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Journal of Food Protection, Vol. 73, No. 8, 2010, Pages 14931501

Copyright G, International Association for Food Protection

Food-Grade Antioxidants and Antimicrobials To Control Growth and Ochratoxin A Production by Aspergillus Section Nigri on Peanut Kernels
C. L. BARBERIS, A. L. ASTORECA, A. M. DALCERO,
AND

C. E. MAGNOLI*

a e Inmunolog a, Facultad de Ciencias Exactas, F sico-Qu micas y Naturales, Universidad Nacional de R o Cuarto, Ruta Departamento de Microbiolog o Cuarto, Co ficas y Te Nacional No. 36 Km 601 (5800), R rdoba, Argentina; and Consejo Nacional de Investigaciones Cient cnicas, Buenos Aires, Argentina MS 09-539: Received 30 December 2009/Accepted 19 March 2010

ABSTRACT
Each year, a significant portion of the peanuts produced cannot be marketed because of fungal disease at the postharvest stage and mycotoxin contamination. Antioxidants could be used as an alternative to fungicides to control ochratoxigenic fungi in peanuts during storage. This study was carried out to determine the effect of the antioxidant butylated hydroxyanisole (BHA) and the antimicrobial propyl paraben (PP) on the lag phase before growth, growth rate, and ochratoxin A (OTA) production by Aspergillus section Nigri strains in peanut kernels under different conditions of water activity (aw) and temperature. At 20 mM/g BHA, 18uC, and 0.93 aw, complete inhibition of growth occurred. For PP, there was no growth at 20 mM/g, 18uC, and 0.93, 0.95, and 0.98 aw. BHA at 20 mM/g inhibited OTA production in peanuts by Aspergillus carbonarius and Aspergillus niger aggregate strains at 0.93 aw and 18uC. PP at 20 mM/g completely inhibited OTA production at 18uC. The results of this work suggest that PP is more appropriate than BHA for controlling growth and OTA production by Aspergillus section Nigri species in peanut kernels.

Mycotoxin contamination is a serious problem in developing countries where climatic conditions and agricultural and storage practices are conducive to fungal growth and toxin production. At present, Argentina does not control either the storage conditions or fungal contamination in cereals, oil seeds, and other raw materials. Peanut (Arachis hypogaea L.) is one of the most important agricultural products in Argentina. The centralsouth region of Co rdoba province produces 94% of the countrys peanuts. The peanut industry exports 90% of its product, with Argentina second in the world in peanut exports (5). Each year, a significant portion of the peanuts produced cannot be marketed because of fungal disease at the postharvest stage and mycotoxin contamination (24). Ochratoxin A (OTA) is an important mycotoxin for human and domesticated animals with a diverse range of toxicological effects, including renal toxicity, mutagenicity, teratogenicity, and immunotoxicity (15). OTA has been classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer (7, 19). In previous studies, 27 to 32% of Argentinean strains in the Aspergillus section Nigri produced OTA, and 32 to 50% of the stored peanut samples were contaminated with OTA in the first (2003) and second (2004) year of sampling, respectively (10, 11).
* Author for correspondence. Tel: z54 358 4676429; Fax: z54 358 4676231; E-mail: cmagnoli@exa.unrc.edu.ar.

Control of environmental conditions such as water activity (aw) and temperature during storage could prevent Aspergillus section Nigri growth and OTA production in several agricultural products. For example, an aw of less than 0.93 and a temperature of less than 15uC prevented Aspergillus section Nigri strains from growing in peanuts, maize kernels, and dried grapes (3). Storage fungi are often managed by use of synthetic chemical products (fungicides), but these products may have adverse effects such as carcinogenicity, teratogenicity, and residual toxicity (14). Food-grade antioxidants, e.g., butylated hydroxyanisole (BHA) and butylated hydroxytoluene, and the antimicrobial agent as propyl paraben (PP) have been used safely as alternatives to fungicides to control fungal species in various food and agricultural products (2, 22). These additives can limit fungal growth and the production of fumonisin and aflatoxin on natural substrates (6, 16, 17, 25). These alternative treatments also have antifungal effects against Aspergillus section Flavi strains on peanuts (17, 18). Under some conditions, BHA and PP can inhibit the growth of strains from Aspergillus section Nigri species and inhibit OTA accumulation on peanut-based medium (4, 12). No data are available on the impact of these food-grade compounds on fungal growth and OTA production on peanuts. These additives could be used as alternative treatments for the control ochratoxigenic fungi in peanuts during storage.

