Early expression of the aflatoxin gene cluster in Aspergillus nomiae isolated from Brazil nut.

Aspergillus nomiae is the most important contaminant in Brazil nut due to its high incidence in these nuts and its strong production of carcinogenic metabolites: aflatoxins (AF). Aflatoxin biosynthesis pathway in A. nomiae is poorly studied. Thus, in present investigation, aflatoxin production and gene cluster (aflC, aflQ, aflU, and aflX) expression profile were evaluated on two strains of A. nomiae isolated from Brazil nut samples collected in the Amazon region, and cultivated on Brazil nut-based medium. The results showed that the expression of the aflatoxin gene cluster in A. nomiae, started at day 2 and occurred before the production of aflatoxins. Aflatoxin production (AFB1 and AFG1) was detected on day 3 on both strains. From day 4 onwards, all four toxins were detected and their production kept at similar proportions (AFG1>AFB1>AFG2>AFB2). Thus, the anticipated information obtained through early expression profile results of aflC, aflQ, aflU, and aflX gene cluster in A. nomiae may foresee aflatoxin production before its detection in Brazil nuts.

A non-toxigenic Aspergillus flavus strain prevents the spreading of Fusarium verticillioides and fumonisins in maize.

We evaluated Fusarium verticillioides and fumonisin contamination in maize samples after application of a non-aflatoxigenic Aspergillus flavus strain in the field. The sampling was performed 150 days after planting. The results showed a reduction in F. verticillioides frequency, as well as in fumonisin levels when samples were obtained from field areas treated with non-aflatoxigenic A. flavus strain. These results suggested a competition for substrate or space between fungi reducing the frequency of F. verticillioides and, consequently, fumonisin production.

Characterization of Aspergillus section Flavi isolated from organic Brazil nuts using a polyphasic approach.

Brazil nut (Bertholletia excelsa), an important non-timber forest product from Amazonia, is commercialized in worldwide markets. The main importers of this nut are North America and European countries, where the demand for organic products has grown to meet consumers concerned about food safety. Thus, the precise identification of toxigenic fungi is important because the Brazil nut is susceptible to colonization by these microorganisms. The present study aimed to characterize by polyphasic approach strains of Aspergillus section Flavi from organic Brazil nuts. The results showed Aspergillus flavus as the main species found (74.4%), followed by Aspergillus nomius (12.7%). The potential mycotoxigenic revealed that 80.0% of A. flavus were toxin producers, 14.3% of which produced only aflatoxin B (AFB), 22.85% of which produced only cyclopiazonic acid (CPA), and 42.85% produced both them. All strains of A. nomius were AFB and AFG producers and did not produce CPA. There is no consensus about what Aspergillus species predominates on Brazil nuts. Apparently, the origin, processing, transport and storage conditions of this commodity influence the species that are found. The understanding about population of fungi is essential for the development of viable strategies to control aflatoxins in organic Brazil nuts.

Fumonisin B1 in cereal mixtures marketed in Brazil.

The aim of this study was to detect and quantify fumonisin B1 (FB1) in cereal mixtures marketed in Brazil. Fifteen samples from different lots were acquired each month by internet from supermarkets during seven months, adding up to 105 analysed samples. The unit sample constituted of an original package with a minimum of 250 g. Extraction and clean-up of samples for FB1 determination were carried out using immunoaffinity columns. Identification and quantification of FB1 were performed by high performance liquid chromatography. Eighty-eight (83.8%) samples were contaminated with FB1 and four (3.8%) presented levels above 500 µg kg(-1) (634, 703, 1269 and 1876 µg kg(-1)). Maximum FB1 + FB2 levels allowed by Brazilian regulations will reach 1500 µg kg(-1) for corn flour in 2016 and 1000 µg kg(-1) for others corn products. This study showed that even at levels below the legislative limits, human exposure to this toxin can occur constantly.

Mycobiota and mycotoxins in Brazil nut samples from different states of the Brazilian Amazon region.

The objective of this study was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in Brazil nut samples collected in different states of the Brazilian Amazon region: Acre, Amazonas, Amapá, and Pará. A total of 200 husk samples and 200 almond samples were inoculated onto Aspergillus flavus-parasiticus agar for the detection of fungi. Mycotoxins were analyzed by high-performance liquid chromatography. The mycobiota comprised the following fungi, in decreasing order of frequency: almonds - Phialemonium spp. (54%), Penicillium spp. (16%), Fusarium spp. (13%), Phaeoacremonium spp. (11%), and Aspergillus spp. (4%), husks - Phialemonium spp. (62%), Phaeoacremonium spp. (11%), Penicillium spp. (10%), Fusarium spp. (9%), and Aspergillus spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins were detected in 22 (11%) of the 200 almond samples, with 21 samples presenting aflatoxin B(1) levels above 8µg/kg, the limit established by the European Commission for Brazil nuts for further processing. Nineteen (9.5%) of the 200 husk samples contained aflatoxins, but at levels lower than those seen in almonds. Cyclopiazonic acid (CPA) was detected in 44 (22%) almond samples, with levels ranging from 98.65 to 161.2µg/kg. Aspergillus nomius and A. flavus were the most frequent Aspergillus species. The presence of fungi does not necessarily imply mycotoxin contamination, but almonds of the Brazil nut seem to be a good substrate for fungal growth.

