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Benedito Corrêa - One of the best experts on this subject based on the ideXlab platform.
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Effect of temperature on growth, gene expression, and aflatoxin production by Aspergillus nomius isolated from Brazil Nuts
Mycotoxin Research, 2020Co-Authors: Nathália B. S. Yunes, Rodrigo C. Oliveira, Arianne Costa Baquião, Liliana O. Rocha, Tatiana A. Reis, Benedito CorrêaAbstract: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.
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occurrence and fumonisin b2 producing potential of aspergillus section nigri in Brazil Nuts
Mycotoxin Research, 2017Co-Authors: Larissa De Souza Ferranti, Maria Helena Pelegrinelli Fungaro, Fernanda Pelisson Massi, Christopher Phippen, Benedito Corrêa, Beatriz T Iamanaka, Jens Christian Frisvad, Marta Hiromi TaniwakiAbstract: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.
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molecular and mycotoxigenic biodiversity of aspergillus flavus isolated from Brazil Nuts
Food Research International, 2016Co-Authors: Arianne Costa Baquião, Evandro Luiz Lopes, Benedito CorrêaAbstract:Abstract The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B1 (AFB1) and CPA production in Aspergillus flavus strains isolated from Brazil Nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25 °C for 10 days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB1 biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB1 and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil Nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil Nuts.
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expression of genes by aflatoxigenic and nonaflatoxigenic strains of aspergillus flavus isolated from Brazil Nuts
Foodborne Pathogens and Disease, 2016Co-Authors: Arianne Costa Baquião, Patricia Zorzete, Evandro Luiz Lopes, Aline Guedes Rodriges, Sabina Moser Tralamazza, Benedito CorrêaAbstract:Abstract 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 ...
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Characterization of Aspergillus section Flavi isolated from organic Brazil Nuts using a polyphasic approach.
Food Microbiology, 2014Co-Authors: Tatiana A. Reis, Arianne Costa Baquião, Danielle Diniz Atayde, F. Grabarz, Benedito CorrêaAbstract: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.
Arianne Costa Baquião - One of the best experts on this subject based on the ideXlab platform.
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Effect of temperature on growth, gene expression, and aflatoxin production by Aspergillus nomius isolated from Brazil Nuts
Mycotoxin Research, 2020Co-Authors: Nathália B. S. Yunes, Rodrigo C. Oliveira, Arianne Costa Baquião, Liliana O. Rocha, Tatiana A. Reis, Benedito CorrêaAbstract: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.
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molecular and mycotoxigenic biodiversity of aspergillus flavus isolated from Brazil Nuts
Food Research International, 2016Co-Authors: Arianne Costa Baquião, Evandro Luiz Lopes, Benedito CorrêaAbstract:Abstract The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B1 (AFB1) and CPA production in Aspergillus flavus strains isolated from Brazil Nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25 °C for 10 days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB1 biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB1 and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil Nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil Nuts.
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expression of genes by aflatoxigenic and nonaflatoxigenic strains of aspergillus flavus isolated from Brazil Nuts
Foodborne Pathogens and Disease, 2016Co-Authors: Arianne Costa Baquião, Patricia Zorzete, Evandro Luiz Lopes, Aline Guedes Rodriges, Sabina Moser Tralamazza, Benedito CorrêaAbstract:Abstract 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 ...
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Characterization of Aspergillus section Flavi isolated from organic Brazil Nuts using a polyphasic approach.
Food Microbiology, 2014Co-Authors: Tatiana A. Reis, Arianne Costa Baquião, Danielle Diniz Atayde, F. Grabarz, Benedito CorrêaAbstract: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.
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monitoring and determination of fungi and mycotoxins in stored Brazil Nuts
Journal of Food Protection, 2013Co-Authors: Arianne Costa Baquião, Maitê Martins Melo De Oliveira, Patricia Zorzete, Danielle Diniz Atayde, T A Reis, Benedito CorrêaAbstract:Brazil nut (Bertholletia excelsa) is an important commodity from the Brazilian Amazon, and approximately 37,000 tons (3.36 × 10⁷ kg) of Brazil Nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs, with subsequent production of mycotoxins. In this context, the objective of the present investigation was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in 110 stored samples of cultivated Brazil nut (55 samples of Nuts and 55 samples of shells) collected monthly for 11 months in Itacoatiara, State of Amazonas, Brazil. The samples were inoculated in duplicate onto Aspergillus flavus and Aspergillus parasiticus agar and potato dextrose agar for the detection of fungi, and the presence of mycotoxins was determined by high-performance liquid chromatography. The most prevalent fungi in Nuts and shells were Aspergillus spp., Fusarium spp., and Penicillium spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins and cyclopiazonic acid were not detected in any of the samples analyzed. The low water activity of the substrate was a determinant factor for the presence of fungi and the absence of aflatoxin in Brazil nut samples. The high frequency of isolation of aflatoxigenic Aspergillus section Flavi strains, mainly A. flavus, and their persistence during storage increase the chances of aflatoxin production on these substrates and indicates the need for good management practices to prevent mycotoxin contamination in Brazil Nuts.
