International Journal of Food Microbiology 83 (2003) 319 – 324

www.elsevier.com/locate/ijfoodmicro

Short communication
Potential use of antioxidants for control of growth and fumonisin
production by Fusarium verticillioides and Fusarium proliferatum
on whole maize grain
A.M. Torres a, M.L. Ramirez a, M. Arroyo b, S.N. Chulze a, N. Magan b,*
a
Departamento de Microbiologia e Inmunologia, Facultad de Ciencias Exactas Fisico-Quı́micas y Naturales,
Universidad Nacional de Rio Cuarto, Rio Cuarto, Cordoba, Argentina
b
Applied Mycology Group, Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedford MK 45 4 DT, UK
Received 28 September 2001; received in revised form 13 July 2002; accepted 29 August 2002

Abstract

The effect of interactions between two food grade antioxidants butylated hydroxyanisole (BHA) and propyl paraben (PP,
100, 200, 500 Ag g 1) and water activity (aw, 0.995, 0.98, 0.95) of irradiated maize on lag phase prior to growth, growth rate
and fumonisin production by Fusarium verticillioides and Fusarium proliferatum was evaluated at 25 jC. Both antioxidants
had an effect on growth characteristics, and fumonisin production. However, this was dependent on the dose used and the aw
treatment. At 500 Ag g 1 BHA and PP increased the lag phase prior to growth, and reduced the growth rate of both Fusarium
species significantly, especially at 0.95 aw. Both antioxidants significantly reduced the production of fumonisin by both
Fusarium species, especially at 0.98 and 0.95 aw. These results suggest that these antioxidants have potential for treatment of
maize grain for controlling growth of these mycotoxigenic species and prevent fumonisin accumulation.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Antioxidants; Water activity; Growth; Fumonisins; Maize

1. Introduction Ganaderı́a Pesca y Alimentación, 1997). Ear rot dis-

Maize (Zea mays L) is one of the main crops in
Argentina, with a total production of approximately
19 million tons. Half of the production is exported and
the rest is devoted to the internal market both for
animal feed and foodstuffs (Secretarı́a de Agricultura,
* Corresponding author. Tel.: +44-1525-863539; fax: +44-
1525-863540.
E-mail address: n.magan@cranfield.ac.uk (N. Magan).
eases of maize are caused by different Fusarium
species. The Fusarium species predominantly respon-
sible for ear rot in Argentina are Fusarium verticil-
lioides (Sacardo) Nirenberg, Fusarium proliferatum
(Matsushima) Nirenberg, and Fusarium subglutinans
(Wollenweber et Reinking) Nelson, Tousson, and
Marassas. These three species have also been isolated
from asymptomatic maize kernels both in the field and
during postharvest storage (Chulze et al., 1996; Gon-
zalez et al., 1995; Ramirez et al., 1996; Sydenham et
al., 1993; Kedera et al., 1999).

