toxins Toxins Toxins Toxins 2072-6651 MDPI 10.3390/toxins2061399 toxins-02-01399 Article Effect of Antioxidant Mixtures on Growth and Ochratoxin A Production of Aspergillus Section Nigri Species under Different Water Activity Conditions on Peanut Meal Extract Agar Barberis Carla Astoreca Andrea Fernandez-Juri María Guillermina Dalcero Ana María Magnoli Carina * Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional Nº 36 Km 601 (5800) Río Cuarto, Córdoba, Argentina; Email: cbarberis@exa.unrc.edu.ar (C.B.); aastoreca@exa.unrc.edu.ar (A.A.); jfernandezj@exa.unrc.edu.ar (M.G.F.-J.); adalcero@exa.unrc.edu.ar (A.M.D.). Author to whom correspondence should be addressed; Email: cmagnoli@exa.unrc.edu.ar; Tel.: +54-358-4676429; Fax: +54-358-4676231. 10 06 2010 062010 2 6 1399 1413 22 04 2010 26 05 2010 08 06 2010 © 2010 by the authors; licensee MDPI, Basel, Switzerland 2010

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

The effect of mixtures of antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) on lag phase, growth rate and ochratoxin A (OTA) production by four Aspergillus section Nigri strains was evaluated on peanut meal extract agar (PMEA) under different water activities (aw). The antioxidant mixtures used were: BHA + PP (mM), M1 (0.5 + 0.5), M2 (1.0 + 0.5), M3 (2.5 + 0.5), M4 (0.5 + 1.0), M5 (1.0 + 1.0), M6 (2.5 + 1.0), M7 (5.0 + 2.5) and M8 (10 + 2.5). The mixture M8 completely suppressed mycelial growth for all strains. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest aw; whereas M6, M7 and M8 completely inhibited OTA production in all strains assayed; except M6 in A. carbonarius strain (RCP G). These results could enable a future intervention strategy to minimize OTA contamination.

 butylated hydroxyanisol propyl paraben growth parameters ochratoxin A Aspergillus carbonarius A. niger aggregate peanut meal extract agar 1. Introduction

Peanut (Arachis hypogaea L.) is an important oilseed crop, and a major food legume, cultivated in over 100 tropical and subtropical countries. The seed has several purposes as whole seed or processed to make peanut butter, oil, soups, stews and other products. The cake has several uses in feed and infant food formulations. The protein, oil, fatty acid, carbohydrate and mineral content of this nut become sensitive to fungal contamination, in pre and post-harvest stage. The fungal contamination is one of the main problems when inappropriate processing and storage conditions occur [1].

This oilseed is one of the most important agricultural products in the Argentinean economy. The center-south region of Córdoba province produces 94% of the country’s production. The peanut industry exports 90% of its product, with Argentina being the second in the world in peanut exports. This activity is not a production chain, but meets all characteristics of a cluster such as geographical proximity, expertise and innovation [2]. At the post-harvest stage around 8% loss of the total production by peanut disease and mycotoxins contamination has been reported in recent years [3].

Aspergillus species are important contaminants of several pre, post harvest and stored cereal and oilseed grains. Furthermore toxigenic species of Aspergillus section Flavi and Nigri and the main mycotoxins (aflatoxins and ochratoxin A) have been detected in different nuts e.g., peanut kernels [4,5,6,7]. Aspergillus section Nigri species have acquired interest by their ability to produce ochratoxin A (OTA), a potent nephrotoxin known for the teratogenic, immunosuppressive and carcinogenic effects. It has been classified by the International Agency for Research on Cancer [8] as a possible human carcinogen (group 2B) based on sufficient evidence of carcinogenicity for animals and inadequate evidence in humans [9].

In our region, the presence of potential OTA-producer species has been recently detected in wine grapes, dried vine grapes, corn and stored peanut kernels [6,7,10,11,12].

Synthetic antioxidants, namely, food grade antioxidants and antimicrobials [13], are products widely used as preservatives especially in foods that contain oils or fats because they exhibit an exceptional stress oxidative protection. At present, butylated hydroxyanisole (BHA) and propyl paraben (PP) are permitted for use as antimicrobial agents in different foods and are on the list generally regarded as safe (GRAS) chemicals of the Food and Drug Administration in the USA. Several phenolic antioxidants showed in vitro biocidal action against yeast [14] and filamentous fungi [15]. These compounds showed the capacity to control mycotoxigenic fungi growth and mycotoxin accumulation in synthetic media and agricultural products such as corn and peanut kernels [16,17,18,19,20,21,22,23,24,25,26,27].

In previous studies, the effectiveness of BHA, butylated hydroxytoluene (BHT), and PP as fungal inhibitors in relation to A. flavus and A. parasiticus strains and their toxin accumulation on peanut meal extract agar has been determined. In these studies a fungal control was observed when these antioxidants and antimicrobial were applied in binary mixtures [23,24]. On the other hand, Reynoso et al. [17] observed that the binary mixtures of BHA and PP were effective to reduce the growth rate and fumonisin production by Fusarium verticillioides and F. proliferatum in corn meal extract agar.