Maria Helena Pelegrinelli Fungaro - One of the best experts on this subject based on the ideXlab platform.
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occurrence and fumonisin b2 producing potential of aspergillus section nigri in Brazil Nuts
Mycotoxin Research, 2017Co-Authors: Larissa De Souza Ferranti, Maria Helena Pelegrinelli Fungaro, Fernanda Pelisson Massi, Christopher Phippen, Benedito Corrêa, Beatriz T Iamanaka, Jens Christian Frisvad, Marta Hiromi TaniwakiAbstract: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.
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identification of aspergillus nomius in bees visiting Brazil nut flowers
Microbes and Environments, 2015Co-Authors: Fernanda Pelisson Massi, Marcelo Casimiro Cavalcante, Helena Viaro, Larissa De Souza Ferranti, Josué José Da Silva, Rafael Elias Silva Penha, Maria Helena Pelegrinelli FungaroAbstract: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.
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identification of aspergillus nomius in floral visitors of Brazil Nuts
XII Latin American Congress on Food Microbiology and Hygiene, 2014Co-Authors: Fernanda Pelisson Massi, Helena Viaro, Marcelo Casimiro, Larissa De Souza Ferranti, Josué José Da Silva, Rafael Elias Silva Penha, Maria Helena Pelegrinelli FungaroAbstract:Fernanda Massi, Rafael Penha, Marcelo Casimiro, Helena Viaro, Josue Silva, Larissa Ferranti, Maria Helena Fungaro.Identification of Aspergillus Nomius in Floral Visitors of Brazil Nuts. In: Anais do 12o Congresso Latinoamericano de Microbiologia e Higiene de Alimentos MICROAL 2014 [= Blucher Food Science Proceedings, num.1, vol.1]. Sao Paulo: Editora Blucher, 2014. DOI 10.5151/foodsci-microal-071 Identification of Aspergillus Nomius in Floral Visitors of Brazil Nuts
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Brazil Nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius
International Journal of Food Microbiology, 2014Co-Authors: Fernanda Pelisson Massi, Maria Lucia Cameiro Vieira, Carla De Freitas Munhoz, Josué Maldonado Ferreira, Daniele Sartori, Rafael Elias Silva Penha, Beatriz T Iamanaka, Marta Hiromi Taniwaki, Jens Christian Frisvad, Maria Helena Pelegrinelli FungaroAbstract:Abstract 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 β-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 β-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.
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the biodiversity of aspergillus section flavi in Brazil Nuts from rainforest to consumer
International Journal of Food Microbiology, 2013Co-Authors: Thaiane Ortolan Calderari, Maria Helena Pelegrinelli Fungaro, Daniele Sartori, Beatriz T Iamanaka, Jens Christian Frisvad, John I Pitt, Jose Luiz Rezende Pereira, Marta Hiromi TaniwakiAbstract:A total of 288 Brazil nut samples (173 kernel and 115 shell) from the Amazon rainforest region and Sao 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 Sao 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.
Marta Hiromi Taniwaki - One of the best experts on this subject based on the ideXlab platform.
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occurrence and fumonisin b2 producing potential of aspergillus section nigri in Brazil Nuts
Mycotoxin Research, 2017Co-Authors: Larissa De Souza Ferranti, Maria Helena Pelegrinelli Fungaro, Fernanda Pelisson Massi, Christopher Phippen, Benedito Corrêa, Beatriz T Iamanaka, Jens Christian Frisvad, Marta Hiromi TaniwakiAbstract: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.
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Brazil Nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius
International Journal of Food Microbiology, 2014Co-Authors: Fernanda Pelisson Massi, Maria Lucia Cameiro Vieira, Carla De Freitas Munhoz, Josué Maldonado Ferreira, Daniele Sartori, Rafael Elias Silva Penha, Beatriz T Iamanaka, Marta Hiromi Taniwaki, Jens Christian Frisvad, Maria Helena Pelegrinelli FungaroAbstract:Abstract 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 β-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 β-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.
