Unveiling ochratoxin A and ochratoxigenic fungi in Brazilian artisanal Cheeses: Insights from production to consumption.

Ochratoxin A (OTA) is a toxin produced by several Aspergillus species, mainly those belonging to section Circumdati and section Nigri. The presence of OTA in cheese has been reported recently in cave cheese in Italy. As artisanal cheese production in Brazil has increased, the aim of this study was to investigate the presence of ochratoxin A and related fungi in artisanal cheese consumed in Brazil. A total of 130 samples of artisanal cheeses with natural moldy rind at different periods of maturation were collected. Of this total, 79 samples were collected from 6 producers from Canastra region in the state of Minas Gerais, since this is the largest artisanal cheese producer region; 13 samples from one producer in the Amparo region in the state of São Paulo and 36 samples from markets located in these 2 states. Aspergillus section Circumdati occurred in samples of three producers and some samples from the markets. A. section Circumdati colony counts varied from 102 to 106 CFU/g. Molecular analysis revealed Aspergillus westerdijkiae (67 %) as the most frequent species, followed by Aspergillus ostianus (22 %), and Aspergillus steynii (11 %). All of these isolates of A. section Circumdati were able to produce OTA in Yeast Extract Sucrose Agar (YESA) at 25 °C/7 days. OTA was found in 22 % of the artisanal cheese samples, ranging from 1.0 to above 1000 µg/kg, but only five samples had OTA higher than 1000 µg/kg. These findings emphasize the significance of ongoing monitoring and quality control in the artisanal cheese production process to minimize potential health risks linked to OTA contamination.

Assessment of early harvest in the prevention of aflatoxins in peanuts during drought stress conditions.

The present study aimed to evaluate the effectiveness of early harvest in preventing aflatoxins in peanuts under drought-stress conditions. A field experiment was conducted on the 2018-2019 and 2019-2020 growing seasons in a greenhouse with an irrigation system to induce three drought stress conditions: no stress, mild, and severe stress. In addition, three harvest dates were proposed: two weeks earlier, one week earlier, and ideal harvest time. The mean peanut yield was 2634 kg/ha, considering the two growing seasons, and the drought stress conditions and harvest dates did not influence significantly. The shelling percentage was significantly higher in samples harvested at ideal harvest (77.7 %) than two weeks earlier (76.2 %) and was not influenced by drought stress conditions. Although a low mean percentage of grains with insect damage was identified, this percentage was statistically higher under severe stress (0.4 %) compared to no-stress conditions (0.2 %). The soil contamination ranged from 2.52 × 103 to 1.64 × 104 CFU/g of Aspergillus section Flavi, and the drought stress resulted in significantly higher concentrations in mild and severe stressed samples. A. section Flavi was found to infect all the peanut kernel samples. The drought stress resulted in higher percentages of A. section Flavi infections in samples from mild and severe stress conditions. The harvest date did not influence the soil and peanut kernel occurrence of A. section Flavi. A total of 435 and 796 strains of A. section Flavi were isolated from soil and peanut kernels, respectively. The potential of aflatoxin production by soil isolates was 31, 44, and 25 % for aflatoxin non-producers, aflatoxin B producers, and aflatoxin B and G producers, respectively, while in peanut kernel isolates were 44, 44, and 12 %. Three different A. section Flavi species were identified from peanut kernels: A. flavus, A. parasiticus, and A. pseudocaelatus. The mean aflatoxin concentration in peanut kernels was 42, 316, and 695.5 µg/kg in samples under no stress, mild stress, and severe stress conditions, respectively. Considering the harvest time, the mean aflatoxin concentration was 9.9, 334.3, and 614.2 µg/kg in samples harvested two weeks earlier, one week earlier, and in ideal harvest, respectively. In conclusion, the early harvest proved to be a viable, cost-free alternative for controlling aflatoxin in the peanut pre-harvest, resulting in a safer product and a better quality for sale and economic gain.

