Brazil nuts: determination of natural elements and aflatoxin

Castanha do Brasil: determinação de elementos naturais e aflatoxinas

QUÍMICA, ALIMENTOS, MICROBIOLOGIA

Brazil nuts: determination of natural elements and aflatoxin

Castanha do Brasil: determinação de elementos naturais e aflatoxinas

Maristela Martins^I; Ariane Mendonça PachecoI^*; Ana Cyra Santos Lucas^I; Avacir Casanova Andrello^II; Carlos Roberto Appoloni^II; Jose Junior Mendonça Xavier^III

^I Faculty of Pharmaceutical Sciences, Federal University of Amazonas, Manaus, Amazonas, Brazil, 69010-330

^II Department of Physical, State University of Londrina, Londrina, Paraná, Brazil, 86051-990

^III Applied Biosystems of Brazil, São Paulo, Brazil, 04311-000

ABSTRACT

A study was carried out to evaluate the association of levels of radioactivity, selenium and aflatoxin in shelled Brazil nuts, which were classified in different sizes, for export. The selenium determinations were performed by inductively coupled plasma optical emission spectrometry (LOQ = 3.0 µg g^-1), and aflatoxins were detected by Liquid chromatography-mass spectrometry (LOQ = 0.85 µg kg^-1), recovery rates were between 92 and 100%. Radioactivity was measured by high-resolution gamma spectrometry. The selenium mean concentration was (22.7 ± 7.4) µg g^-1. (n = 30). Mean activities determined for the following radium isotopes were: 15.77 Bq kg^-1 for ²²⁴Ra, 104.8 Bq kg^-1 for ²²⁶Ra and 99.48 Bq kg^-1 for ²²⁸Ra. For ²²⁶Ra, the levels did not vary significantly with nut sizes, although such differences were observed for ²²⁴Ra and ²²⁸Ra. There was no statistically significant association between the level of selenium and the activity of radionuclides, however, there was correlation between the radionuclides. Aflatoxins above the quantification limit were not found.

Keywords: radioactivity; selenium; Bertholletia excelsa.

RESUMO

Um estudo foi realizado para avaliar a associação dos níveis de radioatividade, selênio e aflatoxinas em castanha-do-Brasil descascada, que foram classificadas em diferentes tamanhos, para exportação. As determinações de selênio foram realizadas por espectrometria de emissão óptica com plasma indutivamente acoplado (LOQ=3,0 µg kg^-1) e aflatoxinas foram detectadas por LC-MSMS (LOQ=0,85 µg kg^-1), as taxas de recuperação ficaram entre 92 e 100%. A radioatividade foi medida por espectrometria gama de alta resolução. A concentração média de selênio foi de (22,7 ± 7,4) µg g^-1. (n = 30). A atividade média determinada para os radioisótopos foram: 15,77 Bq kg^-1 para o ²²⁴Ra, 104,8 Bq kg^-1 para ²²⁶Ra e 99,48 Bq kg^-1 para ²²⁸Ra. Para ²²⁶Ra, os níveis não variaram significativamente com o tamanho das nozes, embora tais diferenças foram observadas para ²²⁴Ra e ²²⁸Ra. Não houve associação estatisticamente significativa entre o nível de selênio e a atividade de radionuclídeos, no entanto, houve correlação entre os radionuclídeos. Não foi encontrado aflatoxinas acima do limite de quantifcação.

Palavras-chave: radioatividade, selênio, Bertholletia excelsa.

INTRODUCTION

The contamination by fungi and their metabolites in food has been focused on the food science to identify risks and prevent diseases. However, the natural properties of some foods, such as radioactivity and their chemical compositions, have been assessed as being active in themselves, but not deeply studied in tree nuts, for example.

The Brazil nut (Bertholletia excelsa HBK) is well-known as a relevant food with nutritional properties, such as a high protein content (15-20%) and sulfur amino acids, 60-70% lipids (Essential fatty acids), vitamin E (Chunhieng et al. 2008) and antioxidant properties due to its selenium content (Ryan et al. 2006).

