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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

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
*
Author to whom correspondence should be addressed.
Submission received: 20 April 2010 / Revised: 26 May 2010 / Accepted: 8 June 2010 / Published: 10 June 2010

Abstract

:
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.

Graphical Abstract

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).
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.
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 variationDf aLag phaseGrowth rate
MS bF cMS bF c
I3118472.0110.24*47.2415789.44*
M717221856.841493.79*399.3699999.99*
aw21278196.03108.61*40.3313472.66*
I × M2860021.365.15*31.0010584.69*
I × M × aw78108999.999.33*10.263552.52*
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.
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.
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.
StrainsawLag phase (h)
(BHA + PP) (mM)
0M 1M 2M 3M 4M 5M 6M 7M 8
RCP Ga0.99511 jk21 lmno28 fghi36 h28 fghi41 fgh68 fg--
0.98020 klm30 fghi32 ghi54 defghi40 fgh62 ij79 g--
0.93028 fghi28 fghi57 ij118 de64 fg67 fg97 fg--
RCP 203a0.99515 klmno27 fghi22 ijklm21 lmno45 gh60 ij86 gh117 de-
0.98018 lmno33 fghi31 fghi40 fgh65 fg73 defg86 gh--
0.93046 gh58 ij49 gh79 defg76 defg80 g103 fg--
RCP 42b0.99510 jk26 fghi59 ij53 defghi77 defg89 gh110 d--
0.98016 klmno29 fghi32 fghi39 fgh52 defghi93 fg---
0.93027 fghi51 defghi59 ij59 ij84 g260 a---
RCP 191b0.99510 jk13 ghi18 lmno26 fghi26 fghi40 fgh45 gh216 b -
0.98021 lmno27 fghi23 ijklm29 fghi38 fgh34 fghi60 ij--
0.93027 fghi47 gh42 fgh55 defghi59 ij95 fg131 c--
Figure 1. 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. 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.
Toxins 02 01399 g001
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

Figure 2. 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).
Figure 2. 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).
Toxins 02 01399 g002
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).
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.
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 variationOTA production
Df aMS bF cPr > F
I367.6515159.37*0.0001
M7217.7348788.22*0.0001
aw229.666623.96*0.0001
I × M289.332071.76*0.0001
I × M × aw781.45322.30*0.0001
a Degrees of freedom. b Mean square. c F-Snedecor. * Significant P < 0.0001.
Table 4. Influence of water activity (aw), and antioxidant mixtures (M)on growth parameters and OTA production at 25 °C.
Table 4. Influence of water activity (aw), and antioxidant mixtures (M)on growth parameters and OTA production at 25 °C.
TreatmentsGrowth rate (mm d−1)Lag phase (h)OTA production (ng g−1)
Mean ± SD
Water activity (aw)
0.9950.44 ± 0.37 a1.93 ± 0.68 a1.59 ± 1.37 a
0.9800.41 ± 0.33 a1.97 ± 0.74 a1.50 ± 1.42 ª
0.9300.28 ± 0.28 b2.21 ± 0.76 b1.09 ± 1.09 b
Antioxidant mixtures BHA + PP (mM)
M1 (0.5 + 0.5)5.72 ± 0.26 a1.16 ± 0.55 a2.76 ± 2.02 a
M2 (1.0 + 0.5)3.89 ± 0.16 b1.55 ± 0.46 b2.64 ± 1.74 a
M3 (2.5 + 0.5)1.39 ± 0.21 c1.88 ± 0.58 c1.66 ± 1.47 b
M4 (0.5 +1.0)1.72 ± 0.75 c1.91 ± 0.99 c0.45 ± 0.37 c
M5 (1.0 +1.0)0.87 ± 0.36 d2.02 ± 1.00 c0.11 ± 0.07 d
M6 (2.5 +1.0)0.29 ± 0.16 d2.36 ± 0.59 d0.08 ± 0.02 d
M7 (5.0 + 2.5)0.17 ± 0.14 d2.22 ± 0.49 d0.00 ± 0.00 d
M8 (10 + 2.5)0.00 ± 0.00 d2.05 ± 1.00 e0.00 ± 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

