2. Antifungal activity
3.1. Chemical prole
The percentage yield of essential oil obtained from the rhizomes
of C. longa (CLEO) was 2.60 0.04. The chemical prole of CLEO was
determined by GCeFID analysis and the results are presented in
Table 1. A total of 35 compounds were identied accounting for
97.58%, and major compounds were ar-turmerone (53.1%),
beturmerone (6.42%), a-turmerone (6.15%), ar-curcumene (4.81%),
b-phellandrene (4.39%), a-terpinene (3.28), limonene (3.15%), gterpinene (2.70%) and a-phellandrene (2.15%). Obtained results in
the present study were in line with Singh et al., (2010) who reported the major constituents of C. longa essential were arturmerone (24.4%), alpha-turmerone (20.5%) and beta-turmerone
(11.1%) in fresh rhizome and ar-turmerone (21.4%), alphasantalene (7.2%) and ar-curcumene (6.6%) in dry rhizome. In
another study, Gounder and Lingamallu (2012) reported arturmerone (30.3%), a-turmerone (26.5%) and b-turmerone (19.1%)
were major compounds. Similar ndings were made by Angel,
Menon, Vimala, & Nambisan (2014) who reported, ar-turmerone
(49.8%), beturmerone (7.9%) and a-turmerone (9.1%) as major

In the present study, MIC and MFC values of CLEO on

F. graminearum were determined as 2450 and 3300 mg/mL,
respectively (Fig. 1). The obtained MIC and MFC values of CLEO were
slightly higher compared to reference standards nystatin
(MIC 2200 and MFC 3000 mg/mL) and amphotericin B
(MIC 1400 and MFC 1900). The effect of CLEO on
F. Graminearum mycelial morphology and spore structure was
observed by SEM. Mycelia exposed to MIC and MFC concentrations
of CLEO showed distinct changes in the morphology compared with
CLEO untreated control (Fig. 2). Signicant changes in mycelia
structure, including cell wall, membrane integrity were observed.
In control, hyphae were turgid with a smooth cell wall (Fig. 2a),
whereas the hyphae treated with CLEO exhibited reduced cytoplasmic content and membrane integrity and exhibited craters at
both the MIC and MFC concentrations (Fig. 2b and c). Similarly,
spores also showed signicant variations in morphology compared
to CLEO untreated control (Fig. 2). The control spores were turgid,
smooth and round in shape (Fig. 2d), whereas the spores treated
with MIC and MFC values of CLEO showed distinct morphology

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K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

Fig. 3. Effect of C. longa essential oil (CLEO) on fungal biomass and zearalenone (ZEA) production of F. graminearum. (a) Effect of various concentrations of CLEO on fungal biomass,
(b) calibration curve for ZEA (peak area versus concentration of ZEA), (c) effect of CLEO on production of ZEA in Sabouraud dextrose broth (SDB) and (d) effect of CLEO on ZEA
production in maize grains. Data were analyzed by one-way ANOVA in accordance to the Tukey's test (n 6) and p value was signicant (<0.05).

such as disrupted, wrinkled and dispersed structure (Fig. 2e and f).

These results were in accordance to the previous study of
Yamamoto-Ribeiro et al., (2013) who reported the micro morphological effects of Zingiber ofcinale essential oil, cinnamon oil and
cinnamaldehyde on mycelia of Fusarium verticillioides. The results
of the present study clearly revealed that the antifungal activity of
CLEO on F. graminearum are in line with the previous reports of
Dhingra et al., (2007), Singh et al., (2011) and Sindhu et al., (2011).
Reported antifungal activity of CLEO was attributable to the
presence of terpene and aromatic compounds, including arturmerone, beturmerone, a-turmerone, ar-curcumene, b-phellandrene, a-terpinene, limonene, g-terpinene, a-phellandrene etc.
These compounds are permeable into the cell through the cell wall
and cytoplasmic membrane, thus to alter the cell membrane
integrity. This cascade of events leads to the impairment of membrane uidity, leakage of the cytoplasmic contents, and also causes
osmotic and enzymatic regulation disturbances that are crucial for
ATP synthesis, cell growth and proliferation of fungi (Dalleau,
s, Berjeaud, & Imbert, 2008 and da Cruz et al., 2013).
Cateau, Berge

