Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach / Identificação de espécies toxigênicas de Aspergillus isoladas da dieta de cabras leiteiras utilizando uma abordagem polifásica

Some species of filamentous fungi that infest agricultural commodities are able to produce mycotoxins, contaminating feed and animal products. The aim of this research was to identify the mycoflora present in the feed and forage for dairy goat and to isolate and characterize the Aspergillus flavus and A. parasiticus strains based on a morphological and molecular characterization and mycotoxigenic ability. The goat dairy diets were collected monthly from 11 goat milk farms, totaling 129 and 106 samples of concentrate and forage, respectively. For the isolation of the mycobiota the surface plating method was used. Aspergillus, Penicillium, and Fusarium were the main fungi producing mycotoxins isolated. The morphological and molecular characterization and mycotoxigenic ability were used for A. flavus and A. parasiticus identification. The Aspergillus spp. from feed 39% produced aflatoxins B1 and B2, 17% produced cyclopiazonic acid (CPA), 18% produced both toxins, and 42% had no toxigenic ability. Only 2.0% of the strains produced aflatoxins B1, B2, G1, and G2, but no CPA. The strains from forage were producers of aflatoxins B1 and B2 (37%), CPA (14%), 14% of both mycotoxins, whereas 49% have shown no toxigenic ability. The aflD and aflR genes were used by PCR and PCR-RFLP, respectively. The presence of toxigenic species in samples of feed for lactating goats indicates a potential risk of contamination of dairy products, if they are exposed to environmental conditions favorable to fungal growth and mycotoxin production.

Algumas espécies de fungos filamentosos que infestam os produtos agrícolas e ração são capazes de produzir micotoxinas. O objetivo deste trabalho foi identificar a micoflora presente nos concentrados e volumosos utilizados na dieta de cabras leiteiras e isolar as espécies Aspergillus flavus e A. parasiticus, com base em uma caracterização morfológica e molecular e capacidade micotoxigênica. Os alimentos foram coletados mensalmente em 11 fazendas produtoras de leite de cabra, totalizando 129 e 106 amostras de concentrado e volumoso, respectivamente. Para o isolamento da micobiota, foi utilizado o método de plaqueamento de superfície. Aspergillus, Penicillium e Fusarium foram os principais gêneros de fungos produtores de micotoxinas isolados das amostras. A caracterização morfológica e molecular e capacidade micotoxigênica foram utilizadas para identificação de A. flavus e A. parasiticus. Das cepas Aspergillus spp isoladas do concentrado, 39% produziram aflatoxinas B1 e B2, 17% produziram ácido ciclopiazônico (ACP), 18% produziram ambas as toxinas e 42% não tinham capacidade toxigênica. Apenas 2,0% das cepas produziram aflatoxinas B1, B2, G1 e G2. As cepas de Aspergillus spp. isoladas do volumoso foram produtores de aflatoxinas B1 e B2 (37%), ACP (14%), sendo que 14% produziram ambas toxinas e 49% não foram produtoras. Os genes aflD e aflR foram utilizados para a PCR e a PCR-RFLP, respectivamente. A presença de espécies toxigênicas em amostras de alimentos destinados a caprinos em lactação indica um risco potencial de contaminação dos produtos lácteos por aflatoxinas e ACP, caso estes sejam expostos a condições ambientais favoráveis ao crescimento de fungos e produção de micotoxinas.

Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach / Identificação de espécies toxigênicas de Aspergillus isoladas da dieta de cabras leiteiras utilizando uma abordagem polifásica

Some species of filamentous fungi that infest agricultural commodities are able to produce mycotoxins, contaminating feed and animal products. The aim of this research was to identify the mycoflora present in the feed and forage for dairy goat and to isolate and characterize the Aspergillus flavusand A. parasiticusstrains based on a morphological and molecular characterization and mycotoxigenic ability. The goat dairy diets were collected monthly from 11 goat milk farms, totaling 129 and 106 samples of concentrate and forage, respectively. For the isolation of the mycobiota the surface plating method was used. Aspergillus, Penicillium,and Fusariumwere the main fungi producing mycotoxins isolated. The morphological and molecular characterization and mycotoxigenic ability were used for A. flavusand A. parasiticusidentification. The Aspergillusspp. from feed 39% produced aflatoxins B1 and B2, 17% produced cyclopiazonic acid (CPA), 18% produced both toxins, and 42% had no toxigenic ability. Only 2.0% of the strains produced aflatoxins B1, B2, G1, and G2, but no CPA. The strains from forage were producers of aflatoxins B1 and B2 (37%), CPA (14%), 14% of both mycotoxins, whereas 49% have shown no toxigenic ability. The aflD and aflR genes were used by PCR and PCR-RFLP, respectively. The presence of toxigenic species in samples of feed for lactating goats indicates a potential risk of contamination of dairy products, if they are exposed to environmental conditions favorable to fungal growth and mycotoxin production.(AU)