Selenium (Se) is an essential micronutrient that, once incorporated into selenoproteins, performs important functions in the human body, participating in antioxidant defense, in the immune system and in the regulation of thyroid function (Rayman 2000). Glutathione peroxidase is a selenoprotein that acts as an antioxidant enzyme in plasma and is associated with slowing the aging process, boosting the immune system and protecting against heart disease and certain forms of cancer (Yang 2009). However, at doses above the recommended daily intake of 55 µg per day, Se can be toxic (Yang 2009). Brazil nut trees absorb the natural element Se from soil (Parekh et al. 2008) as along with radionuclides, such as ²²⁶Ra (Tagami and Uchida 2009).

The concentration of radioactive elements in Brazil nuts can reach quantities up to 1000 times greater than in other foods (Gabay and Sax 1969). Due to their high concentration of radionuclides, studies report that Brazil nuts are radioactive (Turner et al. 1958; Smith 1971). The occurrence of cancer is among the risks associated with the ingestion of radioactive foods, and monitoring is a form of health protection (Irigaray et al. 2007; Celik 2008).

The Brazil nut is native to the Amazon region and is economically important. It is collected in indigenous regions and in small communities during the rainy season, and transported to processing plants, to undergo treatment involving the following stages: sorting, drying, breaking and sorting by size. The initial stage of manual/visual sorting is important to remove moldy and stained nuts, and precedes classification by size. At the end of the drying and cooling processes, the product is subjected to vacuum packaging and hot sealing (Figure 1) (Pacheco and Scussel 2009).

Besides the amount exported from Brazil - around 23.600 t in 2008/2009 (MICD 2010) - Brazil nuts have been used in industrial products, such as biscuits, oil, sweets, cereals and bakery products (Souza and Menezes 2004). However, the export of Brazil nuts to Europe has suffered drastic reductions due to the presence of aflatoxins at concentrations exceeding those accepted by European legislation (EU 2003; 4 µg kg^-1 ).

Aflatoxins are carcinogenic substances (IARC 1997) from the secondary metabolism of aflatoxigenic fungi. Brazil nuts and other tree nuts have been studied for their association with these fungi, as well as for levels of aflatoxin contamination (Scussel 2004). Brazil nut production occurs in environments with temperatures of 30-35 °C and with high relative humidity (80-95%). The environmental conditions in the Amazon region influence the level of water activity (Wa) and moisture in Brazil nuts and, favor aflatoxin production by some fungal strains (Johnsson et al. 2008).

Considering these aspects, the aim of this work was to evaluate the association of radioactive and selenium levels concerning the aflatoxins contamination in Brazil-nuts for export.

MATERIALS AND METHODS

Reagents

Reagents used in this study included: methanol, acetonitrile, benzene (HPLC grade, Carlo Erba, Rodano, Italy), ultrapure water (MilliQ system, Millipore, Billerica, USA) and ammonium acetate (analytical grade, Vetec, Rio de Janeiro, Brazil). Aflatoxin standards included: AFB1, AFB2, AFG1 and AFG2 (Sigma , Saint Louis, MO, USA).

Instruments

A liquid chromatograph was used (1100, Agilent, Santa Clara, USA) with a quaternary pump, degasser, autosampler and a 20 mL loop. Reverse phase columns studied included: three C18 (4.5 mm i.d., 150 mm length from Hichrom (Theale, UK) and of 250 mm length (5 and 10 mm i.d.)] from Phenomenex (Torrance, USA), and one C8 (4.6 mm i.d., 150 mm length) from Agilent (Santa Clara, USA). A liquid chromatograph coupled with tandem-mass spectrometer (LC-MS/MS) (Applied Biosystems MDS SCIEX, Foster City, USA) was equipped with an atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) interfaces in positive modes as well as an infusion pump (Harvard Apparatus, Holliston, USA). An inductively coupled plasma optical emission spectrometer (Otima 2000, Perkin-Elmer, Toronto, Canada.), a mill (Romer, Union, USA), an industrial nut-cracker (CIEX, Manaus, Brazil), a HPGe detector (GEM-M-7080 PS, ORTEC, USA), a high voltage power supply (model ORTEC 659, USA), a pulse generator (model ORTEC 419, USA), a preamplifier and linear amplifier (ORTEC model 575, USA), an oscilloscope (model TDS tektronic 220), a multichannel plate (model ORTEC Trump-8K, USA) and a shield (ORTEC, model HPLDS1, USA).