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TABLE 1. Effect of butylated hydroxyanisole (BHA) concentrations on lag phase of Aspergillus section Nigri strains at different aw levels and temperatures
Lag phase (h) at temp and BHA concn of a: 18uC Aspergillus strain aw 0 mM/g 1 mM/g 5 mM/g
DEFGHI FG D O FG CD FGH O D EFGHI O D

25uC 10 mM/g 20 mM/g 0 mM/g 1 mM/g 5 mM/g 10 mM/g 20 mM/g

A. carbonarius RCPG A. carbonarius RCP203 A. niger RCP42

A. niger RCP191

0.98 0.95 0.93 0.98 0.95 0.93 0.98 0.95 0.93 0.98 0.95 0.93

38 44 56 43 48 50 33 39 51 38 40 55

HI EFGHI DEFGHI EFGHI GH DEFG FGHI HI GH HI FGH DEFGHI

38 49 59 46 52 69 33 44 58 40 46 64

56 63 G 126 EFGHI 53 O 66 EFG 190 FGHI 41 EFGHI 52 G 126 HI 47 EFGHI 52 FG 133

HI DEFG

84 91 164 93 89 201 88 91 179 84 103 198

FGH DE CD DE DE C DE DE CD FGH DEF C

196 C 225 A b 200 C 232 A 174 CD 223 A 187 CD 211 B

12 27 34 14 31 36 10 13 23 13 16 25

LMNO IJ FGHI LMNO FGH FGHI JK LMNO FG LMNO GHI FG

14 29 36 15 33 35 23 19 32 22 33 43

LMNO FGH FGHI GHI FGHI FGHI FG GHI FGHI KLM FGHI FGH

20 28 55 19 39 48 23 28 41 30 42 54

KLM FGH DEFGHI GHI FGH DEFGHI FG IJ FGH IJ FGH DEFGHI

28 33 71 33 67 84 32 26 96 36 42 125

FGH FGHI DEFG FGHI DEFGHI DEFG FGHI FG ED FGHI FGH D

36 42 88 40 117 160 46 98 167 46 112 174

FGHI FGH ED FGH D AB HI ED AB HI D AB

a b

Hour values followed by the same letter are not significantly different according to the LSD test (P . 0.001). , under the experimental conditions, strains were not able to reach the exponential phase. The peanut mixtures were kept at 4uC for 48 h with manual periodic shaking to allow absorption and to reach equilibrium. Twenty grams of peanuts was placed as a monolayer into sterile petri dishes and inoculated centrally with 5 ml of a fungal spore suspension (1 | 106 spores per ml) from a 7-day-old culture growing on 2% malt extract agar (20). Aqueous spore suspensions were prepared by adding various amounts of glycerol to reach the desired aw (0.93, 0.95, and 0.98). Inoculated peanuts in plates with the same aw were sealed in plastic containers. Each container held beakers with a glycerol-water solution at the same aw as the peanuts to maintain a constant relative humidity. Four replicate plates per treatment were used and incubated at 18 and 25uC for 4 weeks. The entire experiment was conducted twice (n ~ 8). Control plates without BHA or PP also were prepared. Growth parameters. Assessment of growth was made daily during the incubation period by examination of peanut kernel cultures with a binocular magnifier (|10). Two diameters of the growing colonies were measured at right angles from each other until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression analysis was applied to obtain the growth rate (millimeters per day) as the slope of the line. The length of the lag phase before growth also was determined, with lag phase defined as the time (hours) required for each colony to reach 2.5 mm in diameter. Growth analyses were conducted for 3 aw levels | 2 temperatures | 4 strains | 4 replicates | 1 antioxidant and 1 antimicrobial | 5 treatments per antioxidant and antimicrobial. OTA determination. At the end of the incubation period, three replicates per treatment for A. carbonarius RCPG and A. niger aggregate RCP191 were destructively sampled, dried at 50uC for 24 h, and stored at 220uC until analyzed for OTA. Five grams of a finely ground peanut sample was added to a 250-ml Erlenmeyer flask with 50 ml of an acetonitrile-water mixture (84:16). The contents were shaken in an orbital shaker for 30 min and filtered through filter paper (Whatman no. 1, Whatman, Clifton, NJ). The extract (7 ml) was added to a clean up column (MycoSep 229 Ochra column, MFC, Romer Labs, Inc., Union, MO) and acidified with 70 ml of acetic acid. Four milliliters of the