Recently, the effect of BHA and PP alone over a wide range of concentrations (1 to 20 mM) on the growth rate and OTA production by the Aspergillus section Nigri species on peanut meal extract agar at three water activities was evaluated [28,29]. The results of those studies suggest that growth rate and OTA production by these strains are completely inhibited at concentrations of 20 and 5 mM of BHA and PP, respectively. However, there is no available information on the efficacy of antioxidants binary mixtures to determine the additive or synergistic effects on growth and OTA production by Aspergillus section Nigri strains under different environmental conditions in peanut kernels. Thus, the aim of the present study was to evaluate the effect of binary mixtures of the antioxidant, butylatedhydroxyanisole, and the antimicrobial, propyl paraben, on (i) the lag phase before growth; (ii) growth rates and (iii) OTA production by strains of Aspergillus section Nigri under different water activities on peanut meal extract agar.

 2. Materials and Methods 2.1. Fungal strains

Four Aspergillus section Nigri strains were evaluated: A. carbonarius (RCPG and RCP203), A. niger aggregate (RCP42 and RCP191). All of the strains were isolated previously from peanut kernels in Argentina [7]. The identification of strains was carried out on the basis of macroscopic and microscopic features of the fungal isolates. Only A. carbonarius isolates were identified at species level [30], whilst the other biseriate isolates were, on the whole, classified as A. niger aggregate [31]. Ochratoxin A production was assayed on YES medium (2% yeast extract, 15% sucrose) [7]. Strains were maintained in glycerol (15%) at −80 °C and kept in the culture collection at the Department of Microbiology and Immunology, National University of Río Cuarto, Córdoba, Argentina.

 2.2. Antioxidants

The antioxidant 2(3)-tert-Butyl-4-hydroxyanisole (BHA) and antimicrobial n-propyl 4-hydroxybenzoate (PP) were used and obtained from Sigma-Aldrich Chemical (Dorset, UK). Stock solutions of BHA and PP (1 M) were prepared by dissolving 18 g in 100 mL of ethyl alcohol absolute.

2.3. Culture medium

Peanut meal extract agar (PMEA) was prepared at 3% (w/v).Thirty grams of ground peanuts per liter were boiled for 45 min and filtering the resultant mixture through a double layer of muslin. The volume was made up to 1 L and agar-agar at 2% (w/v) was added [28,29]. The water activity of the basic medium was adjusted to 0.995, 0.980 and 0.930 with known amounts of glycerol [32]. The basic media was autoclaved at 120 °C for 20 min before cooling at 50 °C and poured into 90-mm sterile Petri dishes. From stock solutions of BHA and PP, an appropriate volume was added to autoclaved based media (PMEA) to reach the intended BHA and PP concentrations in each binary mixture: mixture M1 (0.5 + 0.5 mM), M2 (1.0 + 0.5 mM), M3 (2.5 + 0.5 mM), M4 (0.5 + 1.0 mM), M5 (1.0 + 1.0 mM), M6 (2.5 + 1.0 mM), M7 (5.0 + 2.5 mM) and M8 (10 + 2.5 mM). The PMEA medium with the same amount of ethyl alcohol absolute was used as control. Water activity of representative plates of each treatment was checked at the beginning and during the experiment with an AquaLab Series 3 (Decagon Devices, Inc., Pullman, WA, USA).

2.4. Inoculation and incubation conditions

Fungal strains were grown on malt extract agar (MEA) for 7 days at 25 °C to obtain heavily sporulating cultures, and a spot of spores suspended in soft agar was inoculated in the center of each plate. The plates were incubated under all the assayed conditions. Petri dishes of the same aw levels were sealed in polythene bags for maintaining constant aw levels. Four replicate plates per treatment were used and incubated at 25 °C for 4 weeks; all the experiments were repeated twice.

 2.5. Growth parameters

Assessment of growth was made daily during the incubation period, with peanut meal extract agar cultures being examined using a binocular magnifier (10×). Two diameters of the growing colonies were measured at right angles to each other until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression applied to obtain the growth rate (mm/day) as the slope of the line. Lag phase before growth (h) in each treatment was determined as the abscissa from the growth rate curves.

Number of growth and lag phase analysis = 3, aw × 1, T × 4, strains × 4, rep × 1, antioxidant and antimicrobial mixture × 9, treatments (8 mixtures and 1 control).

 2.6. Ochratoxin A extraction

At the end of the incubation period (28 days), OTA was determined following the methodology proposed by Bragulat et al. [33] with some modifications. From the plates of each treatment, three agar plugs were removed from different points of the colony and extracted with 1 mL of methanol. The mixture was centrifuged at 14,000 rpm for 10 minutes. The solutions were filtered, evaporated to dryness, re-dissolved in 200 µL of mobile phase (acetonitrile-water-acetic acid, 57: 41: 2) and the extract analyzed by high performance liquid chromatography (HPLC).

2.7. Detection and quantification of ochratoxin A

The production of OTA was detected and quantified by the methodology proposed by Scudamore & McDonald, [34] with some modifications, the reversed phase high performance liquid chromatography (HPLC). The HPLC system consisted of a Hewlett Packard model 1100 pump (Palo Alto, CA, USA) connected to a Hewlett Packard 1100 Series with fluorescence detector (λexc 330 nm; λem 460 nm) and a data module Hewlett Packard Kayak XA (HP ChemStation Rev. A.06.01). The C18 column (Supelcosil LC-ABZ, Supelco; 150 × 4.6 mm, 5 μm particle size), connected to a pre-column (Supelguard LC-ABZ, Supelco; 20 × 4.6 mm, 5 μm particle size) was used. The mobile phase was pumped at 1.0 mL/min. The injection volume was 100 μl and the retention time was around 4 minutes.