3.3. Effects of CLEO on fungal biomass and ZEA production

3.3.1. In vitro broth culture studies
To know the effects of CLEO on fungal biomass and ZEA production, in vitro broth (SDB) culture experiments were carried out.
The fungal biomass was decreased with increasing the concentration of CLEO in SDB and effect of CLEO on fungal biomass was
represented in Fig. 3a. The fungal biomass of the untreated CLEO
control sample was recorded as 65.0 mg/100 mL, whereas the
mycelial biomass of CLEO treated samples were 63.66, 53.91, 41.26,
27.21 and 16.83 mg/100 mL at CLEO concentrations of 500, 1000,
2000, 2500 and 3000 mg/mL, respectively. Interestingly, no fungal
growth was observed at 3500 mg/mL or above concentrations of
CLEO. The other hand, the concentration of ZEA in CLEO treated and
untreated samples were determined from the calibration curve of
standard ZEA (Fig. 3b). UHPLC analysis revealed that, ZEA concentration of the untreated control sample was 323.83 mg/100 mL,
whereas the concentration of ZEA were 294.58, 237.33, 145.91 mg
and 70.41 mg/100 mL at 500, 1000, 2000 and 2500 mg/mL concentrations of CLEO treated samples (Fig. 3c). Moreover, decrease in

K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

527

Fig. 4. Antifungal activity of C. longa essential oil (CLEO) on F. graminearum. (a) Determination of reactive oxygen species by DCFH-DA, (b) (AeD) phase-contrast image of CLEO
untreated and treated mycelia and (EeH) uorescent images of CLEO untreated and treated mycelia.

concentration of ZEA was observed with increasing the concentration of CLEO at all the studied concentrations and at MFC concentration no toxin was detected. Moreover, no ZEA was detected at
3000 mg/mL of CLEO concentration, but at this concentration a
signicant amount (16.83 mg/100 mL) of fungal biomass was
observed. These ndings clearly indicated that, CLEO was effective
in controlling the ZEA production than the growth of
F. graminearum.
3.3.2. In vivo maize experiments
To check the applicability of the present study an in vivo maize
culture experiments were included, CLEO and fungal treatments
and ZEA analysis were followed as mentioned earlier. The concentration of ZEA in CLEO untreated control maize sample was
determined as 607.16 mg/100 g, whereas the concentration of ZEA in
CLEO treated maize samples were recorded as 579.63, 450.08,
284.45 and 115.95 mg/100 g at 500, 1000, 2000 and 2500 mg/mL of
CLEO concentrations, respectively (Fig. 3d). The observed effects of
CLEO might be due to the presence of phenolic and other chemical
compounds which will be involved the stress induction lead to the
altered metabolic pathway gene regulation thus to decreased toxin
production and fungal growth. The obtained results are supported
by in vitro broth culture assays. Further studies are mandate to
know the exact mechanism of CLEO induced inhibitory effects
against the growth and ZEA production of F. graminearum.
3.4. CLEO induced ROS production in F. graminearum
In the present study to examine the cell death, the production of
reactive oxygen species (ROS) was assessed in CLEO treated
mycelium using DCFH-DA staining followed by van der Weerden,
Lay, and Anderson (2008). The hydrophobic non-uorescent
molecule (DCFH-DA) enters quickly into the cell and hydrolyses

to DCFH molecule by intracellular esterases, which further oxidized

to uorescent molecule 2-electron product 20 ,70 dichlorouorescein
(DCF). The percentage of release of reactive oxygen species
increased with increasing the concentration of CLEO in a dosedependent manner (Fig. 4a). A signicant difference in uorescence intensity was observed upon DCF-DA staining to CLEO
treated and untreated samples under a uorescent microscope
(Fig. 4b). The results of the present study indicated that, the accumulation of free radicals in CLEO treated mycelium may be
responsible for the apoptotic cell death and altered toxin metabolic
pathway gene regulation thus to decrease in toxin production. In
this context, further experiments are needed to conclude these
effects at the cellular level.
4. Conclusion
Antioxidant activity together with inhibitory properties of CLEO
against F. graminearum were studied in the present study, CLEO
might have a potential application in food industries, thus to control the food spoilage and fungal infestation. Also, it may serve as a
potential bio-control agent to control the fungal infections in cereal
crops.
Conict of interest
None.
Acknowledgment
The rst author was thankful to the University Grants Commission [Grant number: F. 2-14/2012(SA-I)] for providing the
fellowship to pursue PhD.

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K. Naveen Kumar et al. / LWT - Food Science and Technology 69 (2016) 522e528

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.lwt.2016.02.005.
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