The aim of the present study was to evaluate the effect of the best food-grade antioxidant, BHA, and the antimicrobial PP on (i) lag phase before growth, (ii) growth rate, and (iii) OTA production by strains of Aspergillus section Nigri on peanut kernels under different environmental conditions. MATERIALS AND METHODS
Fungal strains. Four Aspergillus section Nigri strains were evaluated: A. carbonarius strains RCPG and RCP203 and A. niger aggregates RCP42 and RCP191. All strains were isolated from peanut kernels in Argentina and were identified based on morphological characters (8). OTA production was assayed on YES medium (2% yeast extract and 15% sucrose) (11). Strains were maintained in glycerol (15%) at 280uC and kept in the culture collection at the Department of Microbiology and o Cuarto (Co Immunology of the National University of R rdoba, Argentina). Substrate. Peanut kernels (Runner variety) that had been irradiated (7 kGy) and retained their germinative capacity were used. Kernels were checked for sterility and the absence of OTA (23) and were kept at 4uC until used. Irradiated peanut kernels were rehydrated to 0.93, 0.95, and 0.98 aw by adding sterile distilled water. The aw of representative samples of each treatment was checked at the beginning of the experiment with an AquaLab Series 3 water activity meter (Decagon Devices, Inc., Pullman, WA). Antioxidants. The antioxidant BHA (2(3)-tert-butyl-4-hydroxyanisole) and antimicrobial PP (n-propyl 4-hydroxybenzoate) were obtained from Sigma-Aldrich Chemical Co. (Dorset, UK). Stock solutions of BHA and PP (1 M) were prepared by dissolving 18 g in 100 ml of 95% ethyl alcohol in water (vol/vol). Inoculation and incubation conditions. Peanut kernels in bottles (500 g) were conditioned with an appropriate amount of water for the aw levels. From stock solutions of BHA or PP, 0.5, 2.5, 5, and 10 ml were added to peanuts to reach final concentrations of 1, 5, 10, and 20 mM/g.

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TABLE 2. Effect of propyl paraben (PP) concentrations on lag phase of Aspergillus section Nigri strains at different aw levels and temperatures

