Advances in Microbiology, 2012, 2, 310-316 Published Online September 2012 (
Saffron (Crocus sativus L.) Inhibits Aflatoxin B1
Production by Aspergillus parasiticus
Cryssa Tzanidi, Charalampos Proestos, Panagiota Markaki*
Department of Chemistry, Laboratory of Food Chemistry, National and Kapodistrian University of Athens, Athens, Greece
Email: *
Received May 2, 2012; revised June 1, 2012; accepted June 14, 2012
Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. The aim of the present
study was to investigate the effect of saffron stigmas on A. parasiticus growth and AFB1 production in Yeast Extract
Sucrose (YES) medium. AFB1 was extracted from cultures and purified with immunoaffinity columns followed by
high performance liquid chromatography (HPLC) coupled to fluorescence detector (FL) analysis. Methods’ recovery
and limit of detection were 95.3% and 0.02 ng AFB1·ml–1 of YES respectively. Results indicated that AFB1 produc-
tion in samples of YES inoculated with A. parasiticus after the addition of saffron dried stigmas (100 mg·flask–1) was
significantly lower (p < 0.05) compared to control samples (inoculated without the addition of saffron stigmas)
throughout the entire incubation period (18 days), at the same time as mycelial growth was noticeable. In addition,
mycelial growth was observed and AFB1 was detected, after the 7th day of observation in cultures with saffron alone.
Maximum production was observed on the 12th day (0.018 μg AFB1·flask–1) and on the 9th day (0.051 μg AFB1·flask–1)
for samples of YES with the addition of saffron inoculated with A. parasiticus and samples with saffron alone
(non-inoculated), respectively. In control samples (inoculated without saffron) the maximum production on the
same days 12 and 9 was 75.31 μg AFB1·flask–1 and 64 μg AFB1·flask–1 respectively. Conclusively when saffron
was added to YES inoculated with A. parasiticus, AFB1 production decreased by 99.9% compared to control
cultures without saffron addition. This inhibition can be attributed to the antioxidant capacity of saffron con-
Keywords: Aflatoxin B1; A. parasiticus; HPLC-FL Analysis; Saffron; Antioxidants
1. Introduction
Aflatoxins are mycotoxins of great significance in foods
and feeds and are mainly produced by Aspergillus (A)
flavus, A. parasiticus and A. nomius. Aflatoxin B1 (AFB1)
is usually found at highest concentrations in contami-
nated food and feed. Additionally it is regarded to be
genotoxic and the most potent liver carcinogen for many
animal species as well humans [1,2].
Saffron consists of dried stigmas of the herb Crocus
sativus L. belonging to the Iridaceae family. It is culti-
vated not only in Mediterranean countries such as Greece,
Spain and France but also in India and Iran [3]. There is
limited knowledge about the physiology of vegetative
development of saffron. It has been shown that the corm
size, water availability and cultivation conditions have a
significant effect on vegetative development of saffron.
The biomass contribution of leaves and mother corm for
maintaining the vegetative development of saffron varies
throughout the growing season. Photosynthetic rate is
consistently very high throughout the year but is reduced
in the largest corms [4]. In addition saffron is world’s
most expensive spice and several studies indicate its po-
tential as anticancer, anti-inflammatory and hypolipi-
daemic agent [5-9]. Asdaq and Inamdar [10] investigated
the potential of saffron and its constituent, crocin, as
hypolipidemic and antioxidant agent in rats and reported
that saffron was found to be superior to crocin indicating
the involvement of other potential constituents of saffron
apart from crocin for its synergistic behaviour of quench-
ing free radicals. Moreover according to Kumar et al. [11]
saffron is known to have antioxidant-like properties.
Furthermore, extracts of several spices and herbs have
been shown to reduce A. parasiticus growth and AFB1
production [12]. Moreover, essential oils have been con-
sidered to inhibit A. flavus growth and AFB1 production
[13,14]. Pawar and Thaker [15] investigated 75 essential
oils against the fungus A. niger growth and saffron was
found to have no effect on the fungus. According to
Igawa et al. [16], in addition to production of inhibitors,
the development of plants that can inhibit mycotoxins is
a promising approach.
