Wageningen Academic 

World Mycotoxin Journal, 2015; 8 (3): 283-289 P u b l i s h e r s

Inhibition of ochratoxin A production in Aspergillus carbonarius by hydroxycinnamic

acids from grapes
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2014.1753 - Wednesday, February 14, 2018 2:17:28 AM - University of Guelph IP Address:131.104.97.86

M. Ferrara1*, A. Gallo2, R. Lo Scalzo3, M. Haidukowski1, V. Picchi3 and G. Perrone1

1Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Amendola 122/O, 70126 Bari,
Italy; 2ISPA, CNR, via Provinciale Lecce-Monteroni, 73100 Lecce, Italy; 3Consiglio per la Ricerca e la Sperimentazione
in Agricoltura, Research Unit of Food Technology, via G. Venezian 26, 20133 Milano, Italy; massimo.ferrara@ispa.cnr.it

Received: 25 March 2014 / Accepted: 14 July 2014

© 2014 Wageningen Academic Publishers

RESEARCH ARTICLE
Abstract

Hydroxycinnamic acids (HCAs), phenolic components of wine, are known to have antimicrobial properties.
Aspergillus carbonarius is one of the most important ochratoxin A (OTA) producing fungi in wine. Strategies for
the control and prevention of A. carbonarius contamination are important for the maintenance of wine safety. This
study sought to determine the potential of HCAs, such as caffeic, p-coumaric and ferulic acids, as antifungal natural
compounds for the control of A. carbonarius growth and OTA production. The HCAs were tested at the increasing
concentrations of 0.30, 0.65 and 1.10 mg/ml in minimal medium (MM) and grape juice. Germination of conidia was
not affected in neither of the two media in presence of HCAs. At all the concentrations tested, OTA biosynthesis in
MM was reduced and the dose effect was more evident for p-coumaric and ferulic acids; in grape juice the reduction
trend was confirmed, and ferulic acid showed the highest inhibitory effect. Moreover, the expression level of genes
encoding a polyketide synthase (AcOTApks) and a nonribosomal peptide synthetase (AcOTAnrps) involved in OTA
biosynthesis, was evaluated by real-time PCR in A. carbonarius grown in presence of 0.65 mg/ml of HCAs. From
gene expression analysis only the AcOTApks gene showed a marked reduction of transcription level in presence
of p-coumaric and ferulic acids. On the contrary, caffeic acid seemed to not influence the expression levels of the
genes analysed in this study, suggesting a different mechanism of action on the regulation of OTA biosynthesis.

Keywords: caffeic acid, p-coumaric acid, ferulic acid, gene expression, OTA biosynthesis genes

1. Introduction ester of caffeic acid (caftaric acid, which represents up

Phenolic compounds have been found to inhibit the
production of several mycotoxin, including ochratoxin A
(OTA), fumonisin, tricothecenes and aflatoxins (Beekrum
et al., 2003; Passone et al., 2005; Ponts et al., 2011; Romero
et al., 2009; Samapundo et al., 2007). They are the most
important phytochemicals in grape for their biological
activities and health-promoting benefits; they include
mainly anthocyanins, flavanols, stilbenes (resveratrol)
as well as phenolic acids, such as hydroxybenzoic and
hydroxycinnamic acids (HCAs), which are known for
their antimicrobial properties (Shrikhande, 2000; Spacil
et al., 2008; Waterhouse, 2002). Among HCAs, caffeic,
p-coumaric, and ferulic acids are precursors of most of
the compounds present in grape and wine, like the tartaric
to 50% of total HCAs), the tartaric esters of p-coumaric
acid (p-coutaric acid) and ferulic acid (fertaric acid), and
the trans-p-coumaric glucoside. Depending on grape
variety, tissue portion, growing conditions, climate and
environment, the relative presence of different HCAs
may vary widely. In general, concentrations from 25 to
100 mg/l of HCAs in wines are described, predominantly
caffeic acid and its tartaric ester (caftaric acid) (10-50
mg/l), while for p-coumaric and its ester p-coutaric acid,
and ferulic and its ester fertaric acid values around 10-20
mg/l and 5-15 mg/l are found, respectively (Rentzsch et
al., 2009). In respect to their biological activity, various
studies have demonstrated the effectiveness of HCAs as
antifungal or anti-mycotoxigenic compounds (Harris et
al., 2010; Palumbo et al., 2007), and their potential use