Sampling and preparation

A total of 30 samples were collected from a batch of Brazil nuts for export from the 2009 harvest in a factory from the city of Manaus in the state of Amazonas, Brazil.

The sampling method used was that required by the European Union, (EU 2003). The samples were representatively collected from bags under vacuum/heat sealing of 20 kg. Incremental samples were taken from the bags and homogenized. Final portions of 1 kg were packed and immediately sent to the laboratory. This portion was considered to be representative of < 0.1 t. Frozen samples were finely ground (particle size <100 µm) in a disk mill and were homogenized. Subsequently, 500 g portions were transferred to polyethylene containers with stoppers and stored in a freezer. Portions of 50 and 25 g were used for Se and aflatoxin determinations, in duplicates. For radioactivity analysis, incremental samples were taken from the bags and homogenized; final portions of 2 kg were obtained. Samples, incinerated according to the Association of Official Analytical Chemists (AOAC 2005), were properly packed (to fill a 2 cm tall container - about 50 g sample) in cylindrical plastic containers with a volume of 300 mL. After packing, samples were sealed and remained at rest for a period of forty days to reach secular equilibrium. Measurements were performed at the Laboratory of Applied Nuclear Physics (Physics Department, State University of Londrina) (LFNA/UEL).

Selenium determination

The analysis of selenium was performed by ICP optical emission spectrometry (OES), using the atomic emission (28). The digestion of samples was carried out by acid in microwave in a closed system. Mass of about 0.3 g + 3 mL of concentrated nitric acid and 1 mL of hydrogen peroxide, high pressure system 100 DAK Bergof, irradiation with 200 watts for five minutes and increase 50 watts min^-1 up to 700 watts for fifteen minutes, total time thirty minutes. It was then cooled, depressurized and swelled to 50 mL. The limit of detection (LOD) was 1.50 mg g^-1 and the limit of quantification (LOQ) was 3.00 mg g^-1. The LOQ was defined as the lowest point of the curve with high reproducibility, axial view. The level of recovery was 90% (n=30). The analytical lines used for selenium determination was 196.03 nm. The operational features of the spectrometer were: operating range optics - Used: 170 to 850 nm, power radio frequency = 1300 watts box, plasma gas flow rate = 15 L min^-1 - adjunct = 2 L min^-1 - 0.6 nebulizer mL min^-1, peristaltic pump with adjustable flow rate of 2 mL min^-1.

Aflatoxin Determination

Aflatoxin determination was performed by liquid chromatography (LC) coupled with tandem - mass spectrometry (MS-MS) APCI in the positive detection mode (Xavier and Scussel 2008). The LC conditions (C8 column) involved a mobile-phase with a methanol/water gradient [45% water/55% methanol (tree minutes); from three to five minutes the gradient was changed to 30% water/70% methanol] and a flow rate of 1 mL min^-1. For MS/MS, the parent and two daughter ions (m/z) were selected for each toxin as follows: AFB1, m/z 313.1 (241.10 and 285.10); AFB2, m/z 315 (259.09 and 287.20); AFG1, m/z 329.1 (200.05 and 243.05); and AFG2, m/z 331.2 (245.07 and 231.20). The LOD and LOQ values for LC-MS/MS of AFB1, AFB2, AFG1, AFG2 were 0.05, 0.075, 0.075 and 0.1 μg kg^-1 and 0.15, 0.2, 0.2 and 0.3 μg kg^-1 for each aflatoxin, respectively. The LOD and LOQ values for total aflatoxin were 0.3 and 0.85 μg kg^-1. To obtain those parameters, the finely ground Brazil nuts were homogenized and spiked prior to extraction with aflatoxins at five concentrations between 1 to 10 μg kg^-1. Portions of 25 g were taken for extraction by adding 100 mL of acetonitrile/water (80:20, v/v) to the sample, which was followed by mixing for 2 h and filtration. The LOD method was defined by t times the signal-to noise ratio, and the LOQ method was defined by 6 times the signal-to-noise ratio. Five-point analytical curves were constructed for quantification and for the estimation of LOD and LOQ. Each point corresponded to a mean of five injections of each extract. The recoveries for each aflatoxin (AFB1, AFB2, AFG1, AFG2) were 92.4, 72.5, 99.8, and 97.1%, respectively. The shell/nut ratio used for calculation was that reported by de Mello and Scussel (2007) [60:40 (60% shell/40% nut) with a factor of 1.5, which was considered the standard ratio for normal healthy whole Brazil nuts].