Number of OTA analysis = 3, aw × 1, T × 4, strains × 4, rep × 1, antioxidant and antimicrobial mixture × 9, treatments (8 mixtures and 1 control).

 2.8. Statistical analysis

Data analyses were done by analysis of variance. All data were transformed to lg (x + 1) to obtain the homogeneity of variance. Means were compared by Fisher´s Least Significant Difference Test to determine the influence of aw, antioxidant mixtures concentration and strains on lag phase before growth, growth rate and OTA levels produced by the section Nigri strains. The Pearson correlation coefficient was used to evaluate the strength of the relationship between growth rate and OTA levels produced by the strains. The analysis was conducted using PROC GLM in SAS (SAS Institute, Cary, NC) [35].

3. Results and Discussion 3.1. Effect of antioxidants treatments on lag phase and growth rate

The analysis of variance on the effect of single (strains, aw and antioxidant mixtures concentration) two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P < 0.0001) in relation to lag phases and growth rates forall Aspergillus section Nigri strains assayed (Table 1).

toxins-02-01399-t001_Table 1

Analysis of variance of water activity (aw), antioxidant mixtures (M) and different isolates (I), and their interactions on lag phase and growth rate of Aspergillus section Nigri strains at 25 °C.

Source of variation Df a Lag phase Growth rate
MS b F c MS b F c
I 3 118472.01 10.24* 47.24 15789.44*
M 7 17221856.84 1493.79* 399.36 99999.99*
aw 2 1278196.03 108.61* 40.33 13472.66*
I × M 28 60021.36 5.15* 31.00 10584.69*
I × M × aw 78 108999.99 9.33* 10.26 3552.52*

a Degrees of freedom. b Mean square. c F-Snedecor. * Significant P < 0.0001.

Table 2 shows the effect of mixtures of antioxidants (BHA + PP) in different concentrations on lag phase before growth (h) in four Aspergillus section Nigri strains. In general, all strains showed a similar behavior. Further, the lag phase increased significantly as aW decreased in control and each antioxidant mixture treatments. The lengthened lag phases were observed in the presence of the highest concentrations (M7 and M8) of both antioxidant and antimicrobial combinations for A. carbonarius and A. niger aggregate strains. The mixture M2 and M5 showed that an increase in the doses of PP from 0.5 to 1.0 mM resulted in an increase in the lag phase from 31 to 73 h and from 32 to 93 h at 0.980 aw for A. carbonarius (RCP 203) and A. niger aggregate (RCP 42), respectively (P < 0.0001). The mixture M7 BHA + PP (5.0 + 2.5 mM) showed the highest lag phase before growth, 117 and 216 h at 0.995 aw for A. carbonarius (RCP 203) and A. niger aggregate (RCP 191), respectively. At the highest concentration of antioxidants mixture (M8) BHA + PP (10 + 5.0 mM) showed that all the strains tested were not able to reach the exponential phase.

toxins-02-01399-t002_Table 2

Effect of antioxidant mixtures (BHA, butylatedhydroxyanisol; PP, propyl paraben) and water activity (aw) on the lag phase of Aspergillus section Nigri strains on peanut meal extract agar at 25 °C.

Strains aw Lag phase (h)
(BHA + PP) (mM)
0 M 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8
RCP Ga 0.995 11 jk 21 lmno 28 fghi 36 h 28 fghi 41 fgh 68 fg - -
0.980 20 klm 30 fghi 32 ghi 54 defghi 40 fgh 62 ij 79 g - -
0.930 28 fghi 28 fghi 57 ij 118 de 64 fg 67 fg 97 fg - -
RCP 203a 0.995 15 klmno 27 fghi 22 ijklm 21 lmno 45 gh 60 ij 86 gh 117 de -
0.980 18 lmno 33 fghi 31 fghi 40 fgh 65 fg 73 defg 86 gh - -
0.930 46 gh 58 ij 49 gh 79 defg 76 defg 80 g 103 fg - -
RCP 42b 0.995 10 jk 26 fghi 59 ij 53 defghi 77 defg 89 gh 110 d - -
0.980 16 klmno 29 fghi 32 fghi 39 fgh 52 defghi 93 fg - - -
0.930 27 fghi 51 defghi 59 ij 59 ij 84 g 260 a - - -
RCP 191b 0.995 10 jk 13 ghi 18 lmno 26 fghi 26 fghi 40 fgh 45 gh 216 b -
0.980 21 lmno 27 fghi 23 ijklm 29 fghi 38 fgh 34 fghi 60 ij - -
0.930 27 fghi 47 gh 42 fgh 55 defghi 59 ij 95 fg 131 c - -

Mean in a row with a letter in common is not significantly different according to LSD test (P < 0.0001). (–) Under these conditions the strains were not able to reach the exponential phase.

a A. carbonarius (RCP G and RCP 203); b Aspergillus niger aggregate (RCP 42 and RCP 191).