  1. 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. [Google Scholar] [PubMed]
  2. Bongiovanni, R.M.; Gilleta, M. Asociación Argentina de Economía Agraria. Análisis económico del cultivo de maní bajo diferentes rotaciones y sistemas de labranza. 2009. Available online: http://agro.uncor.edu/~aaea2007/Comunicaciones%20A/Bongiovanni.pdf (Accessed on 15 December 2009).
  3. Secretaría de Agricultura, Ganadería, Pesca y Alimentación (SAGPyA). 2009. Available online: http://www.sagypa.mecon.gov.ar (Accessed on 15 December 2009).
  4. 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. [Google Scholar]
  5. 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. [Google Scholar] [CrossRef]
  6. 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. [Google Scholar]
  7. 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. [Google Scholar]
  8. IARC, Ochratoxin A. In 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, pp. 26–32.
  9. 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. [Google Scholar]
  10. 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. [Google Scholar]
  11. 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. [Google Scholar]
  12. 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. [Google Scholar]
  13. Safety Evaluation of Certain Food Additives. In 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.
  14. 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. [Google Scholar]
  15. 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. [Google Scholar]
  16. 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. [Google Scholar]
  17. 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. [Google Scholar] [CrossRef]
  18. 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. [Google Scholar]
  19. 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. [Google Scholar]
  20. 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. [Google Scholar]
  21. 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. [Google Scholar]
  22. 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. [Google Scholar] [CrossRef]
  23. 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. [Google Scholar]
  24. 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. [Google Scholar]
  25. 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. [Google Scholar] [CrossRef] [PubMed]
  26. 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. [Google Scholar]
  27. 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. [Google Scholar]
  28. 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. [Google Scholar]
  29. 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. [Google Scholar]
  30. 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. [Google Scholar]
  31. Klich, M.A. Identification of common Aspergillus species; Centraalbureau voor Schimmelcultures: Utrecht, The Netherlands, 2002; p. 116. [Google Scholar]
  32. 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. [Google Scholar] [CrossRef] [PubMed]
  33. 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. [Google Scholar]
  34. 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. [Google Scholar]
  35. Quinn, G.P.; Keough, M.J. Experimental Design Data Analysis for Biologists; Cambridge University Press: Cambridge, UK, 2002; p. 537. [Google Scholar]
  36. Nesci, A.; Gsponer, N.; Etcheverry, M. Natural maize phenolic acids for control of aflatoxigenic fungi on maize. J. Food Sci. 2007, 72, 180–185. [Google Scholar]
  37. Smith, J.E.; Moss, M.O. Mycotoxins. Formation, Analyses and Significance; Wiley: Chichester, UK, 1985; pp. 10–31. [Google Scholar]
  38. Lacey, J. Prevention of mould growth and mycotoxin production through control of environmental factors. In Mycotoxins and Phycotoxins. Bioactive Molecules; Natori, S., Hashimoto, K., Ueno, Y., Eds.; Elsevier Science: Amsterdam, The Netherlands, 1989; Volume 10, pp. 161–169. [Google Scholar]
  39. 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. [Google Scholar]
  40. 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. [Google Scholar]
  41. 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. [Google Scholar]
  42. Liewen, M.B. Antifungal food additivies. In Handbook of Applied Mycology. Food and Feeds; Arora, D.K., Mukerji, K.G., Marth, E.H., Eds.; Marcell dekker: New York, NY, USA, 1991; Volume 3, pp. 541–552. [Google Scholar]
  43. 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. [Google Scholar]
  44. 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. [Google Scholar]
  45. 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. Available online: http://www.codexalimentarius.net/web/jecfa (Accessed on 15 April 2010).
  46. Degré, R.; Sylvestre, M. Effect of butylated hydroxyanisole on the cytoplasmic membrane of Staphylococcus aureus Wood 46. J. Food Protect. 1983, 46, 206–209. [Google Scholar]
  47. 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. [Google Scholar]
  48. Eklund, T. Organic acids and esters. In Mechanisms of Action of Food Preservation Procedures; Gould, G.W., Ed.; Elsevier Applied Science: New York, NY, USA, 1989; pp. 181–182. [Google Scholar]
  49. 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. [Google Scholar] [CrossRef] [PubMed]

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MDPI and ACS Style

Barberis, C.; Astoreca, A.; Fernandez-Juri, M.G.; Dalcero, A.M.; Magnoli, C. 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. Toxins 2010, 2, 1399-1413. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins2061399

AMA Style

Barberis C, Astoreca A, Fernandez-Juri MG, Dalcero AM, Magnoli C. 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. Toxins. 2010; 2(6):1399-1413. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins2061399

Chicago/Turabian Style

Barberis, Carla, Andrea Astoreca, María Guillermina Fernandez-Juri, Ana María Dalcero, and Carina Magnoli. 2010. "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" Toxins 2, no. 6: 1399-1413. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins2061399

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