Radioactivity determination by Gamma-Ray Spectrometry

Traces of the radionuclides ²²⁸Ra, ²²⁶Ra and ²²⁴Ra were measured by gamma spectrometry using a HPGe detector (GEM-M 7080-PS, ORTEC) with 66% relative efficiency. Each nut sample was counted for 86,400 seconds. The concentration of 228Ra was determined from the 911, 338 and 969 keV lines for ²²⁸Ac in each sample. The concentration of ²²⁶Ra was determined from the 609, 1120 and 1764.5 keV lines for ²¹⁴Bi, and from the 352 and 295 keV lines for ²¹⁴Pb. The average activity of each of these two radionuclides in all samples was then calculated. Subsequently, an average weighted by the variances of these two values was used to determine the average value of the activity of 226Ra and 228Ra in these nuts. The concentration of ²²⁴Ra was obtaind from the 239 keV line for ²¹²Pb and the 583 keV line for ²⁰⁸Tl. The minimum detectable activity (MDA) for each spectral line is shown in Table 1.

Committed effective doses

The calculation of dose per unit intake is provided by the International Commission on Radiological Protection (ICRP 1996). The potential radiological impact through food intake is assessed from the calculation of committed effective dose (CED) in Sv.a-1, given by the equation: CED = e (g) ATc; where: e (g) is the committed effective dose per unit intake, or the effective dose coefficient, A is the average activity of the radionuclide and Tc is the rate of annual consumption of that food. The values for the coefficient of effective dose [e (g)] are based on models and metabolic data used for the evaluation by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR 2000). These coefficients of effective dose of interest for this work are those defined by the International Atomic Energy Agency (IAEA 1996). The values of the rate of annual consumption (Tc) of nuts for Brazil used in this study were suggested by the Household Budget Survey (POF) from the Brazilian Institute of Geography and Statistics (IBGE 2004) and also suggested by diet Cluster GEMs / Food (Global Environmental Monitoring System) (WHO 2006).

Statistical analysis

Comparison between the sizes was performed using analysis of variance (ANOVA). For correlation analysis, we used the Pearson coefficient. The Student t-test and chi-square test were used for comparison between doses and committed ceiling.

RESULTS AND DICUSSION

Selenium in Brazil nut

Selenium levels ranged from 9.4 to 39.0 µg g^-1, see Table 2. There were no significant differences in Se concentration (p> 0.18) among the different sizes of nuts. Moodley et al. (2007) studied the concentration of Se in different types of tree nuts sold in South Africa and reported a concentration of 36.1 ± 0.4 µg g^-1 in Brazil nuts, and 0.0039 ± 0.0007 µg g^-1 in almonds, but did not detect levels of Se in other types of nuts studied. Rodushkin et al. (2008) reported a wide variation in Se levels among different types of nuts and reported a relatively low concentration in shelled Brazil nuts (1.2 µg g^-1). The levels of Se found in this study were lower than those reported (126 µg g^-1) by Chunhieng et al. (2004). However, our results were similar to those reported by Parekh et al. (2008) who reported that levels of Se were inversely proportional to the concentration of barium in the samples studied. The concentration of Se in Brazil nuts seems to be influenced by the absorption capacity of the tree and may vary according to factors arising from the composition of the soil from which they originate. Levels of 8.0 to 69.7 µg g^-1 in shelled Brazil nuts from different regions of the Amazon have been reported by Pacheco and Scussel (2007). In that study, the authors found also a correlation between levels of Se and aflatoxin: the higher the level of Se, the higher the aflatoxin contamination. Considering the fact that their correlation, as well as the levels obtained in our study is significantly lower than those reported by Pacheco and Scussel (2007), it may be possible to explain the non-detection of aflatoxins in our samples studied by the LOQ.

