Aspergillus section Flavi and aflatoxins in Brazilian cassava (Manihot esculenta Crantz) and products.

Aflatoxins are carcinogenic compounds produced by some species of Aspergillus, especially those belonging to Aspergillus section Flavi. Their occurrence in food may start in the field, in the post-harvest, or during storage due to inadequate handling and storage. Because cassava is a staple food for a high percentage of the Brazilian population, we evaluated the presence of aflatoxin-producing species in cassava tubers, cassava products (cassava flour, cassava starch, sour starch, and tapioca flour), and in soil samples collected from cassava fields. In addition, the levels of aflatoxin contamination in cassava products were quantified. A total of 101 samples were analyzed, and 45 strains of Aspergillus section Flavi were isolated. Among the identified species, Aspergillus flavus, Aspergillus arachidicola, Aspergillus novoparasiticus, and Aspergillus parasiticus were found. The majority of strains (73.3%) tested for their aflatoxin-producing ability in synthetic media was positive. Despite that, cassava and cassava products were essentially free of aflatoxins, and only one sample of cassava flour contained traces of AFB1 (0.35 µg/kg).

Isolation and characterization of Aspergillus flavus strains in China.

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B1 (AFB1) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB1 biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.

Molecular characterization of Aspergillus flavus and aflatoxin contamination of wheat grains from Saudi Arabia.

Twelve species belonging to six fungal genera were found to be associated with wheat (Triticum aestivum L.) grain samples collected from three main regions in Saudi Arabia. The most common genera (average frequency) were Aspergillus (14.3%), Fusarium (29.1%), Penicillium (9.3%), and Alternaria (8.2%). Nineteen isolates of Aspergillus flavus were screened for their ability to produce aflatoxins using HPLC. Thirteen isolates produced aflatoxins ranging from 0.5 to 2.6 µg/kg. Inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of A. flavus to discriminate between aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, useful for genetic characterization. Clustering based on RAPD and ISSR dendograms was unrelated to geographic origin. RAPD and ISSR markers were not suitable to discriminate aflatoxigenic and non-aflatoxigenic isolates, but ISSR primers were better compared to RAPD.

Aflatoxin contamination in different fractions of rice from Pakistan and estimation of dietary intakes.

The purpose of this study was to determine the distribution of aflatoxins in rice milling fractions and to estimate dietary intakes. A total of 413 rice samples (paddy 58, parboiled 69, brown 84, white 93, and broken 109) were analyzed by HPLC with fluorescence detector. The results showed that 64 % paddy (16.35 ± 1.67 µg/kg), 38 % parboiled (14.20 ± 2.04 µg/kg), 33 % brown (9.85 ± 1.25 µg/kg), 42 % white (7.10 ± 1.39 µg/kg), and 50 % broken (8.5 ± 1.71 µg/kg) rice samples were contaminated with aflatoxins. It was found that paddy rice was most contaminated with aflatoxins while white rice was least contaminated. The percentage of samples exceeding EU maximum contents for total aflatoxins in rice (4 µg/kg) varied from 14 to 36 % when compared to the analyzed rice fractions. The total estimated amount of aflatoxin intake for average rice consumers ranged from 19.1 to 26.6 ng/kg body weight/day, much higher than the reference value of 1 ng/kg body weight/day. This is the first report discovering that rice is a major contributor to the dietary intake of aflatoxins in Pakistan.

Natural occurrence of aflatoxin residues in fresh and sun-dried meat in Nigeria.

INTRODUCTION: In recent times, food safety and security have generally remained basic human needs, therefore because of the largely unregulated nature of the Nigerian markets, coupled with the poor housing and feeding conditions to which animals are subjected in the abattoirs, a survey for assessing potential mycotoxin exposure through meat consumption was undertaken. METHODS: Eighty Samples of meat bought randomly from 5 major markets distributed in 5 local government areas of Oyo state , Nigeria were analysed for contaminating mycoflora using the plate count and micromorphological methods, while aflatoxin detection and quantification was by Thin Layer Chromatography (TLC). RESULTS: Mycological analysis of samples revealed a higher contamination level in the sun-dried samples. Eighteen fungi species belonging to 8 genera, namely, Aspergillus, Penicilliu, Alternaria, Cladosporium, Fusarium, Neurospora, Rhizopus and yeast were identified. The predominant genus Aspergillus yielded 7 species while the potential toxicogenic fungi represented 38% of the isolated mycoflora. The genera requiring higher water activity for growth ( Alternaria, Fusarium and yeast) were not obtained from the dried meat. Aflatoxins B1, B2, G1 and G2 were detected in all the samples analysed. The fresh samples with the exception of the total aflatoxin G (AFG) in kidney gave the highest mean concentrations for all aflatoxins, also an exceptionally high aflatoxin content was found in all the kidney samples. CONCLUSION: Aflatoxin detection in meat should be addressed urgently to avert the possible adverse health effects like aflatoxicosis, exacerbated malnutrition, suppression of growth and immune functions on consumers. Also the animal health inspectors should pay more attention to the feeding conditions of the animals on farm and the abattoirs.