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2014.1753283

 M. Ferrara et al.
as effective alternatives to conventional fungicides (Kim
et al., 2004; Passone et al., 2005; Tawata et al., 1996). For
example, gallic acid, a phenolic compound from walnut seed
coats, was recently shown to prevent aflatoxin biosynthesis
by Aspergillus flavus (Mahoney and Molyneux, 2004).
Recently, the anti-mycotoxigenic activity of phenolic acids
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2014.1753 - Wednesday, February 14, 2018 2:17:28 AM - University of Guelph IP Address:131.104.97.86
has also been demonstrated for OTA producing fungi. In a
study of Palumbo et al. (2007), different antioxidants, among
which also some HCAs, showed effects on fungal growth
and OTA production, which differed among Aspergillus
strains and species.
OTA is a well-known nephrotoxin with carcinogenic and
immunosuppressive properties produced by Aspergillus
and Penicillium. It is considered the main contaminant
of wines, and relevant for human health risk. In addition,
OTA has been shown to induce oxidative DNA damage
in mammalian cells and in rat liver and kidney in vivo,
indicating its role in cytotoxicity and tumorigenicity (IARC,
1993; Pfohl-Leskowicz and Manderville, 2007). Among
all OTA producing fungi, Aspergillus carbonarius is the
primary causative agent of OTA contamination of grapes,
grape juice, dried vine fruit, must and wine (Battilani et al.,
2006; Perrone et al., 2007). In this work we investigated the
activity of three hydroxycinnamic acids naturally present
on grape berry skin, i.e. caffeic, p-coumaric and ferulic
acids (Figure 1), and their influence on growth and OTA
production by A. carbonarius in artificial and natural
(grape juice) substrate. Moreover, in order to clarify the
molecular mechanism by which antioxidant compounds
regulate OTA production, the expression levels of the
genes AcOTApks and AcOTAnrps, encoding respectively
a polyketide synthase (PKS) and a nonribosomal peptide
synthetase (NRPS) involved in OTA biosynthesis in A.
carbonarius, were analysed in presence of HCAs.
2. Materials and methods
Fungal strains and culture conditions
A. carbonarius ITEM 7444 (Agri-Food Toxigenic Fungi
Culture Collection of the Institute of Sciences of Food
Production, CNR, Bari, Italy), an OTA producing strain
O
HO OH
HO HO
caffeic acid p-coumaric acid
Figure 1. Chemical structures of the hydroxycinnamic acids used in this study.
284
isolated from grapes, was used throughout this study. It was
routinely grown at 25 °C on potato dextrose agar (Oxoid
Ltd., Basingstoke, UK) for 5 days. Caffeic, p-coumaric and
ferulic acids were provided by Fluka-Sigma Aldrich (St.
Louis, MO, USA). The HCAs were dissolved in H2O +
3% Tris-HCl and added at the increasing concentrations
of 0.30, 0.65 and 1.10 mg/ml in Minimal Medium (MM)
(6 g/l NaNO3, 0.52 g/l KCl, 0.52 g/l MgSO47H2O, 1.52
g/l KH2PO4, pH 6.5, 10 g/l glucose, 2 ml/l Hutner’s trace
elements) or grape juice. The test was conducted in 12 well
plates, each well containing 1 ml of the medium added with
HCAs and inoculated with 10 µl of a conidial suspension
(1×105 conidia/ml). Three replicates were prepared for each
concentration of each phenolic compound tested and for
controls amended with H2O + 3% Tris-HCl. The test was
repeated twice. Plates were incubated at 25 °C in the dark
for 6 days, and conidial germination was monitored after 12,
14 and 36 h by light microscopy analysis, using an inverted
microscope (Leica DM IL; Leica, Wetzlar, Germany). After
6 days, culture filtrate and mycelium were collected for
OTA analysis and RNA extraction.
RNA extraction, cDNA synthesis and qRT-PCR analysis
RNA was extracted from 100 mg of mycelium ground
in liquid nitrogen and homogenised in 1 ml of QIAzol
Lysis Reagent (Qiagen, Hilden, Germany). Samples were
processed according to the QIAzol method and after
recovering the aqueous phase 0.5 vol of 100% ethanol
was added. The next steps were performed according to
the RNeasy Mini Kit (Qiagen) instructions. On-column
DNase treatment was performed according to the
manufacturer’s instructions. RNA samples were further
purified through polypropylene columns (Zymo-Spin™ IV-
HRC Column, Zymo Research, Orange, CA, USA) in order
to remove pigment residues. RNA quantity and quality
were determined by spectrophotometric analysis and gel
electrophoresis (Sambrook and Russel, 2001). RNA aliquots
were stored at -80 °C. First strand cDNA was synthesised
using 3 µg of total RNA, oligo(dT)20 mixed with random
hexamers primers in a 1:1 ratio, and SuperScript III reverse
transcriptase (Invitrogen, San Diego, CA, USA), according
to the manufacturer’s protocol. Transcription profiles of
OH
O
O
OH
OH
O
CH3
ferulic acid