Mixtures (BHA + PP): M1 (0.5 + 0.5 mM), M2 (1.0 + 0.5 mM), M3 (2.5 + 0.5 mM), M4 (0.5 + 1.0 mM), M5 (1.0 + 1.0 mM), M6 (2.5 + 1.0 mM), M7 (5.0 + 2.5 mM) and M8 (10 + 2.5 mM).

Combined effect of butylatedhydroxyanisol (BHA) and propyl paraben (PP) and water activity (aw) on the growth rate of Aspergillus carbonarius RCP G (A), RCP 203 (B) and A. niger aggregate RCP 42 (C) and RCP 191 (D) on peanut meal extract agar at 25 °C.

Figure 1 shows the effect of antioxidant mixtures (BHA + PP) in different concentrations on the growth rate of Aspergillus section Nigri strains at different aW levels at 25 °C. The antioxidant mixture M8 (10 + 2.5 mM) completely suppressed mycelial growth for all Aspergillus section Nigri strains at different aw levels assayed. This behavior was observed with mixture M7 (5.0 + 2.5 mM) on A. carbonarius (RCP G) and A. niger aggregate (RCP 42) at all aw assayed, and on A. carbonarius (RCP 203) at 0.930 and 0.980 and on A. niger aggregate (RCP 191) at the lowest aw. While with mixture M6 completely suppressed mycelial growth was observed only on A. niger aggregate (RCP 42) at 0.930 and 0.980 aw. No significant differences between the mixtures BHA + PP M4 (0.5 + 1.0 mM) and M5 (1.0 + 1.0 mM) on growth rate of A. carbonarius RCP 203 at all aw assayed and between M3 (2.5 + 0.5 mM) and M4 (0.5 + 1.0 mM) for both A. niger aggregate strains at 0.980 and 0.995 aW were observed (P < 0.0001). At 0.980 aw the growth rates of A. carbonarius RCP G strain were significantly highest in mixtures M2 to M6; whereas in A. niger aggregate RCP 191 strain this behavior was observed in control treatment and mixtures M1 to M6 (P < 0.0001).

3.2. Effect of antioxidants treatments on ochratoxin A production

Combined effect of butylatedhydroxyanisol (BHA) and propyl paraben (PP) and water activity (aw) on OTA production by Aspergillus carbonarius RCP G (A), RCP 203 (B) and A. niger aggregate RCP 42 (C), RCP 191 (D) on peanut meal extract agar at 25 °C. Detection limit: 1 ng g−1 (ppb). Antioxidant mixtures (BHA + PP): M1 (0.5 + 0.5 mM), M2 (1.0 + 0.5 mM), M3 (2.5 + 0.5 mM), M4 (0.5 + 1.0 mM), M5 (1.0 + 1.0 mM), M6 (2.5 + 1.0 mM), M7 (5.0 + 2.5 mM) and M8 (10 + 2.5 mM).

Recovery of the toxin was 89.2 ± 9.7% from the peanut meal extract agar at tested OTA levels. The detection limit of the technique was 1 ng g−1. The effect of the concentration range of BHA + PP mixtures/aw treatment at 25 °C on OTA production for all the tested strains is shown in Figure 2. In general, OTA production did not show a similar pattern to the one on the growth rate. The effect of the antioxidants treatments was variable and depended on the species and the water availability assayed. Ochratoxin A production was significantly reduced in respect of control in both A. carbonarius strains with all mixtures. On the other hand, for both A. niger aggregate strains, the response varied to the binary mixture assayed. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest aw, while mixtures M6 (2.5 + 1.0 mM), M7 (5.0 + 2.5 mM) and M8 (10 + 2.5 mM) completely inhibited OTA production in all strains assayed; except to M6 in A. carbonarius strain (RCP G).

The analysis of variance on the effect of single (strains, aW and antioxidant mixture concentration) two- and three- way interaction showed that all factors alone and all interactions were statistically significant (P < 0.0001) in relation to OTA production for all Aspergillus section Nigri strains assayed (Table 3). The Fisher´s Least Significant Difference test (LSD) of data shows the influence of water activity, BHA and PP mixture concentration on growth parameters (lag phase and growth rate) and OTA production. The statistical analysis of all strains showed that all analyzed factors influenced significantly on growth parameters, whereas on OTA production, aw and mixture concentration influenced significantly (P < 0.05) (Table 4).

toxins-02-01399-t003_Table 3

Analysis of variance of water activity (aw), antioxidant mixtures (M) and different isolates (I), and their interactions on OTA production by Aspergillus section Nigri strains at 25 °C.

Source of variation OTA production
Df a MS b F c Pr > F
I 3 67.65 15159.37* 0.0001
M 7 217.73 48788.22* 0.0001
aw 2 29.66 6623.96* 0.0001
I × M 28 9.33 2071.76* 0.0001
I × M × aw 78 1.45 322.30* 0.0001

a Degrees of freedom. b Mean square. c F-Snedecor. * Significant P < 0.0001.

 toxins-02-01399-t004_Table 4

Influence of water activity (aw), and antioxidant mixtures (M)on growth parameters and OTA production at 25 °C.

