Selenium and Aflatoxins in Brazil Nuts

Brazil nuts (Bertholletia excelsa H.B.K.) are considered a high nutritious food. Apart from carbohydrates, lipids, sulphur proteins and minerals, Brazil nuts are known to be rich in selenium Se (Barclay et al., 1995; Coutinho et al., 2002, Souza et al., 2004, Pacheco and Scussel, 2007). Despite of that, when their shell are cracked either when pods fall on the ground, or during pod opening for nut extraction (done by an axe) and exposed to high moisture and temperature of the tropical forest, fungi may grow, leading to nut spoilage. If fungi are toxigenic they may produce aflatoxins (AFLs). Se has been reported to be an antioxidant and studies have reported differences on its levels in Brazil nuts from the two Amazon regions being the Eastern nuts richer in Se than the Westerns (Chang et al., 1995; Pacheco and Scussel, 2007). Its content may vary when grown in different soils of the Amazon basin. The aflatoxigenic Aspergillus species of A. flavus and parasiticus are intimately related to agricultural crops, including tree nuts and their growth are influenced by environmental conditions. Although Brazil nuts have tested positive for A. flavus, less is known about its populations on Brazil nuts, how they grow and vary among the two Amazon regions, in the different stages of nut collection prior reaching the factory and how the processing affect them (Castrillon and Purchio, 1988; Freire et al., 2000; Candlish et al, 2001, Caldas et al., 2002). However, AFLs have been reported contaminating Brazil nuts. Their pods are harvested after they fall onto the forest soil. They stay directly in contact with the soil for several days or weeks prior to collection. It is during that time that pods may get contaminated with Aspergillus sp. and so with AFLs. Post harvesting operations are expected to have major influence on further contamination of the nuts (Bayman, 2002; Campos/Pas, 2004). For fungi growth and for their normal maintenance, a number of metals are required in different amounts. Many microorganisms are known to be able to use Se (i.e., selenite, selenate of other forms) in their metabolism (Roux et al., 2001; Fleet-Stalder et al., 2000). Se has been added to the media as sodium selenite (Na2SeO3), to understand its effect in different concentrations on fungi behaviour. The inhibitory action of Se on the growth rate of various fungi such as Aspergillus, Penicillum and Fusarium was reported by several authors (Ramadan et al., 1983, Ragab, et al., 1986, Zohri et al., 1997, Li et al, 2003). When it was used in Czapek Dox agar medium to evaluate the A. parasiticus behavior concerning morphological growth and toxin production, it was observed that fungus growth decreased by the increasing of the Se concentration (Zohri et al., 1997). In an

experiment carried out by Li et al. (2003) utilizing Na 2 SeO 3 , the authors observed from stimulating to toxic effects on organisms depending on the levels applied in the media and concluded that the presence of Se in high concentration in the growth environment can lead to morphological distortion of the fungi characteristics. Se compounds have been reported also been acting as antagonists to the mutagenic and carcinogenic activities of some agents such as UV light and AFLs (Martin et al., 1984, Gregory, 1984, Overvad et al., 1985. AFLs are produced by Aspergilllus flavus in the Brazil nuts that have Se in their composition and the Amazon forest has optimal conditions for fungi growth such as high temperatures (> 25 ºC) and relative humidity (RH) > 80 % (Bayman et al., 2002;Freire et al, 2000;Scussel, 2004;Arrus et al., 2005a;Arrus et al, 2005b;Pacheco and Scussel, 2006). Considering that Brazil nuts are rich in Se, its concentration may vary among regions, AFL contamination has been reported on Brazil nuts and that Na 2 SeO 3 has either stimulation or toxic effects on organisms depending on its levels in media, a work was carried out in order to evaluate the effect of Brazil nut Se content from different Amazon regions and Na 2 SeO 3 on the aflatoxigenic A. flavus FC1087 in terms of (a) fungal growth behavior, (b) colonies characteristics and (c) AFL production. Sterilized polyethene bags. Round and straight platinum wires, Petri dishes -plates (90x15 mm, volume: 15 ml) and Neubauer counting chamber, Optik.