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aflatoxin evaluation in ready to eat Brazil Nuts using reversed phase liquid chromatography and post column derivatisation
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment, 2014Co-Authors: Beatriz T Iamanaka, Larissa De Souza Ferranti, Felipe Nakano, Daniel P Lemes, Marta Hiromi TaniwakiAbstract:A high-performance liquid chromatography-fluorescence (HPLC-FD) method for aflatoxin quantification in Brazil Nuts was developed. Samples of Brazil Nuts collected in Brazilian markets were extracted with methanol:water and cleaned using an immunoaffinity column. Aflatoxins were eluted with methanol and a post-column derivatisation was performed with bromine, using a Kobra Cell system. The optimised method for total aflatoxins was sensitive, with detection and quantification limits of 0.05 and 0.25 µg kg⁻¹, respectively. The method was accurate, with recovery values of 87.6%; 85.3% and 85.0% for 0.5, 5.0 and 14.6 µg kg⁻¹ spiked levels, respectively. It was shown that the method was applicable to Brazil Nuts. From a total of 95 Brazil nut samples analysed from 21 Sao Paulo supermarket samples and 51 Manaus and 23 Belem street markets samples, 37.9% showed detectable levels of aflatoxins and three exceeded the recommended Codex Alimentarius limit of 10 µg kg⁻¹ for ready-to-eat Brazil Nuts.
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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
International Journal of Food Microbiology, 2014Co-Authors: Marta Hiromi Taniwaki, Beatriz T Iamanaka, Rudi F Vogel, Ludwig NiessenAbstract:Abstract 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.
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the biodiversity of aspergillus section flavi in Brazil Nuts from rainforest to consumer
International Journal of Food Microbiology, 2013Co-Authors: Thaiane Ortolan Calderari, Maria Helena Pelegrinelli Fungaro, Daniele Sartori, Beatriz T Iamanaka, Jens Christian Frisvad, John I Pitt, Jose Luiz Rezende Pereira, Marta Hiromi TaniwakiAbstract:A total of 288 Brazil nut samples (173 kernel and 115 shell) from the Amazon rainforest region and Sao 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 Sao 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.
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survival of salmonella enteritidis phage type 30 on Brazil Nuts and pumpkin seeds stored at 8 23 and 37 c
Journal of Food Protection, 2021Co-Authors: Bukola OnarindeAbstract:Experiments were performed to assess the survival of Salmonella on whole Brazil Nuts and pumpkin seeds stored at 8oC, 23oC, and 37oC. Brazil nut kernels and pumpkin seeds were inoculated with bacterial inoculum containing 10.4 Log 10 CFU/ml of Salmonella Enteritidis Phage Type 30 ( S E PT30) and then aseptically dried at room temperature for 24 hrs. After the drying step, levels of Salmonella recovered from Brazil Nuts and pumpkin seeds were 8.67±0.01 and 9.27±0.03Log 10 CFU/g, respectively. The survival of Salmonella and change in water activity was assessed over a total of 413 days. Although Salmonella survived throughout the storage period, significant differences were recorded between the storage temperatures. Results showed that the survival of S E PT30 was more enhanced at 8oC as compared to storage at 23oC and 37oC. Comparing the survival of Salmonella on the two products at different storage temperatures, there was no significant difference between the means of Salmonella counts for the two products. Although results show that Salmonella survived longer on pumpkin seeds stored at 8oC (P=0.53, as compared to Brazil Nuts) and at 23 and 37oC Salmonella survived longer on Brazil Nuts (P=0.12, as compared to pumpkin seeds). The highest and lowest survival of Salmonella was observed on pumpkin seeds with decay rates of -0.003±0.001 and -0.015±0.001 Log10 cfu/g per day for pumpkin seeds stored at 8 and 37oC, respectively. The water activity values recorded at day 2 and day 413 for Brazil Nuts stored at 8oC, 23oC, and 37oC were 0.424 and 0.434, 0.383 and 0.385 and, 0.372 and 0.256, respectively. For pumpkin seeds stored at 8oC, 23oC, and 37oC water activity values recorded at day 2 and day 413 were 0.754 and 0.412, 0.627 and 0.350 and, 0.787 and 0.205, respectively. The data obtained in this study provide useful insight on the influence of temperature on the survival of Salmonella on the surface of Brazil Nuts and pumpkin seeds.