Algumas espécies de fungos filamentosos que infestam os produtos agrícolas e ração são capazes de produzir micotoxinas. O objetivo deste trabalho foi identificar a micoflora presente nos concentrados e volumosos utilizados na dieta de cabras leiteiras e isolar as espécies Aspergillus flavuse A. parasiticus, com base em uma caracterização morfológica e molecular e capacidade micotoxigênica. Os alimentos foram coletados mensalmente em 11 fazendas produtoras de leite de cabra, totalizando 129 e 106 amostras de concentrado e volumoso, respectivamente. Para o isolamento da micobiota, foi utilizado o método de plaqueamento de superfície.Aspergillus, Penicilliume Fusariumforam os principais gêneros de fungos produtores de micotoxinas isolados das amostras. A caracterização morfológica e molecular e capacidade micotoxigênica foram utilizadas para identificação de A. flavus e A. parasiticus.Das cepas Aspergillusspp isoladas do concentrado, 39% produziram aflatoxinas B1 e B2, 17% produziram ácido ciclopiazônico (ACP), 18% produziram ambas as toxinas e 42% não tinham capacidade toxigênica. Apenas 2,0% das cepas produziram aflatoxinas B1, B2, G1 e G2. As cepas de Aspergillusspp. isoladas do volumoso foram produtores de aflatoxinas B1 e B2 (37%), ACP (14%), sendo que 14% produziram ambas toxinas e 49% não foram produtoras. Os genes aflD e aflR foram utilizados para a PCR e a PCR-RFLP, respectivamente. A presença de espécies toxigênicas em amostras de alimentos destinados a caprinos em lactação indica um risco potencial de contaminação dos produtos lácteos por aflatoxinas e ACP, caso estes sejam expostos a condições ambientais favoráveis ao crescimento de fungos e produção de micotoxinas.(AU)

Fungicidal activity and constituents of Ageratum conyzoides essential oil from three regions in São Paulo state, Brazil

The chemical composition and antifungal activity of the Ageratum conyzoidesessential oils, obtained from the leaves collected in Ibiúna (1), Ribeirão Pires (2) and Campinas (3) in the São Paulo state, Brazil, were investigated. The essential oils were obtained from A. conyzoidesleaves by hydrodestilation and analyzed by GC/MS. The chemical composition of the A. conyzoides oil collected in Ribeirão Pires and Ibiúna consisted mainly of precocene I and II. The essential oil from leaves collected in Campinas had only traces of precocene II and a highest proportion of precocene I, -humulene and (E)-caryophyllene than the oils (1) and (2). The Aspergillus flavus growth was inhibited by essential oils (1) and (2) at 60 and 64%, respectively, and the oil (3) was inactive. On the other hand, the three essential oils inhibited the sporulation of the fungus for more than 120 days. The essential oils of leaves collected in sites that show similarities in the latitude, altitude and average temperatures, as Ribeirão Pires and Ibiúna, showed chemical composition and antifungal activity similar, either, which showed the importance of geo-ecological factors in production of metabolites of the plant.

A composição química e a atividade antifúngica do óleo essencial de folhas de Ageratum conyzoides, coletadas em Ibiúna (1), Ribeirão Pires (2) e Campinas (3) no estado de São Paulo, foram investigadas. Os óleos essenciais foram obtidos a partir de folhas de A. conyzoides por hidrodestilação e analisados por CG/EM. A composição química do óleo essencial de A. conyzoides coletadas em Ribeirão Pires e Ibiúna consiste principalmente de precoceno I e II. O óleo essencial das folhas coletadas em Campinas possui apenas traços de precoceno II e uma maior proporção de precoceno I, -humuleno e (E)-cariofileno quando comparado com os óleos (1) e (2). O crescimento de Aspergillusflavus foi inibido pelos óleos essenciais (1) e (2) em 60 e 64%, respectivamente, enquanto que o óleo (3) foi inativo. Por outro lado, os três óleos essenciais inibiram a esporulação do fungo por mais de 120 dias. Os óleos essenciais de folhas coletadas em locais que mostram semelhanças na latitude, altitude e temperatura média, como Ribeirão Pires e Ibiúna, mostraram composição química e atividade antifúngica semelhante. Isso mostrou a importância dos fatores geo-ecológicos na produção de metabólitos da planta.