Methods
a. Brazil nut preparation: 1 kg of in-shell Brazil nuts, of each Se content, after de-shelling was finely grounded (particle size <100 µm), homogenized and three portions of 250 g were separated for Se, AFLs and the mycology tests. b. Se analysis: the method used was that reported by US EPA (1996) using atomic emission spectrometry-ICP-OES. The limit of detection (LOD) was 2.00 mg/kg and the limit of quantification (LOQ) was 3.50 mg/kg. LOQ was defined as the lowest point of the calibration curve with high repeatability-axial view. Three replicates each (n=3). c. AFLs analysis: by AOAC (2005) Table 1. e. Spore suspension preparation: the suspension concentration of the A. flavus spores was set using a Neubauer counting chamber and diluting the reference strain original suspension in 0.2% aqueous tween 80 to get a final concentration of 3.0 x 10 spores/mL. f. Fungal total count: the suspension was spread on the media plates (Group I; Group II and Group III) previously prepared and incubated for 5 days at 28 C. After that period the total colonies were counted utilizing the colony counter. Note: to observe further colony behavior and development, the plates were kept incubated up to 14 days at the same temperature. Four replicates each (n=4). g. The effect of Se on A. flavus FC1087:(g.1) growth and colony features: the fungi spore suspension was also inoculated on another set of the three Groups media, by means of a straight wire to give a single point inoculum on the plate centre, to evaluate the colony radius growth (by a fine measuring scale) and other features behaviour such as shape and colour. After fungi inoculation, dishes were incubated for 14 days at 28 C. Fungi growth rate related to Se content was checked every day by measuring their radius, examining their morphological changes and their verse and reverse colour. At the end of the incubation period, the morphological colonies features (size, colour, shape, reverse colour) were registered (Table 1). Colony colour was defined according to the Methuen Handbook of Colour (Kornerup and Wanscher, 1989). g.
(2) AFL production: The strain AFL production related to Se concentration was evaluated in each media by examining the AFL characteristic fluorescence (white/bluish) development. Each media fluorescence positive had their AFLs extracted (Moss and Badii, 1982) and analyzed as in (c). Four replicates each (n=4). h. Statistical analysis: the data were statistically analyzed using analysis of variance (ANOVA).

Results and discussion
The influence of Se on fungi growth, colony diameter, verse/reverse colour, fluorescence development and AFL production are shown in Table 1. The data obtained shows that Se affected the A. flavus F1087 strain proliferation and AFL production when inoculated in media added either of ground Brazil nuts or Na 2 SeO 3 at different concentrations.
3.1 Effect of Se concentration on the total fungi growth A. flavus FC1087 was able to grow in presence of both Brazil nut Se concentrations (18.4 and 43.5 mg/kg: Western and Eastern region nuts, respectively) -Group I. It was observed that the highest total A. flavus colonies count and growth rate was obtained in the media containing the Western Brazil nuts (Se: 0.02 mg/kg in the plate with 2.3 x 10 cfu) and so for the Control (2.8 x 10 cfu) -Group III. The Eastern nuts media with higher Se content (0.04 mg/kg) presented lower number of colonies than the Western's (1.2 x 10 cfu). Similar to the Eastern nut total colonies count, was observed in the tested Na 2 SeO 3 media concentrations (0.01 to 0.20 mg/kg) -Group II, with total counts of 1.5, 1.3, 1.3 and 1.1 x 10 cfu, respectively. Growth decreased from Na 2 SeO 3 level of 0.20 to 0.4 mg/kg reaching 0.9 x 10 cfu. At the highest Na 2 SeO 3 content (0.60 mg/kg) fungi failed completely to grow, probably because of its toxic level to the strain. These findings are corroborated by previous authors (Badii et al, 1986;Aboul-Dahad 1991;Zohri et al, 1997) that have also found reduction on fungi growth as the concentration of Na 2 SeO 3 increase in the media. The toxic Se effect at higher doses reduces microorganism growth due to the detoxification process that takes place. That involves fungi transformation of the inorganic Se (more toxic) to organics (less toxic). It also involves the reduction of the Se oxyanions, Na 2 SeO 3 and selenate to inert elemental Se within the mycelium. The toxic action of Se in fungi is believed to be due to its incorporation into the protein amino acids instead of sulfur which can lead to alteration of the tertiary structure thus to dysfunction of the fungi proteins and enzymes (Gharieb and Gadd, 1998). Figure 1 shows the A. flavus FC1087 growth from Day 5 to Day 14 of incubation in the media containing Se Western and Eastern Brazil nuts Se.

