Nuclear Magnetic Resonance Approach in Brazil Nut Oil and the Occurrence of Aflatoxins.

Carcinogenic metabolites of fungi such as aflatoxins play a toxic role in some tree nuts and need to be monitored in their by-products, such as oil. In this context, Brazil nut (Bertholletia excelsa) oil, which is a commodity of great economic importance to Brazil, requires attention to monitor the presence of these toxic agents. Therefore, this study aimed to evaluate the presence of aflatoxins in Brazil nut oil and relate it to the presence of fatty acids in the oil as a surveillance tool for food safety. Brazil nut oil samples (n= 25) were acquired in northern Brazil as (a) non-branded products (n= 07) produced by local farmers using artisanal methods from nuts to be discarded by the industry and (b) industrialized products (n= 18). The samples were analyzed for total aflatoxin content by high-performance liquid chromatography and fatty acid content by nuclear magnetic resonance imaging. Seven (28%) samples were positive for the aflatoxin fractions (B1 + B2 + G1 + G2), ranging from undetected (<2.32) to 50.87 µg/kg. Of the aflatoxin positive samples evaluated by NMR analysis, it was not possible to state that the presence of a particular fatty acid can interfere or influence aflatoxin contamination. This was the first study with data on aflatoxin occurrence in Brazil nut oil. Nevertheless, further research is required to relate saturated or unsaturated fatty acid content with aflatoxin levels. We also suggest the implementation of systems to prevent contamination of the raw materials (seed) and detoxification of the oil to guarantee the product's safety and quality.

Design of Nanostructured Lipid Carriers Containing Cymbopogon martinii (Palmarosa) Essential Oil against Aspergillus nomius.

Palmarosa essential oil (PEO) is an alternative to synthetic fungicides to control the contamination by food-deteriorating fungi, such as Aspergillus nomius. Nonetheless, the low long-term stability and volatility hamper its utilization. Thus, this study aimed to develop nanostructured lipid carriers (NLCs) containing PEO to improve its stability and consequently prolong the activity against A. nomius. A mixture design was applied to find the best preparation conditions for antifungal activity. The characterization analyses included size measurements, zeta potential (ζ-potential), entrapment efficiency (EE), and antifungal activity (by inhibition of mycelial growth (IMG) and/or in situ test (pre-contaminated Brazil nuts) tests). The nanocarriers presented particle sizes smaller than 300 nm, homogeneous size distribution, ζ-potential of -25.19 to -41.81 mV, and EE between 73.6 and 100%. The formulations F5 and F10 showed the highest IMG value (98.75%). Based on the regression model, three optimized formulations (OFs) were tested for antifungal activity (IMG and in situ test), which showed 100% of inhibition and prevented the deterioration of Brazil nuts by A. nomius. The preliminary stability test showed the maintenance of antifungal activity and physicochemical characteristics for 90 days. These results suggest a promising system as a biofungicide against A. nomius.

Efficacy of sodium hypochlorite and peracetic acid against Aspergillus nomius in Brazil nuts.

The objective of this work was to assess the efficacy of sodium hypochlorite and peracetic acid for sanitization of Brazil nuts. To evaluate the natural microbiota of the nuts, the total bacteria and fungi as well as the Aspergillus section Flavi were counted. The moisture, water activity and the presence of aflatoxins was quantified. The response surface method was used to determine the influence of exposure time and sanitizers concentration on the reduction of Aspergillus nomius inoculated on the nuts. Microbiological, sensory and quantification analyzes of aflatoxins were performed under optimum conditions The evaluation of the initial contamination of the nuts, despite presenting high microbiological contamination, humidity and water activity, was not detected aflatoxins in any samples. In artificially inoculated samples, the response surface and the desirability function were obtained to determine the optimal point of use for each sanitizer. The nuts had high microbiological contamination, moisture content and water activity. Aflatoxins were not detected in any samples. The response surface and desirability function indicated the optimal sanitization conditions were 250 mg/L and 8.5 min and 140 mg/L and 15 min for sodium hypochlorite and peracetic acid, respectively. Reductions greater than 2 log CFU/g were obtained with sodium hypochlorite and of 1 log CFU/g for peracetic acid. In the tests performed with new Brazil nuts samples under the optimized conditions, reductions of less than 2 log CFU/g were obtained. Aflatoxin B1 was detected in one untreated sample (1.51 µg/kg), one sample treated with sodium hypochlorite (0.60 µg/kg) and two samples treated with peracetic acid (0.64 and 0.72 µg/kg). Demonstrating that the sanitizers in the concentrations used had no action on aflatoxins, despite being efficient for fungal control. The treatments did not cause an unacceptable sensorial impact on the samples.