purified extract was removed, evaporated to dryness, redissolved in 200 ml of the mobile phase (acetonitrilewateracetic acid, 57:41:2), and injected into the high-performance liquid chromatography (HPLC) system, which consisted of an HP model 1100 pump (Hewlett Packard, Palo Alto, CA) connected to a Hewlett Packard 1100 Series variable wavelength detector and a data module Hewlett Packard Kayak XA (HP ChemStation revision A.06.01). OTA levels in each treatment were measured as previously described (23) with minor modifications. HPLC with a fluorescence detector (lexc 330 nm, lem 460 nm) was applied. A C18 column (150 by 4.6 mm inside diameter, 5-mm particle size; Supelcosil LC-ABZ, Supelco, Bellefonte, PA) was used. The mobile phase was pumped at 1.0 ml/min, the injection volume was 100 ml, and the retention time was 4 min. The detection limit was 1 ng/g. Toxin analyses were conducted for 3 aw levels | 2 temperatures | 2 strains | 4 replicates | 1 antioxidant and 1 antimicrobial | 5 treatments per antioxidant and antimicrobial. Assay of spiking and recovery of OTA. A stock solution of 4 ml of OTA in methanol with at 5 mg/ml was prepared for recovery determination. Each OTA-free finely ground peanut sample (5 g) contained in a 250-ml Erlenmeyer flask was spiked with OTA at an equivalent of 0.1, 0.5, 1.0, and 1.8 mg/g. Spiking was carried out in triplicate, and a single analysis of the blank sample was carried out. After 16 h for the solvent to evaporate, extraction solvent (acetonitrile-water, 84:16, vol/vol) was added, and the OTA concentration was determined using the protocol previously described. Statistical analysis. Data were analyzed with an analysis of variance. All data were transformed to log(x z 1) to homogenize the variance. Means were compared using Fishers protected least significant difference (LSD) test to determine the influence of the abiotic factors (water activity, temperature, antioxidant and antimicrobial concentrations, and strains), growth rate, length of lag phase, and OTA concentration produced by the species tested. The analysis was conducted using the PROC GLM in SAS (SAS Institute, Cary, NC) (21).

20 mM/g

DEFG

FGHI

FGH

FGH

ED

ED

ED

ED DEFGHI FGH GHI KLM

FG

DEFGHI

DEFGHI

10 mM/g

DEFGHI

DEFG

DEFG

FGHI

FGHI

FGH

ED

DEFGHI

DEFGHI

DEFGHI

DEFGHI

5 mM/g

1 mM/g

Lag phase (h) at temp and PP concn of a:

LMNO

LMNO

LMNO

0 mM/g

LMNO

FGHI

FGHI

FGH

FG

12 27 34 14 31 36 10 13 23 13 16 25 A. niger RCP191 A. niger RCP42 A. carbonarius RCPG A. carbonarius RCP203

a b

FG

JK

IJ

19 33 40 17 38 39 30 20 44 26 22 50 Hour values followed by the same letter are not significantly different according to the LSD test (P . 0.001). , under the experimental conditions, strains were not able to reach the exponential phase.
FGH FGH FGH FGH EFGHI FGH G EFG EFG EFG DEF CD DE FG DEFGHI DEFGHI DEFG FGHI EFG EFG DEF DEFGHI EFGHI FGHI FGH FGH EFG EFG GH FG DEFGHI

20 mM/g

10 mM/g

5 mM/g

18uC

Effect of BHA and PP treatments on lag phase and growth rate. In control treatments, the lag-phase length increased as aw and the temperature decreased for all strains assayed. In general, all strains behaved similarly at a given temperature (Tables 1 and 2). From 10 mM/g BHA or PP, the increase in the length of the lag phase was significant for both BHA and PP (P , 0.001) under all conditions assayed with respect to the control. At 20 mM/g BHA and 18uC, the strains did not reach the exponential phase at 0.93 aw, whereas at 0.95 and 0.98 aw, the lag phase increased significantly in comparison to the control assay (63%) (Table 1). With the PP treatment, at 18uC and 20 mM/g, the strains were not able to reach the exponential phase at any aw assayed, whereas at 25uC and 20 mM/g, longer lag phases at 0.93 and 0.95 aw were observed (Table 2). BHA at 20 mM/g, 18uC, and 0.93 aw completely inhibited growth of all strains (Fig. 1). At this same temperature with 5 and 10 mM/g BHA, the growth rate was reduced only for the A. carbonarius strains at all aw assayed (Fig. 1A and 1B). At 25uC and 0.95 and 0.98 aw, a significant reduction in growth rate was observed with 5 and