Deep Genotypic Species Delimitation of Aspergillus Section Flavi Isolated from Brazilian Foodstuffs and the Description of Aspergillus annui sp. nov. and Aspergillus saccharicola sp. nov.

Aspergillus section Flavi is a fungal group that is important in food because it contains spoilage and potentially aflatoxigenic species. Aflatoxins are metabolites that are harmful to human and animal health and have been recognized as the primary natural contaminant in food. Therefore, recognizing the biodiversity of this group in food is necessary to reduce risks to public health. Our study aimed to investigate the diversity of Aspergillus section Flavi isolated from Brazilian foodstuffs such as cassava, sugarcane, black pepper, paprika, Brazil nuts, yerba-mate, peanuts, rice, and corn. A polyphasic approach integrating phenotypic data and multilocus genotypic analyses (CaM, BenA, and RPB2) was performed for 396 strains. Two new species in the Aspergillus subgenus Circumdati section Flavi are proposed using maximum-likelihood analysis, Bayesian inference, and coalescence-based methods: Aspergillus saccharicola sp. nov. and Aspergillus annui sp. nov. A. saccharicola sp. nov. belongs to the series Flavi, is a potentially aflatoxigenic species (B1, B2, G1, and G2), closely related to Aspergillus arachidicola, and was found mostly in sugarcane. A. annui sp. nov. was isolated from samples of sweet paprika. To accommodate A. annui sp. nov., a new series Annuorum was proposed.

Low-cost, specific PCR assays to identify the main aflatoxigenic species of Aspergillus section Flavi.

Aflatoxins are fungal metabolites that are present as contaminants in food globally. Most aflatoxigenic species belong to Aspergillus section Flavi, and the main ones are grouped in the A. flavus clade, where many cryptic species that are difficult to discriminate are found. In this study, we investigated inter- and intraspecific diversity of the A. flavus clade to develop low-cost, species-specific PCR assays for identifying aflatoxigenic species. A total of 269 sequences of the second largest subunit of RNA polymerase II (RPB2) locus were retrieved from GenBank, and primer pairs were designed using data mining to identify A. flavus, A. parasiticus, and A. novoparasiticus. Species-specific amplicons of approximately 620, 350, and 860 bp enabled identification of target species as A. flavus, A. parasiticus, and A. novoparasiticus, respectively.

Diversity within Aspergillus niger Clade and Description of a New Species: Aspergillus vinaceus sp. nov.

Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).

Modeling aflatoxin B1 production by Aspergillus flavus during wheat malting for craft beer as a function of grains steeping degree, temperature and time of germination.

This study aimed to model the aflatoxin B1 (AFB1) production by A. flavus in wheat grains during malting for craft beer. A total of sixty-four different combinations of grains steeping degree (ST; 41, 43, 45 and 47%), temperature (13, 15, 17 and 19 °C) and time of germination (48, 72, 96 and 120 h), comprising the range of malting conditions that allow the production of quality malt, were assayed. AFB1 was produced in a range of 15.78 ± 3.54 µg/kg (41% ST, 13 °C for 48 h) to 284.66 ± 44.34 µg/kg (47% ST, 19 °C for 120 h). The regression model showing an acceptable fit to the experimental data (adjusted R2 0.84) for AFB1 as a function of grains steeping degree, temperature and time of germination. Results showed that AFB1 levels in wheat malt increase with increase of the temperature or time of germination. Within the range of tested malting conditions, no significant effects were observed for steeping degree on AFB1 levels in wheat malt. The generated model is useful to estimate the AFB1 levels in wheat malt. Findings highlight overall that if wheat grains are contaminated with A. flavus, AFB1 might be produced in malt in levels above the limits set by regulatory agencies, regardless the steeping conditions used.

Fungal communities in rice cultivated in different Brazilian agroclimatic zones: From field to market.