Effect of Se concentration on A. flavus FC1087 colony features
When A. flavus strain was inoculated in a single point on the media that contained either the two Brazil nut Se concentrations (Group I) or the six increasing Na 2 SeO 3 concentrations (Group II), it was possible to observe their diameter, colours (verse/reverse) and fluorescence variations. Their diameters increased (52 to 194 mm) with the reduction of Na 2 SeO 3 concentration (0.4 to 0.01 mg/kg). The highest colony diameter was obtained in the Western Brazil nut media with 315 mm. Eastern nuts colonies diameter was smaller with only 182 mm. Most of the colonies grown in Na 2 SeO 3 media presented a characteristic green colour with the exception of the ones grown in the lowest Na 2 SeO 3 concentration (0.01 mg/kg) that had only the centre green with a white, broad, non sporulating margin. That non sporulating margin was also observed in the Brazil nut both regions media colonies. On the other hand, the colonies reverse colour changed as the selenite concentration increased in the media from brown (lower Na 2 SeO 3 concentration = 0.01 and 0.05 mg/kg) to orangered (higher Na 2 SeO 3 concentration = 0.2 and 0.4 mg/kg). Some authors have studied and reported these colour changes. McCready et al. (1966) reported that orange intracellular granules in bacteria (Salmonella Heidelberg), when grown in the presence of Na 2 SeO 3 , as amorphous red elemental Se. Badii et al in 1986, also observed a deep orange pigment on the www.intechopen.com undersurface of the A. parasiticus colonies in presence of Na 2 SeO 3 . Aboul-Dahab (1991) found that these colours were due to the reduction of Na 2 SeO 3 with deposition of elemental Se within the fungal cells, as well as, in the growth media. Presumably, the fungus metabolizes Se to produce strong reducing and oxidizing agents into the media environment. Moss et al. (1987) reported that A. parasiticus is able to reduce also sodium bis selenite with the deposition of granules of elemental Se within the mycelium, being that redorange colour a biological reduction of Se compounds into elemental colloidal Se. As far as the fluorescence of possible AFL presence is concerned, all media, containing Se despite of its origin or concentration, presented the characteristic white/bluish fluorescence and so the Control plates (except at the selenite highest concentration). That was an indication of the AFL presence, thus they were submitted to AFL analysis. However, it was observed that the fluorescence intensity reduced as the Na 2 SeO 3 level increased in the media, reaching the highest concentration (0.60 mg/kg) with no fluorescence at all. In fact, A. flavus FC1087 failed to grow at that concentration caused probably by the toxic effect of the Na 2 SeO 3 (Zohri, et al., 1997, Li et al., 2003. Figure 2 shows