1 mM/g

0 mM/g

Aspergillus strain

0.98 0.95 0.93 0.98 0.95 0.93 0.98 0.95 0.93 0.98 0.95 0.93

aw

38 44 56 43 48 50 33 39 51 38 40 55

DEFGHI

EFGHI

EFGHI

DEFG

FGHI

GH

GH

HI

HI

HI

40 52 69 49 61 72 35 42 55 43 44 62

FG

55 69 132 50 72 168 35 56 98 49 57 70

GH

CD

RESULTS

EFG

66 73 141 69 79 168 71 82 101 77 83 113

DEFGHI

FGHI

KLM

FGH

FGH

FGH

GHI

GHI

IJ

IJ

20 44 60 18 49 55 32 32 57 31 40 61

DEFGHI

25uC

FGHI

FGHI

KLM

HI

IJ

24 49 73 30 52 79 33 49 96 35 55 101

ED

FG

24 83 97 35 99 144 37 87 140 40 95 163

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FIGURE 1. Effect of butylated hydroxyanisole (BHA) on growth rate (millimeters per day) of Aspergillus carbonarius RCPG (A) and RCP203 (B) and Aspergillus niger aggregates RCP42 (C) and RCP191 (D) at aw levels of 0.98 (&), 0.95 ( ), and 0.93 (%) and different temperatures. Mean values with a letter in common are not significantly different according to the LSD test (P . 0.001).

10 mM/g BHA in all strains (P , 0.001). At the lowest level of BHA (1 mM/g) and 25uC, fungal growth was stimulated at 0.95 aw in both A. niger aggregate strains (Fig. 1C and 1D) (P , 0.001). PP completely inhibited growth at the highest level used at 18uC under all aw conditions for all strains (Fig. 2). Complete growth inhibition also occurred with 10 mM/g at 0.93 aw for both A. carbonarius strains (Fig. 2A and 2B). At 25uC, mycelial growth was significantly reduced at 20 mM/ g PP at all aw levels and for all strains assayed. For A. niger aggregate strains, at 0.98 aw significant differences in growth rate were observed from 5 to 20 mM/g PP, whereas at 0.95 aw, a significant increase in growth rate occurred at 1 mM/g PP (P , 0.001) (Fig. 2C and 2D). Effect of BHA and PP treatments on OTA production. Recovery of the toxin was 97.3% 7.4% from the peanuts at different OTA levels tested. In general, OTA production did not follow the same pattern as the growth rate. BHA at 20 mM/g completely inhibited OTA production at 0.93 aw and at 18uC in both strains (Fig. 3A and 3C). At the same temperature and 0.95 aw, a reduction

to around 94% in toxin production was observed at 5, 10, and 20 mM/g in the A. carbonarius RCPG strain (Fig. 3A). At the same BHA concentrations, a reduction in OTA production in the A. niger aggregate strains was observed only at 0.95 aw (Fig. 3C). Toxin production at 25uC had a different pattern than that observed at 18uC. In general, at 25uC, BHA did not reduce OTA production under any of the conditions assayed. At 20 mM/g BHA and 0.93 aw, a 300% increase in OTA accumulation occurred for A. carbonarius RCPG; whereas for A. niger RCP191, this increase was observed at 5 mM/g and 0.98 aw (Fig. 3B and 3D). PP at 20 mM/g and 18uC completely inhibited OTA production in both assayed strains under all aw conditions (Fig. 4A and 4C). OTA production also was reduced at 10 mM/g and 0.95 aw in the A. carbonarius strain. For the A. niger aggregate strain at 10 mM/g PP and 18uC, a significant reduction (70%) in OTA production was observed at all aw levels assayed (Fig. 4A and 4C). At 25uC, the OTA production profile of both strains changed. PP at 20 mM/g and 25uC completely inhibited OTA production at 0.93 aw in the A. carbonarius strain (Fig. 4B). For the A. niger aggregate strain at all PP concentrations and

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FIGURE 2. Effect of propyl paraben (PP) on growth rate (millimeters per day) of Aspergillus carbonarius RCPG (A) and RCP203 (B) and Aspergillus niger aggregates RCP42 (C) and RCP191 (D) at aw levels of 0.98 (&), 0.95 ( ), and 0.93 (%) and different temperatures. Mean values with a letter in common are not significantly different according to the LSD test (P . 0.001).