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0168-1605(02)00380-X
320 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324
F. verticillioides and F. proliferatum are responsi-
ble for the production of fumonisins, which have
become common contaminants of maize. They are
of concern due to their toxicological implications to
different biological systems, and cause several myco-
toxicosis such as leucoencephalomalacia in horses,
pulmonary edema in pig and liver cancer in rats
(Gelderblom et al., 1988; Ross et al., 1990; Thiel et
al., 1992). Also these mycotoxins have shown phyto-
toxic effects (Abbas et al., 1999). Epidemiological
studies also suggest that fumonisins could be associ-
ated with human esophageal cancer in some regions
with high intake rate of maize contaminated with
fumonisins (Franceschi et al., 1990).
Several approaches have been taken to try and
diminish the human and animal exposure to these
mycotoxins. In the field, the use of cultivars less
susceptible to toxin production, control of insects
and weeds, and cultural practices are all been exam-
ined. Antioxidants have shown some effects on con-
idial germination, growth and mycotoxin production
by aflatoxigenic species of Aspergillus flavus and
Aspergillus parasiticus (Thompson, 1991). Later stud-
ies by Thompson (1992, 1994) and Thompson et al.
(1993) showed that antioxidants also could inhibit the
growth and moniliformin production by F. verticil-
lioides and Penicillium species. However, in their
studies no account was taken of the effect of inter-
action of antioxidants with natural grain substrates
and with key environmental factors such as water
activity (aw) or temperature. Although propionic acid-
based preservatives are recommended for maize,
recent studies suggest that they are not effective at
controlling Fusarium species or fumonsin production
over a range of environmental conditions (Marin et
al., 2000). Thus, alternative compounds are needed as
part of a corrective strategy if fumonsin enters the raw
material preharvest or during initial postharvest drying
and storage. Recent in vitro studies demonstrated that
the food-grade antioxidants propyl paraben and buty-
lated hydroxyanisole could control both growth and
fumonisin production by F. verticillioides and F.
proliferatum in 2% maize meal extract agar over a
range of water availability regimes (Etcheverry et al.,
2002).
The aim of this study was to evaluate the effect of
the two best food grade antioxidants: butylated
hydroxyanisole (BHA), and propyl paraben (PP) on
(a) the lag phase prior to growth, (b) growth rates and
(c) control of fumonisin production by strains of F.
verticillioides, F. proliferatum under different aw con-
ditions on irradiated maize kernels at 25 jC.
2. Materials and methods
2.1. Fungal strains
F. verticillioides (RC 2000) and F. proliferatum
(ITEM 2443) were isolated from maize in Argentina.
These strains have been demonstrated to be good
fumonisin producers. The strains were maintained in
glycerol (15%, Sigma-Aldrich, Dorset, UK) at 80
jC and kept in the culture collection at the Depart-
ment of Microbiology and Immunology, Universidad
Nacional de Rio Cuarto, Cordoba, Argentina, and the
Institute Tossine e Micotossine da Parassiti Vegetali,
Bari, Italy.
2.2. Substrate and antioxidants
Irradiated maize kernels (12 kGy) with retained
germinative capacity were used in these studies. The
maize kernels were check for sterility and kept to 4
jC. The antioxidants used were butylated hydroxya-
nisole (BHA), and propyl paraben (PP), obtained from
the Sigma. Stock solutions of each antioxidant were
prepared in 95% ethyl alcohol.
2.3. Inoculation and incubation conditions
Irradiated maize was rehydrated to 0.995, 0.98 and
0.95 aw by the addition of sterile distilled water and
reference to a moisture adsorption curve. The aw of
the grains was determined with a Thermoconstanter
Novasina TH 200 (Novasina, Zurich, Switzerland).
The cultures were incubated to 25 jC. The required
concentration of each antioxidant was added to reach
a final concentration of 100, 200 and 500 Ag g 1 of
maize. The maize kernels were allowed to equilibrate
at 4 jC for 48 h with periodic shaking to allow
absorption and equilibrium. The rehydrated maize
kernels were dispensed as a monolayer (approxi-
mately 20 g) in sterile 9 cm Petri dishes.
The plates were inoculated centrally with a 5-mm
diameter mycelial disk taken from the margin of a 7-
 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324 321

day-old culture of each isolate grown on corn-meal and FB3 (Division of Food Science and Technology,
agar (Oxoid, Basingstoke, Hampshire, UK). Plates Pretoria, South Africa) in acetonitrile/water (1:1 v/v)
with the same aw were enclosed in plastic containers at concentrations of 100 Ag ml 1 for FB1 and 50 Ag
together with beakers of a glycerol – water solution at ml 1 for FB2 and FB3, respectively. The detection
the same aw as the plates, to maintain a constant ERH limit of the analytical method for the three fumonisins
inside the boxes. The time periods prior to growth was was 1 Ag g 1.
monitored. Fungal colonization of the grains was
evaluated in two diameters at right angles to each
other every day until the colony reached the edge of 3. Results and discussion
the plate, approximately 15 days. The experiments
were all carried out with three separate replicates per Table 1 shows the effect of antioxidant treatment at
treatment. The radius of the colony was plotted different aws on the lag phase prior to growth (h). This
against time, and the slope of the mycelial extension shows that aw increased the lag phase, but combina-
determined by linear regression. The lag phase was tions of 500 Ag g 1 of either butylated hydroxyani-
obtained by extrapolation of the line to the x-axis for sole (BHA) or propyl paraben (PP) at 0.95 aw resulted
each replicate of each treatment. At the end of the in the longest lag phases prior to growth for both
incubation period the grain samples were analysed for species (60 – 75 h).
fumonisin content. The effect of BHA and PP on control of mycelial
growth of F. verticillioides and F. proliferatum
2.4. Fumonisin analysis showed most efficacy at 0.95 aw and 500 Ag g 1
(Fig. 1). Overall, PP was more effective than BHA,
Briefly, the mycotoxins were extracted with aceto- almost completely inhibiting growth of both species at
nitrile/water (1:1 v/v) by shaking the grains and 500 Ag g 1 concentration. However, at 100 –200 Ag
solvent for 30 min on an orbital shaker and the g 1 and 0.995 or 0.98 aw the antioxidants were
extracts filtered through filter paper (Whatman No. relatively ineffective against both F. verticillioides
4, Whatman International, Maidstone, UK). An ali- and F. proliferatum.
quot of the extracts (1000 Al) was taken and diluted
with acetonitrile/water as necessary for High Perform-
Table 1
ance Liquid Chromatographic (HPLC) analysis. Effect of antioxidants on the lag phase (h) prior to growth of F.
Fumonisin content in the grains was evaluated by verticillioides and F. proliferatum on maize grain at different water
HPLC of the orthophthaldehyde derivative (OPA) as activities and 25 jC
described by Shephard et al. (1990). The HPLC Strain Antioxidant Water activity
system consisted of a Hewlett Packard 1050 pump concentration (Ag/g)
0.995 0.98 0.95
(Palo Alto, CA, USA) connected to a Hewlett Packard F. verticillioides Control 10 17 37
3395 integrator. Chromatographic separations were BHA 100 15 23 28
performed on a stainless steel Supercosil LC-ABZ, 200 12 28 36
C18 reversed-phase column (150  4.6 mm I.D., 5 Am 500 28 48 63
particle size; Supelco, Sigma-Aldrich). Methanol/0.1 F. proliferatum Control 24 19 28
BHA 100 28 17 39
M sodium dihydrogen phosphate (75:25 v/v, Sigma- 200 29 35 42
Aldrich) solution adjusted to pH 3.35 with orthophos- 500 33 55 73
phoric acid (Sigma-Aldrich) as mobile phase was used F. verticillioides Control 10 17 37
at a flow rate of 1.5 ml min 1. Fluorescence of PP 100 17 34 40
fumonisin OPA derivatives was recorded at excitation 200 14 35 19
500 12 39 34
and emission wavelengths of 335 and 440 nm, F. proliferatum Control 24 19 28
respectively. Fumonisin quantitation was performed PP 100 34 30 37
by peak height measurements and comparison with 200 34 32 35
reference standard solutions. The standard solution 500 26 31 74
was obtained by dissolving pure fumonisins FB1, FB2 Data are means of three replicates per treatment.
322 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324