Rice is one of the most consumed cereals in Brazil and around the world. Due to the major health impact of rice consumption on populations, studies about its quality have great importance. The present study determined the mycobiota of soil, field, processing and market rice samples from two production systems in Brazil, dryland in the state of Maranhão and wetland in the state of Rio Grande do Sul. These areas are distinct agroclimatic zones. A total of 171 rice and 23 soil samples were analyzed. A high differentiation was observed in the composition of the fungal communities found in the two production systems, as the wetland presented greater fungal incidence and biodiversity. It was observed that toxigenic species from Aspergillus section Flavi and Fusarium, present in the field, may infect rice grains pre or postharvest and may persist into the final product.

Understanding Mycotoxin Contamination Across the Food Chain in Brazil: Challenges and Opportunities.

Brazil is one of the largest food producers and exporters in the world. In the late 20th century, the European Union program for the harmonization of regulations for contaminants in food, including mycotoxins, led to the examination of mycotoxin contamination in foods at a global level. The problem of the rejection of food by the European Union and other countries became a Brazilian national priority because of economic and food safety aspects. Ochratoxin A in coffee and cocoa and aflatoxins in Brazil nuts are examples of the impact of technical trade barriers on Brazilian foods. To overcome these threats, several strategies were undertaken by Brazilian and international organizations. In this context, the Codex Commission on Food Contaminants (CCCF) has emerged as a forum to discuss with more transparency issues related to mycotoxins, focusing on establishing maximum levels and codes of practices for some commodities and mycotoxins to ensure fair trade and food safety. Our experience in investigating and understanding mycotoxin contamination across the food chains in Brazil has contributed nationally and internationally to providing some answers to these issues.

Occurrence and diversity of Aspergillus in commercial yerba mate elaborated for the Brazilian beverage 'chimarrão'.

Dried leaves and stems of Ilex paraguariensis St. Hil. (yerba mate) are used to make a popular beverage in some countries of South America, commonly known as "chimarrão". The present study was designed to evaluate the occurrence of toxigenic Aspergillus in yerba mate in order to define the mycotoxin risk associated with this foodstuff. All samples tested were positive for fungal contamination, and the fungal load per sample ranged from 2.0 × 102 to 1.6 × 104 CFU/g. Aspergillus section Nigri was found in all samples and represented 76.5% of the total fungi isolated. Aspergillus section Circumdati, Aspergillus section Flavi and Aspergillus section Cremei were found at low frequencies. Thirteen different Aspergillus species were identified. The most common species found was A. luchuensis, which does not produce any harmful toxin for humans. A. niger, A. welwitschiae, A. flavus and A. novoparasiticus, all potentially toxigenic species, were found only in small quantities. The A. niger and A. welwitschiae strains were cultured to test for ochratoxin A and fumonisin B2 production. Only one strain producing ochratoxin A was found, but approximately 29% of the strains were positive for fumonisin B2. The A. flavus and A. novoparasiticus strains were tested for aflatoxins production, and 63% were positive. A. pallidofulvus, recently assigned to A. section Circumdati, was reported for the first time in herbs. All A. pallidofulvus strains analyzed in this study were negative for ochratoxin A production. In conclusion, A. section Nigri occurs with high frequency in yerba mate, and A. luchuensis is the predominant species. Although toxigenic species were found in this herb, the incidence was low.

Occurrence of Aspergillus section Flavi and aflatoxins in Brazilian rice: From field to market.