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds.

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds.

Effects of gamma radiation on maize samples contaminated with Fusarium verticillioides.

The efficacy of gamma-irradiation as a method of decontamination of maize containing Fusarium verticillioides under controlled conditions of relative humidity (RH) (97.5%) and water activity has been studied. Maize grains inoculated with a spore suspension of F. verticillioides were irradiated to 2, 5, and 10 kGy. Thereafter, the irradiated and control samples were analyzed for the presence of fumonisins, their viable cells were counted, and their morphology was investigated by electronic microscopy. It was found possible to decrease the risk of exposure to fumonisins by irradiating maize to 5 or 10 kGy. However, at the dose of 2 kGy, the survived fungi (36%) can produce more fumonisins than the fungi in the control unirradiated samples under the same conditions.

Effects of prolonged administration of aflatoxin B1 and fumonisin B1 in laying Japanese quail.

In the present study, 288 8-wk-old Japanese quail were randomly distributed into 6 experimental groups (48 birds per group) and fed the following diets for 140 d: 1) 0 (control); 2) 10 mg of fumonisin B1 (FB1); 3) 50 microg of aflatoxin B1 (AFB1); 4) 50 microg of AFB1 + 10 mg of FB1; 5) 200 microg of AFB1; and 6) 200 microg of AFB1 + 10 mg of FB1/kg of feed. Each treatment consisted of 4 replicates of 12 quail. Egg production and individual egg weight were checked daily. Feed intake and feed conversion were determined weekly. Results showed that by the end of the fifth cycle, average egg weight was lower (P < 0.05) in groups fed 10 mg of FB1/kg, 50 microg of AFB1/kg, 200 microg of AFB1/kg, and 10 mg of FB1 + 50 microg of AFB1/kg of feed. Egg production decreased (P < 0.05) in birds fed 10 mg of FB1/kg by the third, fourth, and fifth cycles. Feed intake was lower (P < 0.05) in birds fed 10 mg of FB1/kg by the fourth and fifth cycles, and in birds fed 50 and 200 microg of AFB1/kg in the fifth cycle. Birds fed 10 mg of FB1 + 50 microg of AFB1/kg consumed less feed (P < 0.05) in the first, second, and fifth cycles. Results indicated that prolonged administration of FB1 and AFB1, singly or in combination at the levels evaluated, may cause economic losses to quail egg producers.

Effect of low levels of dietary aflatoxin B1 on laying japanese quail.

In the present study, 256 7-wk-old Japanese quail were randomly distributed into four experimental groups (64 birds per group) and given rations containing 0 (controls), 25, 50, or 100 (g aflatoxin B, (AFB1)/kg feed for 168 d. Each treatment consisted of four replicates of 16 quail. Egg production and individual egg weight were checked daily. Feed consumption and feed use were determined weekly. Eggs laid in the last day of each 28-d laying period were collected and subjected to individual analysis for specific gravity, Haugh units, shell thickness and percentage eggshell. Results showed that average egg production, feed use, and body weights were not affected (P > 0.05) by AFB1. However, feed consumption was lower (P < 0.05) for groups fed 50 or 100 microg AFB1/kg. Egg weight was significantly lower (P < 0.05) only for groups exposed to 50 and 100 microg AFB1/kg. Average egg specific gravity, Haugh units, and shell thickness were not affected (P > 0.05) by AFB1. Percentage eggshell was higher (P < 0.05) in the group fed the ration containing 100 microg AFB1/kg. Treatment associated lesions were observed only in the liver. Hepatic cell vacuolation with fatty infiltration was observed in all liver samples of quail fed AFB1-contaminated rations. Bile duct proliferation and trabecular disorder were found only in livers of quail on the 100-microg AFB1/kg treatment. Results indicated that chronic exposure to AFB1 at levels above 50 microg/kg could adversely affect quail performance, emphasizing the importance of controlling aflatoxin contamination in quail rations.

Aflatoxin B1 residues in eggs of laying hens fed a diet containing different levels of the mycotoxin.

The present study was carried out to evaluate the excretion of aflatoxin B1 residues in eggs of young laying hens fed aflatoxin B1-contaminated rations for 8 weeks. To this end, 96 twenty-week-old hens were randomly distributed into four experimental groups (24 birds per group) and given rations containing either 0 (controls), 100 micrograms, 300 micrograms or 500 micrograms aflatoxin B1/kg feed. Egg aflatoxin B1 residues were determined by thin layer chromatography; two samples per treatment per week were used for analysis. Egg production and average egg weights were not affected (p < 0.05) in the groups receiving aflatoxin B1-contaminated rations. Residues of aflatoxin B1 were detected only in the eggs of hens given 500 micrograms/kg feed, at levels that ranged from 0.05 to 0.16 microgram/kg (average: 0.10 microgram/kg). The results indicate that the feed to eggs aflatoxin B1 transmission ratio was approximately 5000:1, emphasizing the importance of controlling aflatoxin levels in rations for laying hens.