Silver nanoparticle from whole cells of the fungi Trichoderma spp. isolated from Brazilian Amazon.

Metal nanoparticles are a promising approach for the development of new antimicrobial systems. Silver nanoparticles (AgNP) have a significant antibacterial activity through bacterial surface adsorption and oxidative stress induction, as indicated by recent observations. This research aimed to use endophytic fungi from the genus Trichoderma spp. isolated from the Bertholletia excelsa (Brazil-nut) seeds and the soil to biosynthesize AgNPs and also test their antibacterial activity. The use of these fungi for this purpose not only valorizes the Amazon biodiversity but it also uses cleaner and cheaper processes, being part of the Green Chemistry concept. The particles were analyzed through Ultraviolet-Visible Spectroscopy and ZetaSizer and the band of absorption at 420 nm was analyzed through Localized Surface Plasmon Resonance. After characterization, the AgNP were tested for antibacterial activity against several bacterial strains, when it was observed that their antibacterial activity was superior in Gram-negative bacteria.

Effect of temperature on growth, gene expression, and aflatoxin production by Aspergillus nomius isolated from Brazil nuts.

Aspergillus nomius is a potent producer of aflatoxins B and G and is one of the most common species of fungi found in Brazil nuts. Temperature is considered a major abiotic factor that influences fungal colonization and aflatoxin production in nuts during pre- and post-harvest. Therefore, assessment of the response of aflatoxigenic species to different temperatures is important to add information about the understanding of aflatoxin production by Aspergillus nomius and may help in the development of new strategies to prevent aflatoxin contamination. The aim of this study was to evaluate the effect of temperature (25, 30, and 35 °C) on the radial growth, aflatoxin production (B and G), and aflatoxin gene expression of seven A. nomius strains isolated from Brazil nuts. The optimal temperature for growth was 30 °C and was also the best condition for the expression of the aflR, aflD, and aflQ genes. However, maximum production of aflatoxins B and G occurred at 25 °C. Interestingly, high expression of the structural gene aflQ was observed in the maximum aflatoxin production condition (25 °C). The present study demonstrates that temperature may influence aflatoxin production by A. nomius. The combination of molecular and physiological data aids the understanding of the aflatoxigenic species response to different temperatures and can assist in predicting the driving environmental factors that influence aflatoxin contamination of Brazil nuts.

Qualidade microbiológica de castanha-do-brasil (Berthollethia excelsa H B K) comercializada na Amazônia Ocidental / Microbiological quality of castanha-do-brasil (Berthollethia excelsa H B K) marketed in the Western Amazon

A castanha-do-Brasil é um produto extrativista de grande relevância econômica. Condições ambientais, manipulação impertinente e características do fruto oferecem subsídios para o crescimento de microrganismos contaminantes. O objetivo desse trabalho foi avaliar a qualidade higiênico-sanitária de Castanha-do-Brasil comercializada na Amazônia Ocidental através da pesquisa de Salmonella sp. e quantificação de bactérias heterotróficas aeróbias mesófilas. Foram coletadas duas amostras de Castanha-do-Brasil de três diferentes locais de comercialização: Boca do Acre (AM), Cujubim e Ji-Paraná (RO), totalizando seis amostras. A partir das análises realizadas foi identificado a presença de Salmonella sp. em duas amostras demonstrando que o consumo deste produto poderia trazer risco a saúde do consumidor.

