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-

eases of maize are caused by different Fusarium
Maize (Zea mays L) is one of the main crops in species. The Fusarium species predominantly respon-
Argentina, with a total production of approximately sible for ear rot in Argentina are Fusarium verticil-
19 million tons. Half of the production is exported and lioides (Sacardo) Nirenberg, Fusarium proliferatum
the rest is devoted to the internal market both for (Matsushima) Nirenberg, and Fusarium subglutinans
animal feed and foodstuffs (Secretarı́a de Agricultura, (Wollenweber et Reinking) Nelson, Tousson, and
Marassas. These three species have also been isolated
from asymptomatic maize kernels both in the field and
* Corresponding author. Tel.: +44-1525-863539; fax: +44- during postharvest storage (Chulze et al., 1996; Gon-
1525-863540. zalez et al., 1995; Ramirez et al., 1996; Sydenham et
E-mail address: [email protected] (N. Magan). 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- (a) the lag phase prior to growth, (b) growth rates and
ble for the production of fumonisins, which have (c) control of fumonisin production by strains of F.
become common contaminants of maize. They are verticillioides, F. proliferatum under different aw con-
of concern due to their toxicological implications to ditions on irradiated maize kernels at 25 jC.
different biological systems, and cause several myco-
toxicosis such as leucoencephalomalacia in horses,
pulmonary edema in pig and liver cancer in rats 2. Materials and methods
(Gelderblom et al., 1988; Ross et al., 1990; Thiel et
al., 1992). Also these mycotoxins have shown phyto- 2.1. Fungal strains
toxic effects (Abbas et al., 1999). Epidemiological
studies also suggest that fumonisins could be associ- F. verticillioides (RC 2000) and F. proliferatum
ated with human esophageal cancer in some regions (ITEM 2443) were isolated from maize in Argentina.
with high intake rate of maize contaminated with These strains have been demonstrated to be good
fumonisins (Franceschi et al., 1990). fumonisin producers. The strains were maintained in
Several approaches have been taken to try and glycerol (15%, Sigma-Aldrich, Dorset, UK) at 80
diminish the human and animal exposure to these jC and kept in the culture collection at the Depart-
mycotoxins. In the field, the use of cultivars less ment of Microbiology and Immunology, Universidad
susceptible to toxin production, control of insects Nacional de Rio Cuarto, Cordoba, Argentina, and the
and weeds, and cultural practices are all been exam- Institute Tossine e Micotossine da Parassiti Vegetali,
ined. Antioxidants have shown some effects on con- Bari, Italy.
idial germination, growth and mycotoxin production
by aflatoxigenic species of Aspergillus flavus and 2.2. Substrate and antioxidants
Aspergillus parasiticus (Thompson, 1991). Later stud-
ies by Thompson (1992, 1994) and Thompson et al. Irradiated maize kernels (12 kGy) with retained
(1993) showed that antioxidants also could inhibit the germinative capacity were used in these studies. The
growth and moniliformin production by F. verticil- maize kernels were check for sterility and kept to 4
lioides and Penicillium species. However, in their jC. The antioxidants used were butylated hydroxya-
studies no account was taken of the effect of inter- nisole (BHA), and propyl paraben (PP), obtained from
action of antioxidants with natural grain substrates the Sigma. Stock solutions of each antioxidant were
and with key environmental factors such as water prepared in 95% ethyl alcohol.
activity (aw) or temperature. Although propionic acid-
based preservatives are recommended for maize, 2.3. Inoculation and incubation conditions
recent studies suggest that they are not effective at
controlling Fusarium species or fumonsin production Irradiated maize was rehydrated to 0.995, 0.98 and
over a range of environmental conditions (Marin et 0.95 aw by the addition of sterile distilled water and
al., 2000). Thus, alternative compounds are needed as reference to a moisture adsorption curve. The aw of
part of a corrective strategy if fumonsin enters the raw the grains was determined with a Thermoconstanter
material preharvest or during initial postharvest drying Novasina TH 200 (Novasina, Zurich, Switzerland).