Treatments Growth rate (mm d−1) Lag phase (h) OTA production (ng g−1)
Mean ± SD
Water activity (aw)
0.995 0.44 ± 0.37 a 1.93 ± 0.68 a 1.59 ± 1.37 a
0.980 0.41 ± 0.33 a 1.97 ± 0.74 a 1.50 ± 1.42 ª
0.930 0.28 ± 0.28 b 2.21 ± 0.76 b 1.09 ± 1.09 b
Antioxidant mixtures BHA + PP (mM)
M1 (0.5 + 0.5) 5.72 ± 0.26 a 1.16 ± 0.55 a 2.76 ± 2.02 a
M2 (1.0 + 0.5) 3.89 ± 0.16 b 1.55 ± 0.46 b 2.64 ± 1.74 a
M3 (2.5 + 0.5) 1.39 ± 0.21 c 1.88 ± 0.58 c 1.66 ± 1.47 b
M4 (0.5 +1.0) 1.72 ± 0.75 c 1.91 ± 0.99 c 0.45 ± 0.37 c
M5 (1.0 +1.0) 0.87 ± 0.36 d 2.02 ± 1.00 c 0.11 ± 0.07 d
M6 (2.5 +1.0) 0.29 ± 0.16 d 2.36 ± 0.59 d 0.08 ± 0.02 d
M7 (5.0 + 2.5) 0.17 ± 0.14 d 2.22 ± 0.49 d 0.00 ± 0.00 d
M8 (10 + 2.5) 0.00 ± 0.00 d 2.05 ± 1.00 e 0.00 ± 0.00 d

Data were transformed to lg (x + 1). a, b, c, d, e Groups with different letters are significantly different according to Fisher’s LSD test (P < 0.05). SD: standard deviation.

A significant (p < 0.05) correlation (r = 0.525, r = 0,715, r = 0,469, r = 0,630 for RCP G, RCP 203, RCP 42 and RCP G, respectively) between the growth rate and OTA production in all Aspergillus section Nigri strains was found (data not shown).

In this study, it was evaluated whether the different binary mixtures of BHA and PP might be able to inhibit the Aspergillus section Nigri species growth and OTA production at 25 °C on peanut meal extract agar.

In previous works carried out with these strains and PMEA, it was observed that 20 mM of BHA or 5 mM of PP at 25 °C, completely inhibited both the growth rate and OTA production in all aW condition assayed [28,29]. The results obtained in the present work showed that the combinations of BHA and PP at lower concentrations (M7: 5.0 + 2.5 and M8: 10 + 2.5) than those previously used showed that all the strains tested were not able to reach the exponential phase, completely inhibiting the growth rate of all Aspergillus section Nigri strains. In addition, the combination of BHA and PP at lower concentrations than each antioxidant separately (M6, M7 and M8) reduced OTA production significantly in all environmental conditions assayed. To discern the effect of antioxidants on lag phase of these fungal species is important because it could prevent the visible fungal growth on grains and consequent mycotoxin production.

4. Discussion

The results obtained regarding OTA production with mixtures M1 to M5 in A. niger aggregate strains suggest that the growth control does not necessarily mean that toxin production is also inhibited. In previous works, stimulation production of other mycotoxin in presence of low concentrations of antioxidants has been observed. Fumonisin stimulation has been informed with mixture of BHT + PP (1 mM) by F. verticillioides and F. proliferatum at 0.995 aw on maize based media [17]. Similar effects have been observed by Nesci et al. [36], who observed stimulation of AFB1 production by A. flavus and A. parasiticus strains with some combinations of subinhibitory concentrations of natural phytochemicals (ferulic and cinamic acids) on maize grains at different aW conditions. It has been reported that subinhibitory doses together with inadequate distribution of preservative, especially at low water activities, could enhance fungal growth [37,38]. This behavior was also observed in population of Aspergillus species with some concentration of propionate mixtures [39], trihydroxybutyrophenone (THB) [23], peppermint and boldus [40], quercetin and caffeic acid [41]. This behavior suggests that the combinations of antioxidants should be assayed at several concentrations, with various strains and environmental conditions to find safe concentrations and prevent undesirable effects.

Some authors [25], evaluated the effects of phenolic antioxidant in OTA production by A. carbonarius on synthetic medium. They showed that some antioxidants, e.g., gallic, 4-hydroxybenzoic and chlorogenic acids tended to inhibit OTA production. In other study with other phenolic compounds, Romero et al. [41], observed a significant reduction in growth rate and OTA production with 250 mg L−1 of caffeic acid, rutin and quercetin. Recently, these authors [27] reported that the highest concentration of gallic acid (500 mg L-1) had significant effect on lag phase and growth rate in A. carbonarius strains. Whereas, a significant effect on OTA production was observed even at the lowest concentration (100 mg L-1) assayed.

Many food preservatives and fungicides have been used in combination. Indeed, some works showed that low concentrations of both antioxidants may be more effective together than either one alone. Reynoso et al. [17] demonstrated a synergistic effect of the mixture BHA + PP at 0.5 + 1.0 mM to control the growth rate and fumonisin production by F. verticillioides and F. proliferatum at 0.995 and 0.980 aW on maize based media. In another study, [24] showed that higher concentrations of BHA + PP (20 + 10 or 10 + 20 mM) at 0.982 aw and BHA + PP (10 + 20 or 20 + 10 mM) at 0.955 aw completely inhibited growth rate of A. flavus and A. parasiticus strains on peanut seeds. In addition, all antioxidant mixtures (BHA + PP, 10 + 10, 10 + 20, 20 + 10 and 20 + 20 mM) significantly reduced AFB1 accumulation after 11 and 35 days at 0.982 aw levels for all Aspergillus section Flavi strains assayed. The results of the present work concur with results of the above mentioned authors who claimed that AFB1 production depends on aw levels, incubation time and applied antioxidant mixtures (BHA + PP). In other studies, other antioxidants in combination have been tested as potent fungal inhibitors, and it was observed that benzoic and sorbic acids together are better than alone, inhibiting spoilage yeasts and filamentous fungi [42]. Khan et al. [43] showed that the combination of BHA (5 mM) and imazalil (250 mM) reduced the development of anthracnose lesions by 65% after inoculation of banana fruit with Colletotrichum musae conidia.

