Aflatoxin evaluation in ready-to-eat brazil nuts using reversed-phase liquid chromatography and post-column derivatisation.

A high-performance liquid chromatography-fluorescence (HPLC-FD) method for aflatoxin quantification in brazil nuts was developed. Samples of brazil nuts collected in Brazilian markets were extracted with methanol:water and cleaned using an immunoaffinity column. Aflatoxins were eluted with methanol and a post-column derivatisation was performed with bromine, using a Kobra Cell system. The optimised method for total aflatoxins was sensitive, with detection and quantification limits of 0.05 and 0.25 µg kg⁻¹, respectively. The method was accurate, with recovery values of 87.6%; 85.3% and 85.0% for 0.5, 5.0 and 14.6 µg kg⁻¹ spiked levels, respectively. It was shown that the method was applicable to brazil nuts. From a total of 95 brazil nut samples analysed from 21 São Paulo supermarket samples and 51 Manaus and 23 Belém street markets samples, 37.9% showed detectable levels of aflatoxins and three exceeded the recommended Codex Alimentarius limit of 10 µg kg⁻¹ for ready-to-eat brazil nuts.

Identification of Genes Differentially Expressed Between Ochratoxin-Producing and Non-Producing Strains of Aspergillus westerdijkiae.

Approximately 70 % of Aspergillus westerdijkiae strains are able to produce ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin which have been found in cereal and food commodities. Despite of its importance there is, up to now, no information available about which genes are differentially expressed between A. westerdijkiae ochratoxin-producing and non-producing strains. Using cDNA RDA approach we successfully sequenced 231 raw ESTs expected to be enriched in the ochratoxin-producing strain. BLASTX searches against the public databases showed that of these, 205 ESTs (79 %) exhibited significant similarities with proteins of known functions, 28 ESTs (11 %) had matches to hypothetical proteins, and the remaining 27 ESTs (10 %) had no significant hits. EST alignment resulted in a total of 14 non-redundant consensus sequences. Three putative genes encoding oxidoreductases were validated as up-expressed in the OTA producer strain using RT-qPCR approach. The expression of the putative genes encoding a cytochrome P450 family protein, 3-hydroxyphenylacetate-6-hydroxylase, and endoplasmic reticulum oxidoreductin were higher (32-, 2.8- and 20-fold respectively) in the OTA producer strain compared to the non-producer strain.