Although Se is a fundamental element that is prized for its antioxidant activity, it is also investigated due to the ambiguity of its actions because both beneficial and toxic effects on organisms have been observed. Its therapeutic range is considered narrow, and its toxicity is partly related to the ability of some compounds containing Se to generate free radicals: therefore, the intake of such compounds should be monitored. The Recommended Dietary Allowance (RDA) for Se is 55 µg day^-1 for adults (NAS 2000), which is based on the amount needed to maximize the synthesis of the selenoprotein glutathione peroxidase (GPx), measured by the plateau in the activity of the plasma isoform of this enzyme. The Tolerable Upper Intake Level (UL) for adults is 400 µg day^-1 based on selenosis, which is the adverse effect resulting from excess intake (NAS 2000). Studies report that consumption of 300 µg day^-1 of Se can have toxic effects on both growth hormone and thyroid hormone synthesis (Kaprara and Krassas 2006). According to Thomson et al. 2008, the consumption of two nuts (mean 100 µg day^-1) would be sufficient to obtain the effects of antioxidants in the body. According to that study, the consumption of peeled Brazil nuts of medium size with an edible portion equal to 4.6 g (De Mello and Scussel 2007) provides a concentration of Se in the range of 43.2 to 179.4 µg g^-1, which covers both the RDA and UL.

Aflatoxins in Brazil nuts

Despite the good sensitivity of the LC-MS/MS method (LOQ = 0.85 µg kg^-1 ΣAFL), aflatoxins were not detected in any sample. One explanation may be that the stripped Brazil nuts underwent three rounds of sorting/screening during processing (Pacheco and Scussel 2009). These steps allow for the disposal of damaged nuts that do not conform to processing standards, greatly reducing the possibility of contamination of batches of the product. Pacheco et al. (2010) reported that samples obtained after drying (i.e., collected at the end of the process) were not contaminated by aflatoxins. Other researchers have reported no detection of aflatoxins in shelled Brazil nuts (Souza and Menezes 2004; Ioannou-Kakouri et al. 1999). According to the report of the Codex Alimentarius Commission (CAC 2010), a change in the limit of aflatoxins from 4 µg kg^-1 to 10 µg kg^-1 in shelled Brazil nuts for consumption was recently established, and this limit was further changed to 15 µg kg^-1 for shelled nuts destined for further processing. Therefore, the samples studied in this work were in accordance with Brazilian law, with a maximum of 30 µg kg^-1 (Brazil 1976) and also met European legislation standards.

²²⁶Ra, ²²⁸Ra and ²²⁴Ra in Brazil nuts

Activities measured for each radionuclide are summarized in Table 3. All samples studied showed activity above the minimum detectable activities for all spectral lines used. The activity of ²²⁶Ra was calculated from the weighted average of the activities of the radionuclides ²¹⁴Pb and ²¹⁴Bi. This was done because ²²⁶Ra, which has a half life of 1620 years, is actually present in Brazil nuts, as was ²³⁸U, which has a half life of 4.5 x10⁹ years. However there is a possibility of the nut seeds to absorbing ²²⁶Ra instead of ²³⁸U; therefore, we attributed the activities of ²¹⁴Pb and ²¹⁴Bi to ²²⁶Ra. In the same way, the activity of ²²⁸Ac was attributed to ²²⁸Ra, and the activities of ²¹²Pb and ²⁰⁸Tl were attributed to ²²⁴Ra.