The peanut grain storage in our country extends from six to eight months and peanuts are subjected to thermal treatment for the production of peanut oil, roasted peanuts and other products, before they reach the consumer. In previous study, Passone et al. [44] showed that the levels of BHA and PP in pods had decreased 66 to 75% and 69 to 76% of the initial levels, respectively. This reduction in the antioxidant concentration during the peanuts storage ensures that the residue of antioxidants in this substrate did not exceed the maximum levels established (200 µg g−1 fat or oil content of the food product) [45].

Current information about action mechanisms of BHA and PP on fungal species is limited. In general, for phenolic compounds several mechanisms of antifungal activity have been proposed. It has been suggested that BHA affects the cell membrane by changing pH values and affecting transduction energy and substrate transport [46,47]. With respect to parabens, the following antimicrobial mechanisms have been determined: enzymatic function inhibition, lipid membrane dissolution, nutrients transport interference, potential membrane destruction and RNA and DNA synthesis alteration [48]. Recently, Kim et al. [49] showed that the antioxidant caffeic acid is a potent antiaflatoxigenic agent in A. flavus strains. The action mode of this anti-aflatoxigenic activity appears to be associated with attenuation of the oxidative stress response of the fungus to organic peroxides. However, there is no information about the anti-ochratoxigenic activity of BHA or PP in Aspergillus section Nigri species.

5. Conclusions

The results obtained in the present study suggest that only determinate combination of BHA and PP antioxidants present additive or synergistic effects on growth rate and OTA production by Aspergillus section Nigri strains. The binary mixture M7 and M8 (BHA + PP: 5.0 + 2.5 mM and 10 + 2.5 mM) could be the appropriate antioxidants combination to control Aspergillus section Nigri strains on synthetic media. This behavior could be due to different action mechanisms of these antioxidants on fungal cell at diverse target levels.

These in vitro studies must be corroborated by in situ conditions on peanut grains before applying the products directly on food commodities. In the future, these antioxidants could be applied in storage peanuts to prolong the hygienic quality of grains and to diminish the entry of ochratoxigenic fungi and OTA into the animal and human food chain.

 Acknowledgements

This work was carried out thanks to grants from the Consejo Nacional de Ciencia y Tecnología (CONICET-PIP), Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto (SECYT-UNRC) and Fondo para la Investigación Científica y Tecnológica (FONCYT-PICTO).