*Corresponding author.
opyright © 2012 SciRes. AiM
Saffron contains chemical constituents such as cro-
cetin, picrocrocin and safranal which are responsible for
color, flavor and aroma respectively. Anthocyanins, fla-
vonoids, vitamins, amino acids, proteins, starch, minerals
and other chemical compounds have also been described
in saffron [15]. It is important to note that under basic
conditions and following the drying process, picrocrocin
is converted to safranal [17]. Additionally crocetin ester
is degraded upon thermal treatment, although it was af-
fected by external factors [18]. Giaccio [3] reported that
crocetin protects against oxidation damage in rats’ pri-
mary hepatocytes, in particular a suppression of AFB1-
induced hepatotoxic lesions by crocetin. The reduction of
toxicity is due to the property of crocetin to stimulate
defense mechanisms in the liver cells with the increase of
the glutathion-S-transferase and also with the decrease of
the alleged AFB1-DNA.
To our knowledge there is little information in the lit-
erature concerning the saffron biological activity as crude
product and its effect on AFB1 production. Hence the aim
of the present study was to investigate the effect of saf-
fron dried stigmas on A. parasiticus growth and AFB1
production in Yeast Extract Sucrose medium.
2. Materials and Methods
2.1. Apparatus and Reagents
A laminar flow (Telstar Bio IIA, Spain), an incubator
WTB Binder (Tuttlinger, Germany), and a centrifuge
Sorvall RC-5B (HS-4) (Norwalk, USA) were used. AFB1
standard was purchased from Sigma-Aldrich Chemical
Co, USA. Millipore filters HVLP (0.45 μm) were pur-
chased from Waters (Millipore, USA). The Aflatest im-
munoaffinity columns were obtained from Vicam, USA.
All reagents used were of analytical grade (Sigma Al-
drich, USA) while HPLC solvents were of HPLC grade
and were purchased from Fisher Scientific, UK. Trifluo-
roacetic acid was from Fluka, The Netherlands.
2.2. Samples
Samples of saffron were collected from the Athens mar-
ket during 2010. Saffron purchased in packages was used
before the expiration date. The packages were little pots
made of glass labeled as “Cooperative de Saffran Ko-
zani”. The content of each pot was 1 g of saffron stigmas.
All samples were stored in dark and cool place prior to
analysis. Just before examination saffron stigmas were
transferred in sterile polyethylene bags. Representative
sub samples of saffron stigmas (100 mg) were collected
aseptically and also used in the present study.
2.3. Media
Aspergillus flavus parasiticus agar (AFPA) was prepared
by dissolving 4 g of yeast extract (Oxoid) (Hamshire,
UK), 2 g of bacteriological peptone (Oxoid) 0.1 g of fer-
ric ammonium citrate, 0.2 ml of Dichloran 0.2% in etha-
nol (Fluka Steinheim, The Netherlands), 0.02 g of chlo-
ramphenicol (Oxoid) and 3 g of agar (Oxoid) in 200 ml
of distilled water, final pH 6.0 - 6.5. Czapek Dox agar
(CZA) was prepared by dissolving 0.4 g of sodium nitrite,
0.1 g of potassium chloride, 0.1 g of magnesium sulfate,
0.002 g of ferric sulfate, 0.2 g of dipotassium phosphate,
6 g of sucrose, 3 g of agar, 0.002 g of zinc sulfate and
0.001 g of copper sulfate in 200 ml of distilled water,
final pH 6.0 - 6.5 [19].
2.4. Preparation of Spore Inoculum
The aflatoxigenic strain A.parasiticus spear (IMI 283883)
utilized throughout this study was obtained from the In-
ternational Mycological Institute (Engham Surrey, UK).