Fig. 1. Effect of concentrations of antioxidants on growth of F. verticillioides and F. proliferatum on layers of irradiated maize grain at different
water activities and 25 jC. Bars indicate least significant difference at P = 0.05.

Fig. 2 shows that F. verticillioides produced fumo-
nisins (FB1, FB2 and FB3) at almost all the BHA
concentrations evaluated, but the amount produced
was dependent on the concentration of antioxidant
and the aw. The lower level of fumonisin or absence of
fumonisin production was only observed in the pres-
ence of BHA (500 Ag g 1 at 0.98 aw). When water
was freely available (0.995 aw) a reduction in fumo-
nisin production was about 20 –30% in the presence
of BHA and PP. At 0.98 aw the reduction ranged
between 94% and 98% at 500 Ag g 1. PP was more
effective against F. proliferatum than F. verticillioides.
Some stimulation of fumonisin production by F.
verticillioides was observed with BHA at 0.995 and
0.95 aw levels and 100 and 500 Ag g 1, respectively.
During ripening maize ears are susceptible to
colonization and infection by F. verticillioides and F.
proliferatum since the water availability changes from
about 50% to 30% ( = 1.0 to 0.98 aw) which is
adequate for germination and growth of F. verticil-
lioides and F. proliferatum. Also at the postharvest
and storage stage, if there is some delay in drying
these species could grow. The results in the present
study showed that the antioxidants BHA and PP are
effective in controlling both growth and toxin produc-
tion on maize grains, but at higher doses than found in
a previous study on maize meal extract agar (Etch-
everry et al., 2002). The different behaviour of the
chemicals on the natural substrate and on culture
media could be related to the distribution of the
antioxidants on the kernel surface and the pericarp
barrier of the kernel that may not allow good contact
between the fungi and the antioxidants.
The effect on fumonisin production is particularly
important as this is of major concern in the food grain
and feed industries. We have demonstrated that these
 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324 323

Fig. 2. Effect of antioxidant treatments on fumonisin production by F. verticillioides and F. proliferatum on layers of irradiated maize grain at
different water activities and 25 jC. Bars indicate least significant difference at P = 0.05.

are effective in decreasing and inhibiting fumonisin
production provided that the right concentration and
coverage can be achieved. It has been suggested that
the Parabens do not have any specific effect on
enzymes but appear to act mainly at the cell mem-
brane eliminating the pH component of the proton
motive force and thus disrupting energy transduction
and substrate uptake (Adams and Moss, 1995). With
regard to phenolic antioxidants various mechanisms
have been proposed for efficacy. Evidence exists that
they interfere with membrane lipid structure, and that
BHA, for example, causes cellular leakage in bacteria
(Degre and Sylvestre, 1983). Increased leakage of
sugars, amino acids and proteins from fungal mycelia
has also been detected, suggesting disruption of the
membranes (Thompson, 1996a,b).
At present BHA and PP are permitted for use as
antimicrobial agents in different foods and are on the
list of generally regarded as safe (GRAS) chemicals of
the Food and Drug Administration in the USA. It may
be possible to examine these types of products as field
sprays or immediately after harvest as the cost of
application is very similar if not cheaper than fungi-
cides. Potential also exists for using mixtures of
antioxidants to try and obtain a synergistic effect on
control of growth and fumonisins by these important
pathogens of maize. Previous studies have found that
combinations of antioxidants can be more effective
than individual ones (Narasimhan et al., 1989), with
Elad (1992) demonstrating that combinations were
better for controlling fungal diseases of pepper and
tomato. This approach is now under investigation.
324 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324
Acknowledgements
We are grateful to the British Council/Foundacion
Antorchas for grant to fund this work.
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