The guarantee of the high quality of rice is of utmost importance because any toxic contaminant may affect consumer health, especially in countries such as Brazil where rice is part of the daily diet. A total of 187 rice samples, from field, processing and market from two different production systems, wetland from the state of Rio Grande do Sul, dryland, from the state of Maranhão and market samples from the state of São Paulo, were analyzed for fungi belonging to Aspergillus section Flavi and the presence of aflatoxins. Twenty-three soil samples from wetland and dryland were also analyzed. A total of 383 Aspergillus section Flavi strains were isolated from rice and soil samples. Using a polyphasic approach, with phenotypic (morphology and extrolite profiles) and molecular data (beta-tubulin gene sequences), five species were identified: A. flavus, A. caelatus, A. novoparasiticus, A. arachidicola and A. pseudocaelatus. This is the first report of these last three species from rice and rice plantation soil. Only seven (17%) of the A. flavus isolates produced type B aflatoxins, but 95% produced kojic acid and 69% cyclopiazonic acid. Less than 14% of the rice samples were contaminated with aflatoxins, but two of the market samples were well above the maximum tolerable limit (5µg/kg), established by the Brazilian National Health Surveillance Agency.

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry.

Aspergillus labruscus sp. nov., a new species of Aspergillus section Nigri discovered in Brazil.

A novel fungal species, Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).

Kinetics of aflatoxin degradation during peanut roasting.

This study investigated aflatoxin degradation during peanut roasting. First, peanuts contaminated with three initial aflatoxin concentrations (35, 332 and 695µg/kg) were roasted at 180°C for up to 20min. The percentage of aflatoxin degradation after 20min were 55, 64 and 81% for peanuts contaminated with aflatoxin at 35, 332 and 695µg/kg, respectively. This difference was statistically significant (p<0.05), showing that initial concentration influences aflatoxin reduction. Thereafter, peanut samples contaminated with an initial aflatoxin concentration of 85µg/kg were roasted at 160, 180 and 200°C for 5, 10, 15, 20 and 25min, then residual concentrations of aflatoxin were determined. Roasting at 160, 180 and 200°C resulted in an aflatoxin reduction of 61.6, 83.6 and 89.7%, respectively. This study has provided quantitative data reinforcing the fact that roasting alone is not enough to control aflatoxins in peanuts.

Occurrence and fumonisin B2 producing potential of Aspergillus section Nigri in Brazil nuts.

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B2 (FB2) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB2: 41 strains (20.5%) were FB2 producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB2, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB2 producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB2.

Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer.

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

Marine-derived fungus Aspergillus cf. tubingensis LAMAI 31: a new genetic resource for xylanase production.

Marine-derived fungi have been reported as relevant producers of enzymes, which can have different properties in comparison with their terrestrial counterparts. The aim of the present study was to select from a collection of 493 marine-derived fungi the best producer of xylanase in order to evaluate the enzymatic production under different conditions. A total of 112 isolates produced xylanase in solid medium containing xylan as the carbon source, with 31 of them able to produce at least 10 U/mL of the enzyme. The best production (49.41 U/mL) was achieved by the strain LAMAI 31, identified as Aspergillus cf. tubingensis. After confirming the lack of pathogenicity (absence of ochratoxin A and fumonisin B2 production) this fungus was submitted to the experimental design in order to evaluate the effect of different variables on the enzymatic production, with the aim of optimizing culture conditions. Three experimental designs (two Plackett-Burman and one factorial fractional) were applied. The best condition for the enzymatic production was defined, resulting in an increase of 12.7 times in comparison with the initial production during the screening experiments. In the validation assay, the peak of xylanase production (561.59 U/mL) was obtained after 96 h of incubation, being the best specific activity achieved after 72 h of incubation. Xylanase from A. cf. tubingensis LAMAI 31 had optimum pH and temperature at 5.0 and 55 °C, respectively, and was shown to be stable at a range of 40-50 °C, and in pH from 3.6 to 7.0. Results from the present work indicate that A. cf. tubingensis LAMAI 31 can be considered as a new genetic resource for xylanase production.

Fungi and mycotoxins in cocoa: from farm to chocolate.