Técnicas de descontaminação de castanhas-do-brasil intencionalmente contaminadas com Aspergillus nomius / Techniques of decontamination of brazil nuts intentionally contaminated with Aspergillus nomius

A castanha-do-brasil é uma matéria-prima de origem amazônica, conhecida por seus benefícios nutricionais. Entretanto, por conta da produção extrativista este produto está susceptivel a vários focos de contaminação durante a cadeia produtiva. Este trabalho teve como objetivos a avaliação de diferentes métodos de descontaminação para controle de Aspergillus nomius produtor de aflatoxinas em castanha-do-brasil. As amostras de castanhas foram inoculadas com suspensão de A. nomius e posteriormente, foram testadas as técnicas de secagem em estufa a 60°C por 8 horas, esterilização em cabine UV por 20 minutos e sanitização com hipoclorito de sódio a 200 ppm por 15 minutos. Todos os tratamentos tiveram ação sob a cepa, sendo obtidos reduções de 3,18; 1,66 e 1,04 RD, respectivamente. A secagem mostrou-se o melhor tratamento de descontaminação, no entanto, mais pesquisas com outros métodos de descontaminação em castanhas são necessárias para controle da contaminação fúngica.

Influence of packaging on the quality of Brazil nuts / Influência da embalagem na qualidade de castanhas-do-brasil

The Brazil nut is an important commodity that grows in the Amazon rainforest and is notable for its high nutritional value. However, the effect that packaging has on product shelf-life is unclear. This study evaluated the microbiological and nutritional quality of unshelled Brazil nuts packed in (a) nylon and polypropylene under vacuum, and (b) in polyethylene terephthalate and polyethylene aluminized films. The nuts were stored for nine months under natural environmental conditions. The most probable number of total and thermotolerant coliforms and Escherichia coli, and counting of molds and yeast were determined in the unshelled nuts shortly after collection and every three months during storage. Moisture, lipids, ash, neutral detergent fiber, and acid detergent fiber contents were measured by the gravimetric method. Crude protein content was measured using the Kjeldahl method. Coliform levels were within legislated standards in both types of packaging. Mold and yeast growth increased with time in both types of packaging (0.1x10¹ to 9.5x10³ colony forming units g-1), but only nuts packaged in aluminum film showed a significant increase after six months of storage. After nine months of storage, both types of packaged nuts had good nutritional quality. Aluminized packaging resulted in better conservation of the lipid fraction and lower permeability to water vapor, however, vacuum packaging was more effective in controlling fungal growth, possibly due to the lack of oxygen.

As castanhas-do-brasil são um importante produto da floresta amazônica e destacam-se pelo seu alto valor nutricional. Contudo, os efeitos da embalagem sobre a vida de prateleira das castanhas não estão completamente esclarecidos. Sendo assim, neste estudo, avaliou-se a qualidade microbiológica e nutricional de castanhas-do-brasil descascadas, embaladas em (a) nylon mais polipropileno a vácuo e (b) filmes aluminizados de polietileno tereftalato (PET) e polietileno (PE). As castanhas foram armazenadas por um período de nove meses, sob condições ambientais naturais. Logo após a coleta, e a cada três meses, foram determinados nas castanhas o número mais provável de coliformes totais, coliformes termotolerantes e Escherichia coli, e contagem total de bolores e leveduras. Também foram medidos os teores de umidade, lipídeos, cinzas, fibra em detergente neutro e fibra em detergente ácido por métodos gravimétricos, e os teores de proteína bruta por meio do método de Kjeldahl. Os níveis de coliformes termotolerantes estiveram dentro dos padrões da legislação nos dois tipos de embalagem. A incidência de bolores e leveduras aumentou ao longo do tempo nas duas embalagens (de 0,1x10¹ a 9,5x10³ unidades formadoras de colônia; UFC g-1), porém somente a embalagem aluminizada apresentou aumento significativo após seis meses de armazenamento. Após nove meses, as castanhas nas duas embalagens apresentaram boa qualidade nutricional, porém a embalagem aluminizada apresentou melhor conservação da fração lipídica e menor permeabilidade a vapores de água. Contudo, a embalagem a vácuo foi mais eficiente no controle do crescimento de fungos, possivelmente devido à ausência de oxigênio.

Fumigation of Brazil nuts with allyl isothiocyanate to inhibit the growth of Aspergillus parasiticus and aflatoxin production.