World Mycotoxin Journal 8 (3)
  Inhibition of OTA production by hydroxycinnamic acids from grapes
AcOTApks and AcOTAnrps in A. carbonarius grown on MM
or grape juice were analysed by using quantitative reverse
transcription PCR (qRT-PCR) which was performed in
a 7500 Fast Real-Time PCR System (Applied Biosystem,
Warrington, UK). For the amplification reaction, a SYBR
Green I assay with 2.5×Real Master Mix SYBR-ROX (5
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PRIME GmbH, Hamburg, Germany) was performed in a
reaction volume of 10 µl with 200 nM of each primer for
target and reference genes. The constitutively expressed
β-tubulin gene was used as internal reference to normalise
gene expression. Primers used for qRT-PCR are listed
in Table 1. The amplification conditions were: an initial
denaturation step at 95 °C for 2 min, followed by 40 cycles
of 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. Specificity
of PCR amplification was confirmed by dissociation curve
analyses. The relative quantification of gene expression
was established using the comparative 2 -ΔΔCT method.
PCR efficiency of each oligonucleotide pair was calculated
from each linear regression of standard curves. Real time
PCR derived data were quantified relatively by using the
‘Relative Expression Software Tool’ (Pfaffl et al., 2002)
taking the different efficiencies into account.
Analysis of ochratoxin A in Aspergillus carbonarius
cultures
OTA levels in MM and grape juice substrates were
determined by high-performance liquid chromatography
coupled with fluorescence detection (HPLC-FLD). The
substrates of MM and grape juice were filtered using RC 0.20
µm filters (Phenomenex, Torrance, CA, USA). A volume of
100 μl was injected into the HPLC apparatus (technology
series 1100, Agilent, Santa Clara, CA, USA) with a full loop
injection system. FLD was set at wavelengths of 340 nm
(excitation) and 460 nm (emission). The analytical column
was a Zorbax SB-C18 (4.6×150×5 mm; Agilent) with a guard
column inlet filter (0.5 µm by 3 mm in diameter; Rheodyne
Inc., Rohnert Park, CA, USA). The mobile phase consisted
of a solution of acetonitrile:water:glacial acetic acid (99:99:2,
v:v:v) at a flow rate of 1 ml/min. OTA was quantified by
measuring peak areas at the retention time of OTA standard
solutions (Sigma-Aldrich, Milan, Italy) and comparing
these areas with the relevant calibration curve at 0.5 to
200 ng/ml. Calibrant solutions for standard calibration
curves were prepared by drying different aliquots of the
OTA stock solution (1 mg/ml) under a nitrogen stream
Table 1. List of primers used in this study.
RT_AcOTApks_F 5’-CGTGTCCGATACTGTCTGTGA-3’
RT_AcOTApks_R 5’-GCATGGAGTCCTCAAGAACC-3’
RT_AcOTAnrps_F 5’-ACGGGTCGCTGCTCTATATC-3’
RT_AcOTAnrps_R 5’-ACTCACCACATCAACCACGA-3’
RT_AcTUB_For 5’-CAAACCGGCCAGTGTGGTA-3’
RT_AcTUB_Rev 5’-CGGAGGTGCCATTGTAAACA-3’
World Mycotoxin Journal 8 (3)
that were successively reconstituted in the HPLC mobile
phase. The detection limit was 0.5 ng/ml, based on a signal
to noise ratio of 3:1.
Statistical analysis
The effect of HCAs concentrations on OTA production was
evaluated by two-way analysis of variance using Statistica
8.0 (StatSoft Inc., Tulsa, OK, USA). Phenolic compounds
and concentrations were considered as analysis factors
and comparison of means between the two factors was
conducted by Duncan test (P<0.05).
3. Results
Effect of hydroxycinnamic acids on ochratoxin A production
The effect of caffeic, p-coumaric and ferulic acids on A.
carbonarius growth and OTA production was evaluated
at concentrations of 0.30, 0.65 and 1.10 mg/l in MM and
grape juice. Conidia germination of A. carbonarius ITEM
7444 exposed to different concentrations of HCAs was
not affected in both MM (Figure 2) and grape juice (data
not shown). In general, not even fungal growth showed
evident differences. As shown in Figure 3A, a significant
reduction of OTA production was observed when HCAs
were added in MM. The dose effect was more evident for
p-coumaric and ferulic acids at the highest concentrations.
The highest inhibition effect in MM was observed at
0.30 mg/ml for caffeic acid (84.8% of reduction of OTA
production), at 0.65 mg/ml for p-coumaric acid (92.7%)
and at 1.1 mg/ml for ferulic acid (87.2%). When added
to grape juice, HCAs showed a different effectiveness in
reducing OTA production (Figure 3B). Specifically, caffeic
and p-coumaric showed the highest OTA reduction rate at
0.65 mg/ml (51.2%) and 1.1 mg/ml (82.8%), respectively.
Figure 2. Conidial germination of Aspergillus carbonarius after
incubation of 14 h in Minimal Medium added with 1.1 mg/ml of
hydroxycinnamic acids. (A) 3% Tris-HCl; (B) caffeic acid; (C)
p-coumaric acid; (D) ferulic acid. Bar = 60 µm.
285
 M. Ferrara et al.