Cocoa is an important crop, as it is the raw material from which chocolate is manufactured. It is grown mainly in West Africa although significant quantities also come from Asia and Central and South America. Primary processing is carried out on the farm, and the flavour of chocolate starts to develop at that time. Freshly harvested pods are opened, the beans, piled in heaps or wooden boxes, are fermented naturally by yeasts and bacteria, then dried in the sun on wooden platforms or sometimes on cement or on the ground, where a gradual reduction in moisture content inhibits microbial growth. Beans are then bagged and marketed. In processing plants, the dried fermented beans are roasted, shelled and ground, then two distinct processes are used, to produce powdered cocoa or chocolate. Filamentous fungi may contaminate many stages in cocoa processing, and poor practices may have a strong influence on the quality of the beans. Apart from causing spoilage, filamentous fungi may also produce aflatoxins and ochratoxin A. This review deals with the growth of fungal species and formation of mycotoxins during the various steps in cocoa processing, as well as reduction of these contaminants by good processing practices. Methodologies for fungal and mycotoxin detection and quantification are discussed while current data about dietary exposure and regulation are also presented.

Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.

Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 105 conidia per reaction with the primer set ID9 for A. nomius and 104 conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61.5% and 66.7% for A. nomius and A. flavus, respectively. When LAMP results were compared with the presence of aflatoxins in corresponding samples, the Negative Predictive Values were 22.2% and 44.4% and the Positive Predictive Values were 52.2% and 78.3% for aflatoxins produced by A. nomius and A. flavus, respectively. The LAMP assays described in this study have been demonstrated to be a specific, sensitive and easy to use tool for the survey of Brazil nuts for contaminations with potential aflatoxin-producing A. nomius and A. flavus in low tech environments where resources may be limited.

The biodiversity of Aspergillus section Flavi in brazil nuts: from rainforest to consumer.

A total of 288 brazil nut samples (173 kernel and 115 shell) from the Amazon rainforest region and São Paulo State, Brazil were collected at different stages of brazil nut production. Samples were analysed for: percentages of aflatoxigenic fungal species and potential for aflatoxin production and presence of aflatoxins. Aspergillus nomius was the most common species found (1235 isolates) which amounted to 30% of the total species with potential to produce aflatoxins. This species is of concern since 100% of all isolates produced aflatoxins B(1), B(2), G(1) and G(2). Aspergillus flavus was almost equally common (1212 isolates) although only 46% produced aflatoxins under laboratory conditions, and only aflatoxins B(1) and B(2). Low number of other species with the potential to produce aflatoxins was isolated: Aspergillus arachidicola and Aspergillus bombycis produced B and G aflatoxins whilst Aspergillus pseudotamarii produced only aflatoxin B(1). The total aflatoxin levels found in samples taken from the rainforests was 0.7 µg/kg, from processing plants before and after sorting 8.0 and 0.1 µg/kg respectively, from street markets in the Amazon region 6.3 µg/kg and from supermarkets in São Paulo State 0.2 µg/kg. Processing, which included manual or mechanical sorting and drying at 60°C for 30 to 36 h, eliminated on average more than 98% of total aflatoxins. These results showed that sorting is a very effective way to decrease aflatoxin content in brazil nuts.

Occurrence of ochratoxin A in cocoa by-products and determination of its reduction during chocolate manufacture.

This work reports an investigation carried out to assess the natural occurrence of ochratoxin A in 168 samples from different fractions obtained during the technological processing of cocoa (shell, nibs, liquor, butter, cake and cocoa powder) and the reduction of ochratoxin A during chocolate manufacture. Ochratoxin A analyses were performed with immunoaffinity columns and detection by high performance liquid chromatography. Concerning the natural ochratoxin A contamination in cocoa by-products, the highest levels of ochratoxin A were found in the shell, cocoa powder and cocoa cake. The cocoa butter was the least contaminated, showing that ochratoxin A seems to remain in the defatted cocoa solids. Under the technological conditions applied during the manufacture of chocolate in this study and the level of contamination present in the cocoa beans, this experiment demonstrated that 93.6% of ochratoxin A present in the beans was reduced during the chocolate producing.