Incidência de aflatoxinas em amostras de amendoim e paçoca comercializadas na cidade de Alfenas-MG, Brasil / Incidence of aflatoxins in samples of peanuts and paçoca marketed in the city of Alfenas-MG, Brazil

Foods are subject to contamination by chemical substances, including aflatoxins, which are potentially carcinogens. The presence of these substances in food is important risk in human population and animals. The aim of this study was to determine the occurrence of aflatoxins B1, B2, G1 and G2 in samples of peanuts and paçoca ramdomly acquired in shops and food market in the city of Alfenas-MG, Brazil, from June 2006 to February 2007. The technique used for the separation, identification and quantification of the substances was a liquid-liquid extraction with subsequent thin layer chromatography (TLC). Results showed that 38 percent of peanuts samples and 13 percent of paçoca samples were contaminated with aflatoxins and the concentrations ranged from 21 to 138 μg kg-1. All positive samples exceeded the maximum level established by Brazilian Legislation (20 μg kg-1). The results obtained in this work confirm the need for frequent monitoring of the presence of aflatoxins in food.

Os alimentos estão sujeitos à contaminação por substâncias químicas, dentre elas as aflatoxinas, as quais são potencialmente carcinogênicas. A presença de tais substâncias nos alimentos constitui importante risco para a população tanto humana quanto animal. O objetivo deste estudo foi determinar a ocorrência de aflatoxinas B1, B2, G1 e G2 em amostras de amendoim e paçoca adquiridas aleatoriamente no comércio da cidade de Alfenas- MG, Brasil, no período de junho de 2006 a fevereiro de 2007. A técnica utilizada para a separação, identificação e quantificação das substâncias foi a cromatografia em camada delgada com prévia extração, líquido-líquido, dos analitos. Os resultados demonstraram que 38 por cento das amostras de amendoim e 13 por cento das amostras de paçoca estavam contaminadas com aflatoxinas, sendo que as concentrações variaram de 21 a 138 μg kg-1. Todas as amostras positivas excederam o limite máximo permitido, preconizado pela legislação brasileira de 20 μg kg-1. Os achados do presente estudo corroboram com os relatados na literatura e confirmam a necessidade do freqüente o nitoramento da presença de aflatoxinas em alimentos.

A sensitive confirmatory method for aflatoxins in maize based on liquid chromatography/electrospray ionization tandem mass spectrometry.

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for measurement of aflatoxins B1, B2, G1, and G2 in maize is described. Aflatoxins (AFs) were extracted from 1 g samples by using tri-portions of acetonitrile/water (80:20, v/v) (10 + 7 + 7 mL), and 2/5 of the extract diluted to 500 mL by water was cleaned up with a 100 mg Carbograph-4 cartridge. After the addition of the internal standard AFM1, the final extract was analyzed by LC/ESI-MS/MS in positive ion mode using multiple reaction monitoring with a triple-quadrupole instrument. A C(18) column thermostatted at 45 degrees C with a mobile phase gradient of acetonitrile/water with 2 mmol/L ammonium formate was used. Although the matrix suppression effect was negligible, quantitation was achieved by an external calibration procedure using matrix-matched standard solutions to improve accuracy. Sample recoveries at four spiking levels ranged from 81 to 101% (relative standard deviation (RSD)

[Determination of aflatoxins in hot chilli products by matrix solid-phase dispersion and liquid chromatography].

A new method based on matrix solid-phase dispersion (MSPD) extraction with neutral alumina and co-column purification with graphitized carbon black has been developed to determine aflatoxins B1, B2, G1, G2 in hot chilli products. The method includes liquid chromatography and fluorescence detection with on-line post-column derivatization with bromine. Optimization of different parameters, such as the type of solid supports for matrix dispersion and co-column clean-up was carried out. The recoveries of aflatoxins B1, B2, G1 and G2 were 95.4%, 87.3%, 91.5% and 92.6%, respectively, with relative standard deviations ranging from 3.3% to 6.1%. The limits of detection were in the range of 0.10 ng/g (B2, G2) to 0.25 ng/g (B1, G1). In addition, the comparison of the extraction and purification effect of MSPD with immunity affinity column showed that, MSPD is a valid method to analyze aflatoxins in hot chilli products.

Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry.

An easy method for the determination of aflatoxins B1, G1, B2 and G2 in Rhammus purshiana by LC coupled to mass spectrometry has been developed. Aflatoxins were extracted with a mixture of methanol and water and then it was purified by solid-phase clean-up using a polymeric sorbent, not described previously, for the determination of these toxins. The eluted extract was injected into the chromatographic system using a reversed-phase C18 short column with an isocratic mobile phase composed of methanol-water (30:70). A single-quadruple mass spectrometry using an electrospray ionization source operating in the positive ion mode was used to detect aflatoxins due to derivatization presenting several disadvantages. Recoveries of the full analytical procedure were 110% for aflatoxin B1, 89% for aflatoxin B2, 81% for aflatoxin G1 and 77% for aflatoxin G2. Detection limit (S/N = 3) was 10 ng and quantification limit (S/N = 10) was 25 ng, calculated as amount in medicinal herb.

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae.

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.

Enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins.

We detected biosynthetic activity for aflatoxins G(1) and G(2) in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G(1) and G(2) were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B(1), aflatoxin B(2), 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G(2) from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G(1) was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G(1) and G(2). Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.