160

Ochratoxin (ng/ml)
120
d
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80

c c
40 c
bc ab ab ab
ab a
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g/m

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Ochratoxin (ng/ml)

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Figure 3. Effect of different concentrations (mg/ml) of caffeic, p-coumaric and ferulic acid on ochratoxin A production (ng/ml)
by Aspergillus carbonarius in (A) minimal medium and (B) grape juice. Bars indicate standard deviations and letters indicate
significantly different means according to Duncan’s test (P<0.05).

Otherwise, ferulic acid was the most effective in reducing
OTA production in grape juice with a reduction of 92.3%,
91.3% and 93.4% at concentrations of 0.30, 0.65 and 1.10
mg/ml, respectively.
Effect of hydroxycinnamic acids on transcription of
AcOTApks and AcOTAnrps genes
showed no significant effect on the transcription of the
pks gene. The expression level of AcOTAnrps gene seemed
to be not greatly influenced by the presence of antioxidant
compounds, exhibiting only slight variations in respect to
the control for all the HCAs tested.
4. Discussion and conclusions

The transcription levels of AcOTApks and AcOTAnrps were
evaluated after six days of incubation of A. carbonarius in
MM added with 0.65 mg/ml of HCAs, as at these conditions
a considerable reduction of OTA production has been
registered. From the results of qRT-PCR analysis shown in
Figure 4, it is evident the effect of p-coumaric and ferulic
acids on the expression level of AcOTApks showing a down-
regulation of about twelve fold in respect to the control
medium without HCAs. On the contrary, caffeic acid
In the present study we analysed the inhibitory effect of
caffeic, p-coumaric and ferulic acids, on OTA production in
A. carbonarius. Results confirmed the ability of antioxidant
compounds in reducing OTA production. However, in our
experiments no evident effect on conidia germination and
the subsequent fungal growth was observed, evidencing that
reduction of toxin biosynthesis is not due to the reduced
growth of the fungus. Similarly, recent works reported that
the use of natural phenolic compounds reduced aflatoxin

286 World Mycotoxin Journal 8 (3)

  Inhibition of OTA production by hydroxycinnamic acids from grapes

A B
1.2 2.0

1.5
Relative expression

Relative expression
0.8

1.0
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0.4
0.5

0.0 0.0
3% Tris-HCl caffeic acid p-coumaric acid ferulic acid 3% Tris-HCl caffeic acid p-coumaric acid ferulic acid

Figure 4. Relative expression of (A) AcOTApks and (B) AcOTAnrps genes in presence of 0.65 mg/ml of caffeic, p-coumaric and
ferulic acids in minimal medium. Gene expression values has been normalised on β-tubulin gene of Aspergillus carbonarius.