High values for the activities of both ²²⁶Ra and ²²⁸Ra in nuts, compared to other foods, were expected (Anderson and Cunningham 2005; Scheibel and Appoloni 2007), because the nut trees from Brazil have a great capacity to absorb radioisotopes from the soil. It is well known that the Brazil nut tree has a high capacity for barium uptake from soil (Hiromoto et al. 1996). Taking into account the similarity of the chemical behaviour of barium and radium, this propertie explains the capacity for radioisotopes absorption.

The low activity concentrations found for ²²⁴Ra agree with the values reported by Parekh et al. (2008). The lowest activity was observed for ²²⁴Ra (11.21 ± 3.18 Bq kg^-1) and the highest was found for ²²⁸Ra (136.10 ± 16.15 Bq kg^-1).

The values of activities for ²²⁶Ra and ²²⁸Ra were higher than those reported by Parekh et al. (2008) and by Hiromoto et al. (1996). Adopting a significance level of α = 0.05, there are no differences in the activity of ²²⁶Ra (p > 0.30) among the different sizes of nuts. However, for ²²⁴Ra and ²²⁸Ra, a similar test showed statistically significant differences between different sizes. In subsequent tests for multiple comparisons with the average of the ranks, increased activity of ²²⁴Ra was detected for small and large nuts. For ²²⁸Ra, the highest activities occurred in small and medium nuts. There may be a pattern of decreased activity of radionuclides with increasing nut size.

Finally, we evaluated the possible relationship between Se concentration and the activity of ²²⁴Ra, ²²⁶Ra and ²²⁸Ra (Figure 2); however, the correlation coefficients were below 50%, allowing us to conclude that there was no relationship in the studied samples, and that the correlation was strongest among radionuclides (Figure 3).

The committed effective doses (CED) were calculated and are presented in Table 4. The CEDs, estimated from the rates of consumption, suggested significant differences between the three radionuclides (p < 0.001). However, based on the results of Table 3, it can be stated that for both estimates of consumption, all CEDs observed in this study show doses below the maximum established by the UNSCEAR (2000) for the radionuclides studied; therefore, no health risk is involved.

The results of this study of radionuclide activity and Se concentration in Brazil nuts can be useful for our understanding of the process of element's absorption by these nuts. Further analysis could provide information on soil characteristics of the region of origin. Another suggestion for future work would be the activity determination of radionuclides of the uranium series which are before ²²⁶Ra, so that we could make an analogy between the activities of these two radionuclides (²³⁸U and ²²⁶Ra). This would also give an idea about the process of absorbing elements from soil by Brazil nut tree.

Thus, it would be possible to make an analysis of the whole series of radionuclides to see whether it is in radioactive equilibrium in Brazil nut. In regard to other variables in the production of aflatoxins, it would be interesting to assess the moisture content and water activity of Brazil nuts throughout the entire production chain. The results of such an investigation could contribute to the search for tools to prevent contamination.

CONCLUSION

The selenium mean concentration was 22.7 ± 7.4. Mean activities determined for the following radium isotopes were: 15.77 Bq kg^-1 for ²²⁴Ra, 104.8 Bq kg^-1 for ²²⁶Ra and 99.48 Bq kg^-1 for ²²⁸Ra. We did not find aflatoxins above the quantification limit. Based on the results, it can be stated that for both estimates of consumption, all CEDs observed in this study show doses below the maximum established by the UNSCEAR (2000) for the radionuclides studied; therefore, no health risk is involved in the Brazil nut consumption according to the evaluated samples.

ACKNOWLEDGMENTS

We are grateful to the Research Foundation of the State of Amazonas - FAPEAM for financial support; to the Coordination of Development of Higher Education - CAPES for the research fellowship; and to Mrs. Viviane Scheibel de Almeida, of the Federal University of Mato Grosso of the South, for information about the committed effective doses.

Recebido em 26/11/2010

Aceito em 27/03/2011

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Publication Dates

• Publication in this collection
  09 Jan 2012
• Date of issue
  Mar 2012

History

• Received
  26 Nov 2010
• Accepted
  27 Mar 2011