An inoculum was obtained by growing the mold on a
slant of stock cultures of CZA, which were maintained at
5˚C. Spore inoculum was prepared by growing A. para-
siticus on CZA for 7 days at 30˚C and spores were har-
vested aseptically using 10 mL of sterile 0.01% v/v
Tween 80 solution. AFB1 carried over from the initial
growth was minimized by centrifuging the spore sus-
pension (1000 g for 10 min) and resuspending the bio-
mass in 10 mL of sterile Tween 80 solution twice. Dilu-
tions (0.1, 0.01, 0.001 and 0.0001) were prepared from
the initial spore in sterile tubes containing 10 mL of
Tween 80 0.05% v/v suspension [19]. The spore concen-
tration was determined by the spread plate surface count
technique, using 0.1 mL of each dilution on four AFPA
plates after incubation at 30˚C for 2 days. The population
size was estimated by the reverse intense yellow/orange
coloration of the colonies. For obtaining an inoculum
containing 100 conidia, plates with 10 - 100 colony form-
ing units (cfu) were selected and the desired 100 spore
quantity used in the present study was estimated.The
quantity of 100 spores flask-1 was chose as it was the
minimum concentration found in the literature producing
a detectable amount of AFB1 by aspergilli [20].
2.5. Inoculation
For each day of observation, 6 flasks containing 10 mL
of YES medium were inoculated with 100 conidia·flask–1
of A. parasiticus. 100 mg of saffron stigmas with natural
microbiota (NM), were added before inoculation into
each of the three flasks for each day of observation. Fur-
thermore, 100 mg of saffron with NM were added to
three additional flasks of non-inoculated YES, for each
day of observation. We should mention that all flasks
with YES were sterilized by autoclaving at 121˚C for 15
min. Saffron was added after sterilization and before in-
oculation. All flasks were incubated under stationary
Copyright © 2012 SciRes. AiM
conditions at 30˚C. Immediately after autoclaving (115˚C,
30 min) for safety reasons [21] the mycelial growth was
determined and AFB1 was assayed on days 0, 3, 7, 9, 12,
15 and 18 of incubation. The experiments were repeated
in triplicate.
2.6. AFB1 Determination
The content of each flask containing YES medium with
saffron and flasks control (without saffron addition) was
mixed with 30 mL of methanol and shaken well for 10
min. After filtration, an aliquot of 1 ml from each flask
was used for AFB1 analysis. The aliquot of 1 ml from the
filtrate was mixed with 10 ml of distilled water. The
mixture was transferred onto an Aflatest immunoaffinity
column with flow rate 6 ml·min–1 and washed twice with
10 ml of distilled water. The column was then allowed
once more to dry by passing air through it. AFB1 was
eluted with 2 ml of acetonitrile (flow rate 0.3 ml·min–1)
[22]. A derivative of AFB1 (AFB2a, hemiacetal of AFB1)
was prepared by adding 200 μl of hexane and 200 μl of
trifluoroacetic acid to the evaporated solution of AFB1
eluate, heating at 40˚C in a water bath for 10 min,
evaporating to dryness under nitrogen, redissolving in
200 - 500 μl in appropriate volume with water/acetoni-
trile 9:1, v/v to give concentration of <5 ng·ml–1 and
analyzing by HPLC (volume injected = 40 μl). AFB2a
shows enhanced fluorescence compared to AFB1 [23].
On the other hand AFB2a is less toxic compared to AFB1
because of its protein binding properties, since it is not
being absorbed from the gastrointestinal tract and there-
fore it is non toxic to experimental animals. Moreover
AFB2a does not interact with nuclear DNA [24].
2.7. Determination of Mycelial Mass in YES
After cooling, mycelia were filtered through filters that
were previously dried (24 h at 80˚C) and weighed. The
mycelium was washed with distilled water and allowed
to dry for 24 h at 80˚C. The dry weight of the mycelium
was then measured.
2.8. HPLC-FL Analysis
HPLC was performed using a Hewlett-Packard 1050
(Waldbornn, Germany) liquid chromatograph equipped
with a JASCO FP-920 (Jasco Ltd, JAPAN) fluorescence
detector and an HP integrator 3395. The HPLC column
used was a C18 Nova-Pak (250 × 4.6 mm, 4 μm particle
size) purchased from Waters (Millipore, USA). For AFB1
determination isocratic elution was employed and mobile
phase consisted of water/acetonitrile/methanol, 20:4:3,
v/v/v. Prior to injection samples were filtered through
Millipore HVLP (0.45 μm) filters. Detection of the AFB1
hemiacetal derivative (AFB2a) was carried out at λex =
365 nm and λem = 425 nm. The flow rate was 1 ml·min–1,
and the retention time for AFB2a was ~15 min.