aw levels assayed, OTA production was not reduced (Fig. 4D). The analysis of variance for the effect of single variables, i.e., strains, aw, temperature, and BHA or PP level, revealed that all variables alone and all interactions were significant (P , 0.001) in relation to lag phases, growth rates, and OTA production (Tables 3 and 4). DISCUSSION This study was conducted to determine whether BHA and PP could inhibit growth and OTA production by Aspergillus section Nigri strains at 18 and 25uC under different aw conditions in peanuts. The lag phase, mycelial growth rate, and OTA production by Aspergillus section Nigri strains were all significantly influenced by BHA and PP treatment, incubation temperature, aw level, and their interactions. Similar results have been obtained in previous studies with BHA and PP in Aspergillus section Flavi and Fusarium strains on maize and peanuts, respectively (6, 17,

18, 25). BHA at 20 mM/liter on peanut-based medium completely inhibited both growth and OTA production at 25uC at all aw levels and regardless of the Aspergillus section Nigri strain assayed (4). In the present study, the complete inhibition of growth on peanuts occurred only at 18uC and 0.93 aw. OTA production did not follow the same general pattern as that observed for growth. OTA production was completely inhibited at the same concentration of BHA (20 mM/g) but under all environmental conditions. In the present study on peanuts, OTA production was observed only at 18uC and at all aw levels assayed, but the response of strains at 25uC was different. These results partially agree with those of Passone et al. (17), who evaluated two concentrations of BHA on aflatoxin B1 (AFB1) production by A. flavus strains growing on peanuts at 28uC. These researchers observed that 20 mM/ g BHA completely inhibited AFB1 production at 0.982 and 0.955 aw at only 11 days of incubation, whereas stimulation of AFB1 production occurred after 35 days of incubation.

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FIGURE 3. Effect of butylated hydroxyanisole (BHA) on ochratoxin A production by Aspergillus carbonarius RCPG at 18uC (A), A. carbonarius RCPG at 25uC (B), A. niger aggregate RCP191 at 18uC (C), and A. niger aggregate RCP191 at 25uC (D) under different aw conditions. Detection limit was 1 ng/g.

Recently, Passone et al. (18) found that BHA at 20 mM/g reduced AFB1 production in peanuts by A. flavus strains in the presence of natural competing mycobiota at 0.982 and 0.955 aw after both 11 and 35 days of incubation, respectively. In another study (12) conducted with PP and Aspergillus section Nigri strains on peanut-based medium, when PP was added at 5 mM/liter none of the strains could reach the exponential growth phase and growth inhibition occurred at all aw levels. OTA production followed the pattern observed for growth parameters. These results partially agree with those of the present study; growth and OTA production were both inhibited at the highest level of PP (20 mM/g) and lower temperature (18uC). Only one previous report was found on the effects of PP on growth parameters and mycotoxin production in Aspergillus species in peanuts. Passone et al. (17) found that PP at 10 and 20 mM/g at 0.955 aw and 30uC inhibited growth by strains of Aspergillus section Flavi by 53 to 100%, whereas AFB1 production was reduced significantly only at 20 mM/g. These results only partially agree with those of the present

study; PP at some aw levels in the A. carbonarius strain did not inhibit OTA production. In the present work, different levels of BHA or PP (5 mM/g PP and 5 mM/g BHA at 18uC, and 5, 10, and 20 mM/g PP at 25uC) stimulated OTA production by A. carbonarius and A. niger aggregate. This stimulation was observed previously (4, 12) in BHA and PP in vitro studies but only at the lowest concentration assayed (1 mM/liter), which stimulated both fungal growth and OTA production by some Aspergillus section Nigri strains. This result also was observed by others, with other antioxidants and antimicrobials. Passone et al. (16) evaluated the effect of various antioxidants on growth and AFB1 production by Aspergillus section Flavi strains and found that subinhibitory concentrations (1 mM/liter) of butylated hydroxytoluene and propyl hydroxybenzoate increased the fungal growth rate and butylated hydroxytoluene at the same concentration stimulated AFB1 production. Torres et al. (25) found stimulation of fumonisin production by Fusarium verticillioides on maize with 0.5 and 2.7 mM/g BHA at 0.995 and 0.95 aw, respectively.