BACKGROUND: Brazil produces approximately 40 000 tons of Brazil nuts annually, which is commonly contaminated with fungi and mycotoxins. Gaseous allyl isothiocyanate (AITC) was used to inhibit the growth of Aspergillus parasiticus and its production of aflatoxins (AFs) in Brazil nuts. RESULTS: Nuts were inoculated with 104 spores g-1 of A. parasiticus and placed in airtight glass jars with controlled relative humidity (RH = 95 or 85%). Samples were treated with 0, 0.5, 1.0 or 2.5 µL L-1 of gaseous AITC and analyzed after 30 days to determine the fungal population and AFs content. Samples were also submitted to sensory evaluation. AITC at 2.5 µL L-1 could completely inhibit the fungal growth and AFs production in both the RH tested. AITC at 0.5 and 1 µL L-1 did not affect the microbial growth at RH = 95%, but 1 µL L-1 reduced the production of AFs by ∼50%. All AITC treatments reduced the fungal population and AFs to undetectable levels at RH = 85%. None of the concentrations altered sensory characteristics of Brazil nuts. CONCLUSION: Gaseous AITC could be used as an alternative to inhibit the growth of A. parasiticus during storage and transport of Brazil nuts. © 2017 Society of Chemical Industry.

Qualidade microbiológica e físico-química de castanhas-do-brasil / Microbiological and physico-chemical quality of brazil nuts

Devido a poucos relatos da qualidade das castanhas-do-brasil oriundas do estado de Mato Grosso avaliaram-se as condições microbiológicas e nutricionais de amêndoas coletadas e beneficiadas, com e sem casca, por produtores de Itaúba/MT. Foram adquiridas 18 amostras em 3 locais distintos (6 amostras por local - 3 com casca e 3 descascadas). Foi investigada a qualidade microbiológica (coliformes totais, termotolerantes, Escherichia coli e bolores e leveduras) e nutricional (peso de 100 amêndoas, umidade, lipídios, proteína bruta e minerais totais). Os níveis de coliformes termotolerantes apresentaram-se abaixo do limite de tolerância preconizado pela legislação brasileira, contudo foi encontrada alta contaminação por bolores e leveduras, sendo as amêndoas sem cascas as mais contaminadas (627,27UFC.g-1). Observaram- se teores apreciáveis de lipídeos (63,25g.100g-1), proteína bruta (15,44g.100g-1) e minerais totais (2,69g.100g-1), porém elevados níveis de umidade (5,55g.100g-1), que podem estar relacionados com a alta contaminação fúngica encontrada. O descascamento influenciou na qualidade nutricional do produto, pois foram observadas diferenças significativas nas castanhas com e sem casca para os teores de lipídios, proteína bruta e cinzas entre os locais de amostragem e as formas de comercialização (teste Tukey p≤0,05).

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.

Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts.

The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p < 0.05) and metalloprotease (p < 0.05) genes were the most expressed by nonaflatoxigenic strains, whereas aflatoxigenic isolates exhibited higher expression of the aflR (p < 0.05) and aflD genes (p < 0.05). Our results suggest that the expression of aflR and aflD is correlated with aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to reduce and prevent aflatoxin contamination in Brazil nuts.

Qualidade higienicos sanitária de produtos sólidos prontos para o consumo (petiscos e similares) comercializados na região de Catanduva – SP / Sanitary hygienic quality of solid products ready for consumption (snacks) marketed in the Catanduva region - SP

Petiscos compreendem sementes comestíveis, salgadas, cruas, doces, torradas, fritas, condimentadas ou não, podendo também ser produtos doces, salgados, fritos, assados, compactados, incluindo torresmos e similares. Na maioria das vezes são consumidos como aperitivos ou acompanhamentos de alguns tipos de pratos, variando conforme sua espécie e região. O objetivo deste trabalho foi avaliar a qualidade higienicossanitária de diferentes amostras de produtos sólidos prontos para o consumo (petiscos e similares), comercializados na região de Catanduva-SP, por meio de metodologias internacionalmente reconhecidas. Para tanto, 20 amostras de diferentes marcas comerciais, dentro do prazo de validade, foram submetidas às seguintes análises: determinação do Número Mais Provável (NMP) de coliformes totais e termotolerantes, pesquisa de Escherichia coli e Salmonella spp. Os resultados obtidos para coliformes termotolerantes revelaram que todas as amostras (100%) encontravam-se de acordo com o padrão federal vigente. No entanto, para Salmonella spp. as análises mostraram que 50% das amostras estavam em desacordo com o padrão federal vigente, podendo ser classificadas como "produtos em condições sanitárias insatisfatórias" e, por conseguinte, "produtos impróprios para o consumo humano".