3. Results and Discussion
Effect of Saffron on Mycelial Growth and AFB1
The in-house characterization of the method for deter-
mining AFB1 in YES medium has been reported in detail
by Vergopoulou et al. [19]. The recovery factor of the
method is 95.3% (RSD 9.6%) and the detection limit of
the derivatized AFB1 (AFB2α) was found to be 0.02 ng
AFB1 ml–1 of YES. In the present study YES medium
was chosen since is an optimum medium for AFB1 bio-
synthesis. Gqaleni et al. [25] reported that in YES liquid
static culture at 30˚C, AFB1 production by A. flavus was
higher compared to agar media. In addition, measuring
AFB1 rather than all four AFBs (AFB1, AFG1, AFB2, and
AFG2) was followed throughout this study. AFB1 is the
prevalent form and also the most potent of these toxins
[26]. A. parasiticus was used as most A. parasiticus
strains are rather stable in aflatoxin production in cul-
tures [27]. Table 1 shows that the mycelial growth was
observed in control cultures inoculated (without saffron
addition) and in cultures with saffron, inoculated and non-
inoculated with A. parasiticus, as well. It is obvious that
molds occur along with other microorganisms in saffron
natural microbiota. The natural microbiota of saffron
stigmas examined, consisted of bacteria (<1000 cfu/gr)
and potentially aflatoxigenic molds (authors unpublished
results). In that case mycelium growth is likely. Palumbo
et al. [28] reported that the growth of A. flavus was com-
pletely inhibited when antagonist bacteria were present in
microbiological media. In the present study microbial
competition in cultures between saffron natural microbi-
ota and A. parasiticus was not proved since the mycelial
growth was just slightly reduced compared to cultures
control (only inoculated with the fungus, Table 1).
The addition of saffron stigmas to cultures inoculated
with A. parasiticus in this study, inhibited significantly
AFB1 production compared to control cultures (inocu-
lated with A. parasiticus without the addition of saffron
(Table 2, Figure 1). AFB1 production in cultures inocu-
lated with A. parasiticus with the addition of saffron was
observed up to the 15th day of incubation, while on the
18th day AFB1 was not detectable. Moreover AFB1 pro-
duction was also observed at lower levels after the 7th day
of incubation in non- inoculated cultures containing saf-
fron (100 mg· f la s k –1). AFB1 in cultures only with saffron
was not detectable the days 0 and 3 of incubation, indi-
cating that the saffron utilized throughout this work was
not originally contaminated with AFB1 at detectable lev-
els. Previously Martins et al. [29] reported that AFB1 was
Copyright © 2012 SciRes. AiM
Table 1. A. parasiticus growth in YES medium against my-
celium growth in YES with saffron addition inoculated and
YES with saffron alone.
Addition of saffron
a 100 mgb 100 mg + 100 conidiac
Mycelium growth
Days g·flask–1 (±SD) g·flask–1 (±SD) g·flask–1 (±SD)
0 0.05 (±0.006) NDd 0.06 (±0.01)
3 0.13 (±0.02) 0.08 (±0.02) 0.12 (±0.02)
7 0.18 (±0.003) 0.16 (±0.04) 0.10 (±0.03)
9 0.21 (±0.04) 0.13 (±0.03) 0.11 (±0.02)
12 0.17 (±0.004) 0.13 (±0.02) 0.08 (±0.03)
15 0.17 (±0.01) 0.19 (±0.006) 0.05 (±0.006)
18 0.15 (±0.02) 0.05 (±0.006) 0.04 (±0.006)
AFB1 (μg/flask)
Days of growth
0 3 7 9 12 15 18
YES inoculated + saffron
YES inoculated (control)
aInoculated without the addition of saffron (control); bAddition of saffron
100 mg·flask–1; cAddition of saffron 100 mg·flask–1 inoculated with 100
conidia of A. parasiticus; dND: not detected.