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FIGURE 4. Effect of propyl paraben (PP) on ochratoxin A production by Aspergillus carbonarius RCPG at 18uC (A), A. carbonarius RCPG at 25uC (B), A. niger aggregate RCP191 at 18uC (C), and A. niger aggregate RCP191 at 25uC (D) under different aw conditions. Detection limit was 1 ng/g.

Because antioxidants and antimicrobials can both promote and inhibit growth and toxin production, determining optimal application doses on natural substrates is very important. High levels of antioxidants or antimicrobials do

not assure inhibition of mycotoxin production and could produce undesirable effects on the organoleptic properties of peanuts. In contrast, subinhibitory doses plus inadequate distribution of the additive, especially at low aw levels, could

TABLE 3. Analysis of variance of effect of water activity (aw), temperature (T), concentration of BHA or PP (C), and different isolates (I) and their interactions on lag phase and growth rate of Aspergillus section Nigri strains
Lag phase Source of variation df a MSb Fc MS Growth rate F

I C aw T I|C I | C | aw I | C | aw | T
a b c

3 4 2 1 28 78 119

118,320.07 17,221,859.92 1,278,237.00 1,399,113.58 60,075.76 108,995.33 103,023.12

10.17* 1,488.66* 107.70* 123.12* 5.13* 9.55* 8.95*

46.52 399.52 42.33 1.94 34.22 10.99 0.38

15,759.66* 99,999.99* 13,452.28* 644.12* 10,629.59* 3,523.52* 177.16*

df, degrees of freedom. MS, mean square. Snedecor F; * P , 0.001.

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TABLE 4. Analysis of variance of effect of water activity (aw), temperature (T), concentration of BHA or PP (C), and different isolates (I) and their interactions on ochratoxin A production of Aspergillus section Nigri strains
OTA production Source of variation df a MSb Fc

5.

6.

I C aw T I|C I | C | aw I | C | aw | T
a

3 4 2 1 28 78 119

72.10 222.98 29.66 6.97 9.22 1.41 1.69

15,168.29* 48,833.58* 6,622.89* 1,549.97* 2,074.63* 318.41* 368.19*

7.

8. 9.

df, degrees of freedom. b MS, mean square. c Snedecor F; * P , 0.001.

10.

stimulate fungal sporulation, growth, and secondary metabolism and increase mycotoxin contamination (13). The mechanism of inhibition of fungal growth and mycotoxin production by BHA and PP is not known. Several antifungal activity mechanisms have been proposed for these phenolic compounds, e.g., impairment of enzymatic processes involved in energy production and structural component synthesis due changes in the cell membrane, alteration of fungal physiological status, and changes in nucleic acid biosynthesis (1, 9). The use of antioxidants and antimicrobials could be an alternative to control ochratoxigenic fungi in peanuts during storage before the product is shipped to internal and external markets and processed. In general, the results of this work suggest that PP is more appropriate than BHA for limiting growth and OTA production by Aspergillus section Nigri strains on peanuts, but the antifungal effect is affected by strain and environmental conditions. Further assays are needed to test the effectiveness of BHA and PP alone or in combination as inhibitors of ochratoxigenic Aspergillus section Nigri strains in peanuts in the presence of natural contaminating fungi. ACKNOWLEDGMENTS
This work was supported by grant FONCYT PICT No. 8-14552 ficas y (2006 and 2007) and Consejo Nacional de Investigaciones Cient Te cnicas (CONICET), Argentina.

11.

12.

13.

14.

15. 16.

17.

18.

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