 References Asis R. Barrionuevo D.L. Giorda L.M. Nores M.L. Aldao M.A. Aflatoxin production in six peanut (Arachis hypogaea L.) genotypes infected with Aspergillus flavus and Aspergillus parasiticus, isolated from peanut production areas of Cordoba, Argentina J. Agric. Food Chem. 2005 53 9274 9280 16277433 Bongiovanni R.M. Gilleta M. Asociación Argentina de Economía Agraria. Análisis económico del cultivo de maní bajo diferentes rotaciones y sistemas de labranza 2009 (Accessed on 15 December 2009) Available online:http://agro.uncor.edu/~aaea2007/Comunicaciones%20A/Bongiovanni.pdf Secretaría de Agricultura Ganadería Pesca y Alimentación (SAGPyA) 2009 (Accessed on 15 December 2009) Available online:http://www.sagypa.mecon.gov.ar Barros G. Torres A. Palacio G. Chulze S. Aspergillus species from section Flavi isolated from soil at planting and harvest time in peanuts-growing regions of Argentina J. Sci. Food Agric. 2003 3 1303 1307 Barros G. Torres A. Chulze S. Aspergillus flavus population isolated from soil of Argentina's peanut-growing region. Sclerotia production and toxigenic profile J. Sci. Food Agric. 2005 85 2349 2353 10.1002/jsfa.2257 Magnoli C. Astoreca A. Ponsone L. Chiacchiera S. Dalcero A. Ochratoxin A and the occurrence of ochratoxin A- producing black Aspergilli in stored peanut seeds from Córdoba, Argentina J. Sci. Food Agric. 2006 86 2369 2373 10.1002/jsfa.2625 Magnoli C. Astoreca A. Ponsone L. Fernández-Juri M.G. Barberis C.L. Dalcero A.M. Ochratoxin A and Aspergillus section Nigri in peanut seeds at different months of storage in Córdoba, Argentina Int. J. Food Microbiol. 2007 119 213 218 10.1016/j.ijfoodmicro.2007.07.05617854935 IARC Ochratoxin A IARC Monographs on the evaluation of carcinogenic risks to human: some naturally occurring substances; food items and constituents, heterocyclic aromatic amines and mycotoxins International Agency for Research on Cancer Lyon, France 1993 56 26 32 Pfohl-Leszkowicz A. Manderville R.A. Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans Mol. Nutr. Food Res. 2007 51 61 99 10.1002/mnfr.20060013717195275 Magnoli C. Astoreca A. Ponsone L. Combina M. Palacio G. da Rocha Rosa C.A. Dalcero A.M. Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets Lett. Appl. Microbiol. 2004 39 326 331 10.1111/j.1472-765X.2004.01583.x15355533 Magnoli C. Hallak C. Astoreca A. Ponsone L. Chiacchiera S. Dalcero A.M. Occurrence of ochratoxin A- producing fungi in commercial corn kernels in Argentina Mycopathologia 2006 161 53 58 10.1007/s11046-005-0237-516389485 Ponsone M.L. Combina M. Dalcero A. Chulze S. Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems Int. J. Food Microbiol. 2007 114 131 135 10.1016/j.ijfoodmicro.2006.07.00117196283 Safety Evaluation of Certain Food Additives WHO Food Additives Series: 47 Fifty-sixth Meeting of the Joint FAO/WHO Expert Committee on Food Additive (JECFA) World Health Organization Geneva, Switzerland February 2001 Rivera-Carriles K. Argaiz A. Palou E. Lopez-Malo A. Synergistic inhibitory effect of citral with selected phenolics against Zygosaccharomyces bailii J. Food Prot. 2005 68 602 606 15771189 Ahn Y.J. Lee H.S. Oh H.S. Kim H.T. Lee Y.H. Antioxidant activity and phenolic composition of citrus peel and seed extracts J. Agric. Food Chem. 2005 46 2123 2129 Etcheverry M. Torres A. Ramirez M.L. Chulze S. Magan N. In vitro control of growth and fumonisin production by Fusarium verticilloides and Fusarium proliferatum using antioxidants under different water availability and temperature regimes J. Appl. Microbiol. 2002 92 624 632 10.1046/j.1365-2672.2002.01566.x11966902 Reynoso M. Torres A. Ramírez M.L. Rodríguez M. Chulze S. Magan N. Efficacy of antioxidant mixtures on growth, fumonisins production and hydrolytic enzyme production by Fusarium verticillioides and F. proliferatum in vitro on maize-based media Mycol. Res. 2002 106 1093 1099 10.1017/S0953756202006135 Torres A.M. Ramírez M.L. Arroyo M. Chulze S. Magan N. Potencial use of antioxidants for control of growth and fumonisin production by Fusarium proliferatum and Fusarium verticilloides on whole maize grain Int. J. Food Microbiol. 2003 83 319 324 10.1016/S0168-1605(02)00380-X12745236 Selvi A.T. Joseph G.S. Jayaprakasha G.K. Inhibition of growth and aflatoxin production in Aspergillus flavus by Garcinia indica extract and its antioxidant activity Food Microbiol. 2003 20 455 460 10.1016/S0740-0020(02)00142-9 Nesci A. Rodriguez M. Etcheverry M. Control of Aspergillus growth and aflatoxins production using antioxidants at different conditions of water activity and pH J. Appl. Microbiol. 2003 95 279 287 10.1046/j.1365-2672.2003.01973.x12859759 Joseph G.S. Jayaprakasha G.K. Selvi A.T. Jena B.S. Sakariah K.K. Antiaflatoxigenic and antioxidant activities of Garcinia extracts Int. J. Food Microbiol. 2005 101 153 160 10.1016/j.ijfoodmicro.2004.11.00115862877 Farnochi C. Torres A. Magan N. Chulze S. Effect of antioxidants and competing mycoflora on Fusarium verticilloides and F. proliferatum populations and fumonisin production on maize grain J. Stored Prod. Res. 2005 41 211 219 10.1016/j.jspr.2004.03.004 Passone M.A. Resnik S.L. Etcheverry M.G. In vitro effect of phenolic antioxidants on germination, growth and aflatoxin B1 accumulation by peanut Aspergillus section Flavi J. Appl. Microbiol. 