Table 2. AFB1 production (μg/flask) by A. parasiticus in
YES medium against AFB1 production in YES with saffron
addition inoculated and YES with saffron alone.
Addition of saffron
a 100 mgb 100 mg + 100 conidiac
AFB1 production
Days μg·flask–1 (±SD) μg·flask–1 (±SD) μg·flask–1 (±SD)
0 0.002 (±0) ND
d 0.004 (±0.001)
3 15.79 (±1.85) ND
d 0.010 (±0.0006)
7 47.54 (±10.77) 0.008 (±0.002) 0.010 (±0.001)
9 64.00 (±0.75) 0.051 (±0.005) 0.012 (±0.004)
12 75.31 (±2.31) 0.043 (±0.005) 0.018 (±0.003)
15 85.47 (±1.76) 0.020 (±0.001) 0.013 (±0.002)
18 90.42 (±1.61) 0.010 (±0.003) NDd
aInoculated without the addition of saffron (control); bAddition of saffron
100 mg/flask; cAddition of saffron 100 mg·flask–1 inoculated with 100
conidia of A. parasiticus; dND: not detected.
found in Portuguese saffron at levels of 1 to 5 μg·kg–1.
Maximum AFB1 production in cultures with saffron
alone and cultures inoculated with 100 conidia of A. pa-
ra s iticu s with saffron, was observed on the 9th day (0.051
μg AFB1·flask–1) and on the 12th day (0.018 μg
AFB1·flask–1) respectively. Maximum AFB1 production
in control samples (inoculated without saffron), on the
same days 12 and 9, was 75.31 μg AFB1·flask–1 and 64
μg AFB1·flask–1 respectively. Therefore AFB1 production
was inhibited in inoculated cultures when saffron was
Figure 1. Saffron (100 mg/flask) significantly inhibits AFB1
production by A. parasiticus in YES medium in comparison
to AFB1 production in YES inoculated with A. parasiticus
without the addition of saffron (control). By the 18th day no
AFB1 was detectable in inoculated cultures with the addi-
tion of saffron.
present. Inhibition amounted to 99.9% in cultures with
saffron inoculated with A. parasiticus for the 15 days of
observation. As mentioned previously AFB1 was not de-
tectable the 18th day (Table 2, Figure 1). In the present
study it was shown that the AFB1 levels for the samples
with saffron only and samples with saffron inoculated
with A. parasiticus, after reaching their maximum on 9th
and 12th day of observation respectively, a decrease was
observed. This decrease is due to the aflatoxin degrada-
tion. According to Doyle et al. [30] as the amount of
AFB1 increases the rate of degradation also increases.
This is in agreement with the results of this work (Table
One concern with the data on AF inhibitors is that the
majority of experiments were conducted in vitro in media
that do not approximate conditions on host plant seeds.
Screening of inhibitors added to media that incorporate
host seed extracts may improve success in identifying
efficacious inhibitors [31]. To our knowledge there is no
information about the saffron biological ability as crude
product. Hence in this work for the first time saffron
stigmas were added to YES media without treatment
containing their natural microbiota. In the present study
100 mg of saffron flask-1 corresponding to 10,000 mg·l–1
were utilized, in view of the fact that saffron consists of
several compounds both active and inactive. Assi-
mopoulou et al. [32] reported previously that methanol
extract of saffron exhibited high antioxidant activity at a
concentration above 2000 mg·l–1. Furthermore saffron at
concentration 100 mg·l–1 (corresponding to 1 mg of saf-
fron stigmas·flask–1 did not have an effect on the AFB1
production (authors’ unpublished results).
Most inhibitors of AF biosynthesis act at one of three
levels: altering the physiological environment or other
signaling inputs perceived by the fungus, interfering with
Copyright © 2012 SciRes. AiM
signal transduction and gene expression regulatory net-
works upstream of AF biosynthesis, or blocking enzy-
matic activity [31].