and storage. Recent in vitro studies demonstrated that The cultures were incubated to 25 jC. The required
the food-grade antioxidants propyl paraben and buty- concentration of each antioxidant was added to reach
lated hydroxyanisole could control both growth and a final concentration of 100, 200 and 500 Ag g 1 of
fumonisin production by F. verticillioides and F. maize. The maize kernels were allowed to equilibrate
proliferatum in 2% maize meal extract agar over a at 4 jC for 48 h with periodic shaking to allow
range of water availability regimes (Etcheverry et al., absorption and equilibrium. The rehydrated maize
2002). kernels were dispensed as a monolayer (approxi-
The aim of this study was to evaluate the effect of mately 20 g) in sterile 9 cm Petri dishes.
the two best food grade antioxidants: butylated The plates were inoculated centrally with a 5-mm
hydroxyanisole (BHA), and propyl paraben (PP) on 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- about 50% to 30% ( = 1.0 to 0.98 aw) which is
nisins (FB1, FB2 and FB3) at almost all the BHA adequate for germination and growth of F. verticil-
concentrations evaluated, but the amount produced lioides and F. proliferatum. Also at the postharvest
was dependent on the concentration of antioxidant and storage stage, if there is some delay in drying
and the aw. The lower level of fumonisin or absence of these species could grow. The results in the present
fumonisin production was only observed in the pres- study showed that the antioxidants BHA and PP are
ence of BHA (500 Ag g 1 at 0.98 aw). When water effective in controlling both growth and toxin produc-
was freely available (0.995 aw) a reduction in fumo- tion on maize grains, but at higher doses than found in
nisin production was about 20 –30% in the presence a previous study on maize meal extract agar (Etch-
of BHA and PP. At 0.98 aw the reduction ranged everry et al., 2002). The different behaviour of the
between 94% and 98% at 500 Ag g 1. PP was more chemicals on the natural substrate and on culture
effective against F. proliferatum than F. verticillioides. media could be related to the distribution of the
Some stimulation of fumonisin production by F. antioxidants on the kernel surface and the pericarp
verticillioides was observed with BHA at 0.995 and barrier of the kernel that may not allow good contact
0.95 aw levels and 100 and 500 Ag g 1, respectively. between the fungi and the antioxidants.
During ripening maize ears are susceptible to The effect on fumonisin production is particularly
colonization and infection by F. verticillioides and F. important as this is of major concern in the food grain
proliferatum since the water availability changes from 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 At present BHA and PP are permitted for use as
production provided that the right concentration and antimicrobial agents in different foods and are on the
coverage can be achieved. It has been suggested that list of generally regarded as safe (GRAS) chemicals of
the Parabens do not have any specific effect on the Food and Drug Administration in the USA. It may
enzymes but appear to act mainly at the cell mem- be possible to examine these types of products as field
brane eliminating the pH component of the proton sprays or immediately after harvest as the cost of
motive force and thus disrupting energy transduction application is very similar if not cheaper than fungi-
and substrate uptake (Adams and Moss, 1995). With cides. Potential also exists for using mixtures of
regard to phenolic antioxidants various mechanisms antioxidants to try and obtain a synergistic effect on
have been proposed for efficacy. Evidence exists that control of growth and fumonisins by these important
they interfere with membrane lipid structure, and that pathogens of maize. Previous studies have found that
BHA, for example, causes cellular leakage in bacteria combinations of antioxidants can be more effective
(Degre and Sylvestre, 1983). Increased leakage of than individual ones (Narasimhan et al., 1989), with
sugars, amino acids and proteins from fungal mycelia Elad (1992) demonstrating that combinations were
has also been detected, suggesting disruption of the better for controlling fungal diseases of pepper and
membranes (Thompson, 1996a,b). tomato. This approach is now under investigation.
324 A.M. Torres et al. / International Journal of Food Microbiology 83 (2003) 319–324

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