2005 99 682 691 10.1111/j.1365-2672.2005.02661.x16108810 Passone M.A. Resnik S.L. Etcheverry M.G. Antiaflatoxigenic property of food grade antioxidants under different conditions of water activity in peanut grains Int. J. Food Microbiol. 2007 118 8 14 10.1016/j.ijfoodmicro.2007.05.01217628129 Palumbo J. O’Keeffe T. Mahoney N. Inhibition of ochratoxin A production and growth of Aspergillus species by phenolic antioxidant compounds Mycopathologia 2007 164 241 248 17874203 10.1007/s11046-007-9057-0 Bisogno F. Mascoti L. Sanchez C. Garibotto F. Giannini F. Kurina-Sanz M. Enriz R. Structure-antifungal activity relationship of cinnamic acid derivatives J. Agric. Food Chem. 2007 55 10635 10640 10.1021/jf072909818038998 Romero S.M. Alberto M.R. Vaamonde G. Effect of gallic acid on Aspergillus carbonarius growth and ochratoxin A production World Mycotox. J. 2010 3 45 48 10.3920/WMJ2009.1181 Barberis C. Astoreca A. Fernández-Juri M.G. Chulze S. Magnoli C. Dalcero A. Use of propyl paraben to control growth and ochratoxin A production by Aspergillus section Nigri species on peanut meal extract agar Int. J. Food Microbiol. 2009 136 133 136 10.1016/j.ijfoodmicro.2009.08.02519744738 Barberis C. Astoreca A. Asili R. Fernández-Juri M.G. Chulze S. Magnoli C. Dalcero A. In vitro control of growth and ochratoxin A production by butylatedhydroxyanisole in Aspergillus section Nigri species Food Contr. 2009 20 709 715 10.1016/j.foodcont.2008.09.003 Samson R.A. Noonin P. Meijer M. Houbraken J. Frisvad J.C. Varga J. Diagnostic tools to identify black Aspergilli Stud. Mycol. 2007 59 129 145 10.3114/sim.2007.59.1318490945 Klich M.A. Identification of common Aspergillus species Centraalbureau voor Schimmelcultures Utrecht, The Netherlands 2002 116 Marín S. Sanchis V. Viñas I. Canela R. Magan N. Effect of water activity and temperature on growth and fuminisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain Lett. Appl. Microbiol. 1995 21 298 301 7576524 10.1111/j.1472-765X.1995.tb01064.x Bragulat M.R. Abarca M.L. Cabañes F.J. An easy screening method for fungi producing ochratoxin A in pure culture Int. J. Food Microbiol. 2001 71 139 144 10.1016/S0168-1605(01)00581-511789931 Scudamore K.A. MacDonald S.J. A collaborative study of an HPLC method for determination of ochratoxin A in wheat using immunoaffinity column clean-up Food Addit. Contam. 1998 15 401 410 10.1080/026520398093746599764209 Quinn G.P. Keough M.J. Experimental Design Data Analysis for Biologists Cambridge University Press Cambridge, UK 2002 537 Nesci A. Gsponer N. Etcheverry M. Natural maize phenolic acids for control of aflatoxigenic fungi on maize J. Food Sci. 2007 72 180 185 10.1111/j.1750-3841.2007.00394.x Smith J.E. Moss M.O. Mycotoxins. Formation, Analyses and Significance Wiley Chichester, UK 1985 10 31 Lacey J. Prevention of mould growth and mycotoxin production through control of environmental factors Mycotoxins and Phycotoxins. Bioactive Molecules Natori S. Hashimoto K. Ueno Y. Elsevier Science Amsterdam, The Netherlands 1989 10 161 169 Marín S. Magan N. Albellana M. Caenal R. Ramos A.J. Sanchis V. Selective effect of propionates and water activity on maize mycoflora and impact on fumonisin B1 accumulation J. Stored Prod. Res. 2000 36 203 214 10.1016/S0022-474X(99)00043-0 Bluma R. Amaiden M.R. Daghero J. Etcheverry M. Control of Aspergillus section Flavi growth and aflatoxin accumulation by plant essential oils J. Appl. Microbiol. 2008 105 203 214 10.1111/j.1365-2672.2008.03741.x18284488 Romero S.M. Alberto M.R. Manca de Nadra M.C. Vaamonde G. Inhibition of growth and ochratoxin A biosynthesis in Aspergillus carbonarius by flavonoid and nonflavonoid compounds Mycotox. Res. 2009 25 165 170 10.1007/s12550-009-0026-y Liewen M.B. Antifungal food additivies Handbook of Applied Mycology. Food and Feeds Arora D.K. Mukerji K.G. Marth E.H. Marcell dekker New York, NY, USA 1991 3 541 552 Khan S.H. Aked J. Magan N. Control of the anthracnose pathogen of banana (Colletotrichum musae) using antioxidants alone and in combination with thiabendazole or imazalil Plant Pathol. 2001 50 601 608 10.1046/j.1365-3059.2001.00599.x Passone M.A. Funes G.J. Resnik S.L. Etcheverry M.G. Residue levels of food-grade antioxidants in postharvest treated in-pod peanuts during five months of storage Food Chem. 2008 106 691 697 10.1016/j.foodchem.2007.06.032 Codex alimentarius, Food additive details. Update up to the twenty-ninth session of the codex alimentarius commission The Joint FAO/WHO Committee on Food Additives. WHO Geneva, Switzerland July 2006 (Accessed on 15 April 2010) Available online:http://www.codexalimentarius.net/web/jecfa Degré R. Sylvestre M. Effect of butylated hydroxyanisole on the cytoplasmic membrane of Staphylococcus aureus Wood 46 J. Food Protect. 1983 46 206 209 Aldunate J. Coloma Torres L. Spenser P. Morello A. Ojeda J.M. Repetto Y. Effects of 2-(3)-tert-butyl-4-hydroxyanisole (BHA) on in situ mitochondria of Tripanosoma cruzi FEBS Lett. 1992 303 73 76 10.1016/0014-5793(92)80480-51592119 Eklund T. Organic acids and esters Mechanisms of Action of Food Preservation Procedures Gould G.W. Elsevier Applied Science New York, NY, USA 1989 181 182 Kim J.H. Yu J. Mahoney N. Chan K.L. Molyneux R.J. Varga J. Bhatnagar D. Cleveland T.E. Nierman W.C. Campbell B.C. Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis Int. J. Food Microbiol. 2008 122 49 60 18166238 10.1016/j.ijfoodmicro.2007.11.058