Previously, Zaica and Buchanan [33] reported that a
large number of compounds were found to inhibit AFB1
biosynthesis mainly through their effect on fungal growth.
In the current study on the 9th and 12th days of maximum
production of AFB1 in samples with saffron alone and
samples with saffron inoculated with A. parasiticus re-
spectively was 1254 - 1751 and 4183 - 5333 folds lower
compared with samples control (inoculated with fungus).
Nevertheless the mycelial mass of the same samples at
the same days was only 1.3 - 1.6 and 1.9 - 2.1 folds lower,
compared to samples inoculated with A. parasiticus
(control). Therefore AFB1 decrease was proved and it is
assumed that is not correlated to fungus growth. Re-
cently López and Gómez-Gómez [34] reported that in-
fection of plants by pathogens activates a complex sys-
tem of signal transduction pathways, which results in the
activation of defense genes. So the authors isolated and
characterized a new chitinase gene from saffron design-
nated Safchi A, which showed chitinase activity in vitro.
Chitin is a major component of fungal walls. Upon
pathogen attack plants produce chitinases that degrade
chitin to chito-oligomers which were shown to elicit strong
defense responses in many plant species [35]. The results
reported by López and Gómez-Gómez [34] indicated that
saffron develops an active defense response to prevent
colonization by the fungus. This statement is in agree-
ment with the results presented in the current study since
A. parsiticus growth was slightly inhibited in the pres-
ence of saffron (Table 1).
The most interesting approach to the study of the in-
hibitory effect of saffron on A. parasiticus growth and
AFB1 production is that saffron acts as an antioxidant
[36]. Oxidative stress has been shown to stimulate afla-
toxin biosynthesis in A. parasiticus [37,38]. The stimula-
tion and suppression of AF biosynthesis by oxidants and
antioxidants, respectively indicates that perturbations in
the oxidative state of the fungal cell influence AF bio-
synthesis. Redox reactions are fundamental to cellular
catabolism and anabolism, and antioxidants may hinder
vital processes. Inhibition of mitochondrial or perox-
isomal b-oxidation by antioxidants may limit the avail-
ability of carbon skeletons for polyketide pathways dur-
ing growth on lipid-rich seeds. Antioxidants could inter-
fere, thereby, reducing the pool of nicotinamide adenine
dinucleotide phosphate (reduced form) available for AF
biosynthetic reactions [39]. Furthermore a survey of in-
hibitors of AF biosynthesis has illustrated that many in-
hibitors have antioxidant activity [31].
In the present study, the significant inhibitory effect of
saffron on AFB1 production should probably be attrib-
uted to a synergistic action of the main bioactive con-
stituents crocin and safranal, which is obtained by picro-
crocin degradation. Assimopoulou et al. [32] reported
that saffron methanol extract exhibited high antioxidant
activity and established that crocin and safranal showed
high scavenging activity which is involved in aging
process, anti-inflammatory, anticancer and wound heal-
ing activity as well. Additionally Papandreou et al. [40]
showed the antioxidant properties of saffron stigmas ex-
tract and its crocin constituents. It is interesting to report
that, in the present study, AFB1 production in cultures
inoculated with A. parasiticus with the addition of saf-
fron before autoclaving, was not significantly different
compared to control cultures (without saffron) (authors
unpublished results). Autoclaving possibly inactivates
crucial antioxidants such as safranal (boiling point 70˚C
at 1 mm Hg) and crocetin as already mentioned previ-
In the present study the significance was given to the
saffron activity as crude material since the antioxidant
activity of the principal components has been already
shown in the literature as mentioned above. Therefore
although saffron consists of many components, both ac-
tive and inactive, in the present study has been revealed
that antioxidant properties dominate. As a result, when
saffron stigmas were added to cultures of YES inoculated
with A. parasiticus, AFB1 production was down 99.9%
compared to control cultures, (without saffron addition)
throughout the period of observation. Conclusively tak-
ing account saffron’s unique properties, it might be util-
ized under specific conditions for processing agricultural
products, to prevent AFB1 production.
4. Acknowledgements
This work was supported in part by the University of
Athens, Special Account for Research Grants (70/4/
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