Snacks include edible seeds, salted, raw, sweet, toast, fried, spicy or not, can also be sweet products, salty, fried, baked, packaged, including greaves and the like. Most often are eaten as appetizers or side dishes, varying according to their species and region. The objective of this study was to evaluate the sanitary quality of different samples of solid products ready for consumption (and similar snacks), marketed in the region Catanduva-SP, through internationally recognized methodologies. For this purpose, samples of different brands within the validity period were subjected to the following tests: determination of Most Probable Number (MPN) of total and thermotolerant coliforms, Escherichia coli and Salmonella spp research. The results obtained for thermotolerant coliforms revealed that all samples (100%) were in accordance with the federal standard force. However, for Salmonella spp. the analysis showed that 50% of the samples weren't in accordance with the current federal standard, which can be classified as "products in poor sanitary conditions" and therefore "products unfit for human consumption".

Occurrence of fungi and aflatoxins B in nuts and products marketed the Brazilian northeastern regions / Ocorrência de fungos e aflatoxinas do tipo B em castanhas e produtos comercializados no Nordeste brasileiro

Aflatoxin contamination has been considered as a public health problem, especially in tropical countries, including Brazil. In order to investigate the presence of type B aflatoxins in products marketed in the city of Fortaleza, 23 samples were analyzed by thin layer chromatography. Visible fungal contamination in food was identified according to their macroscopic and microscopic features. The contamination by aflatoxins was detected in 8.7 % of 23 analyzed samples, and 12.5 % of Brazilian nuts samples were positive for AFB1 (<8 µg/kg) and for AFB2 in a contamination rate above the allowed value (16 µg/kg). Among the peanut samples, 33.3 % were positive to AFB1 and AFB2, also in a contamination rate (317.1 µg/kg) which was higher than that recommended by ANVISA. The isolation and morphological characteristics of fungi detected mainly in Brazilian nuts with peel showed the occurrence of the following species: Aspergillus flavus, Aspergillus niger, Mucor sp, Cladosporium sphaerospermum, Cladosporium cladosporoides, Fusarium sp, Aspergillus terreus and bacteria of Actinomycetes phylum. These findings indicate that it is needed to apply the mostly effective quality monitoring of food available to consumers.

A contaminação por aflatoxinas tem sido considerada um problema de saúde pública principalmente em países tropicais, incluindo o Brasil. Com o intuito de investigar a presença de aflatoxinas do tipo B em produtos comercializados na cidade de Fortaleza, 23 amostras foram analisadas por meio de cromatografia em camada delgada. Os fungos visivelmente presentes em alimentos foram identificados de acordo com suas características macroscópicas e microscópicas. A contaminação por aflatoxina foi detectada em 8,7 % das 23 amostras analisadas; e 12,5 % das amostras de castanha-do-Brasil apresentaram positividade para AFB1 (<8 µg/kg) e para AFB2 com taxa de contaminação acima do valor permitido (16 µg/kg). Quanto às amostras de amendoim, 33,3 % apresentaram positividade para AFB1 e AFB2, também com nível de contaminação (317,1 µg/kg) acima do preconizado pela ANVISA. O isolamento e a caracterização morfológica dos fungos encontrados principalmente nas castanhas-do-Brasil com casca revelaram a presença das espécies: Aspergillus flavus, Aspergillus niger, Mucor sp, Cladosporium sphaerospermum, Cladosporium cladosporoides, Fusarium sp, Aspergillus terreus e bactérias do filo Actinomicetos. Estes resultados demonstram que há necessidade de fiscalização mais efetiva da qualidade dos alimentos oferecidos aos consumidores.

Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin'.

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, ß-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial ß-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype.

Identification of Aspergillus nomius in Bees Visiting Brazil Nut Flowers.

We designed a primer pair (BtubNomF/BtubNomR) specifically for amplifying Aspergillus nomius DNA. In vitro assays confirmed BtubNomF/BtubNomR specificity, corroborating its usefulness in detecting and identifying A. nomius. We then investigated the occurrence of A. nomius in floral visitors of Bertholletia excelsa trees by means of PCR, and A. nomius was detected in the following bees: Xylocopa frontalis, Bombus transversalis, Centris denudans, C. ferruginea, and Epicharis flava. The presence of A. nomius in bees visiting Brazil nuts opens up new avenues for obtaining novel insights into the process whereby Brazil nuts are contaminated by aflatoxin-producing fungi.

Stereo and scanning electron microscopy of in-shell Brazil nut (Bertholletia excelsa H.B.K.): part two-surface sound nut fungi spoilage susceptibility.

This work reports the in-shell Brazil nut spoilage susceptible morpho-histological characteristics and fungi infection (shell, edible part, and brown skin) through stereo and scanning electron microscopies (SEM). The following characteristics related to shell (a) morphology-that allow fungi and insects' entrance to inner nut, and (b) histology-that allow humidity absorption, improving environment conditions for living organisms development, were identified. (a.1) locule in testae-the nut navel, which is a cavity formed during nut detaching from pods (located at 1.0 to 2.0/4th of the shell B&C nut faces linkage). It allows the nut brown skin (between shell and edible part) first contact to the external environment, through the (a.2) nut channel-the locule prolongation path, which has the water/nutrients cambium function for their transport and distribution to the inner seed (while still on the tree/pod). Both, locule followed by the channel, are the main natural entrance of living organisms (fungi and insects), including moisture to the inner seed structures. In addition, the (a.3) nut shell surface-which has a crinkled and uneven surface morphology-allows water absorption, thus adding to the deterioration processes too. The main shell histological characteristic, which also allows water absorption (thus improving environment conditions for fungi proliferation), is the (b.1) cell wall porosity-the multilayered wall and porous rich cells that compose the shell faces double tissue layers and the (b.2) soft tissue-the mix of tissues 2 faces corner/linkage. This work also shows in details the SEM nut spoilage susceptible features highly fungi infected with hyphae and reproductive structures distribution.

Rapid detection of aflatoxin producing fungi in food by real-time quantitative loop-mediated isothermal amplification.

Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (Tt) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A. flavus, A. parasiticus, and A. nomius, respectively. Analysis of contaminated sample materials revealed that the A. nomius specific real-time LAMP assay detected a minimum of 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 10(2) conidia/g and 10(5) conidia/g, respectively in peanut samples as well as 10(4) conidia/g and 10(4) conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain.

Brazil nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius.

The exploitation of the Brazil nut is one of the most important activities of the extractive communities of the Amazon rainforest. However, its commercialization can be affected by the presence of aflatoxins produced by fungi, namely Aspergillus section Flavi. In the present study, we investigated a collection of Aspergillus nomius strains isolated from Brazil nuts using different approaches, including morphological characters, RAPD and AFLP profiles, partial ß-tubulin and calmodulin nucleotide sequences, aflatoxin patterns, as well as tolerance to low water activity in cultured media. Results showed that most of the isolates do belong to A. nomius species, but a few were re-identified as Aspergillus pseudonomius, a very recently described species. The results of the analyses of molecular variance, as well as the high pairwise FST values between A. nomius and A. pseudonomius suggested the isolation between these two species and the inexistence of gene flow. Fixed interspecific nucleotide polymorphisms at ß-tubulin and calmodulin loci are presented. All A. pseudonomius strains analyzed produced aflatoxins AFB1, AFB2, AFG1 and AFG2. This study contains the first-ever report on the occurrence in Brazil nuts of A. pseudonomius. The G-type aflatoxins and the mycotoxin tenuazonic acid are reported here for the first time in A. pseudonomius.

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.