Effect of Se concentration on AFls production
AFls were detected in the media, containing Eastern Brazil nuts and Na 2 SeO 3 media, with the exception of the 0.60 mg/kg Na 2 SeO 3 media, as no A. flavus was able to grow. Only in the Brazil nut medium with 0.04 mg/kg of Se, corresponding to nuts from the Eastern Amazon region, the strain produced AFL at a level of 50.20.6 g/kg for total AFL and 28.50.6 g/kg for AFB 1 . No AFLs were detected in the nuts from Western region, however fungi growth were much abundant in that media, and larger the colonies diameter (315 mm). A. flavus FC1087 was able to produce in the Control media 109.2 0.2 g/kg of AFL. It was observed that, the strain was Na 2 SeO 3 doses-dependent either for its AFL synthesis and growth, as an AFL decrease with the increased of Na 2 SeO 3 concentration occurred, concomitant to the fungi growth (Table 1). Both, the total AFLs (AFB 1 +AFB 2 +AFG 1 +AFG 2 ) and AFB 1 production decreased as the Na 2 SeO 3 levels increased. Similar to the Control plate, tested fungus was able to produce high amounts of AFls total, reaching at the two lowest Se concentrations (0.01 and 0.05 mg/Kg) a total AFLs of 98.70.3 and 102.60.3 g/kg, respectively. In contrary, at the highest concentrations of 0.2 and 0.4 mg/kg, to where fungi were still able to grow, toxin production reduced from 70.30.5 to 30.60.5 g/kg. The higher Se content in the nuts, the less fungi proliferation and rate in the plate. That could be caused by the Se toxic effect on the strain leading to fungi stress thus activating second metabolism of AFL formation. That could be explained for the toxicity of high amount of Se in organism, maybe causing oxidative stress in the strain (Letavayova et al., 2006, Valko et al., 2006. The oxidative stress is a prerequisite for AFL production by A. parasiticus (Jayashree and Subramanyam, 2000). Thus, a Se amount could activate the mechanisms of AFL production and contaminating Brazil nuts. Bronzetti et al (2001) demonstrated Se compounds in yeasts exerted both mutagenic and anti-mutagenic effect at different concentrations. On the other hand, other factors, such as the interaction or the competition with different strains of Aspergillus seem to affect the increasing of AFL production in some substrates (Martins, Martins, Bernardo, 2000).

Brazil nut composition, Se content versus fungi and AFls production
As far as Brazil nuts Se concentration is concerned, it is important to emphasize that there are two approached to take into account related to its benefits: First is the Se content in the nuts for (a) human consumption against diseases and the other is for (b) fungi proliferation and AFLs production in the nuts substrate. (a) Se is important to health as it has been reported being an excellent antioxidant for reducing toxic effects such as the carcinogenicity of some compounds. Its antioxidant protective effect is primarily associated with the presence of glutathione peroxides that protect DNA and other cellular components from damage by oxygen radicals. Se is an essential component of glutathione peroxides (Agar and Alpsoy, 2005). It inclusive, in some concentrations (such as 8 mg/kg), can inhibit the AFB 1 and AFG 1 mutagenic and carcinogenic effects in human blood cell culture (Geyikoglu and Turkez, 2006). (b) Regarding fungi and AFLs, in our study, it was observed that the A. flavus FC1087 on the Western Brazil nuts media presented similar behavior as the Control with 2.3 x 10 total count / 310.5 diameter / green colour / colourless reverse colonies however without AFLs production. On the other hand, the Eastern's media presented lower total A. Flavus FC1087 count, growth rate, and diameter, however with AFL production. Data suggests that: the higher Se content in the nuts, the less fungi proliferation and rate in the plate and high AFLs. That could be caused by the Se toxic effect on the strain leading to fungi stress thus activating the second metabolism of AFL formation. www.intechopen.com

Conclusion
The higher Se content present in the Brazil nuts lead to less fungi proliferation, growth rate in the plate and AFL production. That could be caused by its toxic effect to the fungus provoking stress and activating AFL production. Despite of data obtained, further studies need to be carried out utilizing Brazil nuts media with a more wide Se concentration range to find out the Se role on fungi and AFL production. It would be also necessary to investigated possible interactions and/or competition of different strains of Aspergillus on AFL production in Brazil nuts.