production by Aspergillus flavus without affecting fungal counter the oxidative stress that triggers or enhances
growth (Mahoney et al., 2010; Samapundo et al., 2007). the toxin production by moulds (Hong et al., 2013;
Otherwise, Romero et al. (2009) reported a 10% inhibition Kim et al., 2005; Mahoney et al., 2010). In order to gain
of colony growth of A. carbonarius and a significant OTA preliminary insight on the molecular mechanism by which
reduction in presence of caffeic acid at 250 mg/l, and a antioxidant compounds regulate OTA biosynthesis, we
complete growth inhibition at 500 mg/l. Palumbo et analysed the expression of the OTA biosynthetic genes
al. (2007) reported widely divergent effects of phenolic AcOTApks and AcOTAnrps in presence of phenolic acids.
antioxidants, among which caffeic acid, on OTA production In A. carbonarius, AcOTApks encodes a PKS involved in the
and fungal growth in several ochratoxigenic Aspergilli, first step of formation of dihydroisocoumarin polyketide
including A. carbonarius. In our study, the tested HCAs moiety characteristic of OTA molecular structure,
significantly reduced OTA production, although consistent whereas AcOTAnrps encodes the NRPS responsible for
differences were observed between the two media used. The the ligation of the phenylalanine to the polyketide group
three phenolic acids added at 0.65 mg/ml and 1.1 mg/ml in the penultimate step of biosynthesis pathway (Gallo et
were effective in reducing OTA production in both MM al., 2012, 2014).
and grape juice, whereas a discordant effect was observed
for caffeic and p-coumaric acids when added in grape The study of gene activation of AcOTApks and AcOTAnrps
juice at the lowest concentration. The different response in response to HCAs was focused at 0.65 mg/ml, which
could be due to the heterogenic composition of grape juice, represented an effective dose for all the three HCAs in
which might prevent the effectiveness of compounds when both MM and grape juice. The transcription levels of the
present at low concentrations and interfere with their mode pks gene appeared to be more affected by p-coumaric and
of action. Likely, this interference is overcome when higher ferulic acids, while no significant variation was observed
concentrations are added to grape juice and consequently the for the nrps gene. Surprisingly, caffeic acid did not show any
inhibition effect is restored. Otherwise, ferulic acid showed a direct influence on the expression of both genes, despite
high efficacy in reducing OTA production in A. carbonarius the observed reduction of OTA, although it was not as
in both substrates and at all the tested concentrations. significant as for the other HCAs. The different response
of caffeic acid suggests that the regulatory mechanism of
This is the first known investigation that in vitro inhibition of OTA biosynthesis by antioxidant compounds
demonstrates the strong inhibitory effect of ferulic acid is complex, and likely involves genes upstream of the
on OTA biosynthesis. The presence of natural phenolic biosynthetic cluster, or other genes involved in antioxidant
compounds has been associated to resistance of different response. Further studies are necessary to better understand
crops to fungal invasion (Lattanzio et al., 2006) and also the molecular processes at the basis of antioxidant activity
their role in inhibiting the production of several mycotoxins and whether the mode of action is directly or indirectly
has been well acknowledged (Da Cruz Cabral et al., 2013). related to the primary metabolism or involved in the
Interestingly, the relevant presence of p-coumaric esters in secondary metabolism, or a combination of both.
the skin of some grape varieties (Rodrìguez Montealegre
et al., 2006) may be responsible for an increased resistance The possible role of natural phenolic compounds as
to pathogen attack. The mechanism of action by which antifungal and anti-mycotoxigenic agents, inhibiting fungal
phenolic acids can suppress mycotoxin production has growth and toxin production, has been of recent interest
not yet been established. These compounds are considered as an alternative strategy to the use of chemical fungicides
potent antioxidants and as free radical scavengers could in pre- and post-harvest. However, the in vitro studies

World Mycotoxin Journal 8 (3) 287

 M. Ferrara et al.
must be corroborated by in situ investigation through
direct application of phenolic compounds on grapes. The
concentrations of HCAs used in this work are higher than
those found in the literature as average levels (Waterhouse,
2002). However, depending on the specific variety chosen
(Singleton et al., 1986) and on the grape ripening stage
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(Alonso Borbalan et al., 2003), very high concentration of
HCAs may be found in ripened grapes (up to 5.21 mg/g)
and even higher amounts in early ripening stages (up to
39.9 mg/g), which greatly exceed those used in the present
study. As a consequence, with a proper extraction procedure
for the HCAs from grape or grape pomace, it is possible
to obtain the required concentration of the new potential
products for use in the control of OTA production by
A. carbonarius. In this regard, the determination of optimal
application doses should be taken into account, to avoid
undesirable effects on the grape organoleptic properties
and on its processed products. On the other hand, sub-
inhibitory doses could stimulate fungal growth, sporulation
and secondary metabolites production like mycotoxins
(Marín et al., 2002). Therefore, in order to reduce the doses
of HCAs without losing their effectiveness, it is necessary to
test different combinations of these phenolic antioxidants
on their synergistic effects on growth and OTA production
by A. carbonarius.
Acknowledgements
This research was supported by the Ministerial project
ALISAL-DM 11008/7303/10. We would like to thank
Vincenzo Achille for his technical assistance.
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