Fungicidal activity and constituents of Ageratum conyzoides essential oil from three regions in São Paulo state, Brazil / Atividade fungicida e constituintes químicos do óleo essencial de Ageratum conyzoides de três regiões do estado de São Paulo, Brasil

The chemical composition and antifungal activity of the Ageratum conyzoides essential oils, obtained from the leaves collected in Ibiúna (1), Ribeirão Pires (2) and Campinas (3) in the São Paulo state, Brazil, were investigated. The essential oils were obtained from A. conyzoides leaves by hydrodestilation and analyzed by GC/MS. The chemical composition of the A. conyzoides oil collected in Ribeirão Pires and Ibiúna consisted mainly of precocene I and II. The essential oil from leaves collected in Campinas had only traces of precocene II and a highest proportion of precocene I, -humulene and (E)-caryophyllene than the oils (1) and (2). The Aspergillus flavus growth was inhibited by essential oils (1) and (2) at 60 and 64%, respectively, and the oil (3) was inactive. On the other hand, the three essential oils inhibited the sporulation of the fungus for more than 120 days. The essential oils of leaves collected in sites that show similarities in the latitude, altitude and average temperatures, as Ribeirão Pires and Ibiúna, showed chemical composition and antifungal activity similar, either, which showed the importance of geo-ecological factors in production of metabolites of the plant.(AU)

A composição química e a atividade antifúngica do óleo essencial de folhas de Ageratum conyzoides, coletadas em Ibiúna (1), Ribeirão Pires (2) e Campinas (3) no estado de São Paulo, foram investigadas. Os óleos essenciais foram obtidos a partir de folhas de A. conyzoides por hidrodestilação e analisados por CG/EM. A composição química do óleo essencial de A. conyzoides coletadas em Ribeirão Pires e Ibiúna consiste principalmente de precoceno I e II. O óleo essencial das folhas coletadas em Campinas possui apenas traços de precoceno II e uma maior proporção de precoceno I, -humuleno e (E)-cariofileno quando comparado com os óleos (1) e (2). O crescimento de Aspergillus flavus foi inibido pelos óleos essenciais (1) e (2) em 60 e 64%, respectivamente, enquanto que o óleo (3) foi inativo. Por outro lado, os três óleos essenciais inibiram a esporulação do fungo por mais de 120 dias. Os óleos essenciais de folhas coletadas em locais que mostram semelhanças na latitude, altitude e temperatura média, como Ribeirão Pires e Ibiúna, mostraram composição química e atividade antifúngica semelhante. Isso mostrou a importância dos fatores geo-ecológicos na produ- ção de metabólitos da planta.(AU)

Fungicidal activity and constituents of Ageratum conyzoides essential oil from three regions in São Paulo state, Brazil / Atividade fungicida e constituintes químicos do óleo essencial de Ageratum conyzoides de três regiões do estado de São Paulo, Brasil

The chemical composition and antifungal activity of the Ageratum conyzoides essential oils, obtained from the leaves collected in Ibiúna (1), Ribeirão Pires (2) and Campinas (3) in the São Paulo state, Brazil, were investigated. The essential oils were obtained from A. conyzoides leaves by hydrodestilation and analyzed by GC/MS. The chemical composition of the A. conyzoides oil collected in Ribeirão Pires and Ibiúna consisted mainly of precocene I and II. The essential oil from leaves collected in Campinas had only traces of precocene II and a highest proportion of precocene I, α-humulene and (E)-caryophyllene than the oils (1) and (2). The Aspergillus flavus growth was inhibited by essential oils (1) and (2) at 60 and 64%, respectively, and the oil (3) was inactive. On the other hand, the three essential oils inhibited the sporulation of the fungus for more than 120 days. The essential oils of leaves collected in sites that show similarities in the latitude, altitude and average temperatures, as Ribeirão Pires and Ibiúna, showed chemical composition and antifungal activity similar, either, which showed the importance of geo-ecological factors in production of metabolites of the plant.(AU)

A composição química e a atividade antifúngica do óleo essencial de folhas de Ageratum conyzoides, coletadas em Ibiúna (1), Ribeirão Pires (2) e Campinas (3) no estado de São Paulo, foram investigadas. Os óleos essenciais foram obtidos a partir de folhas de A. conyzoides por hidrodestilação e analisados ​​por CG/EM. A composição química do óleo essencial de A. conyzoides coletadas em Ribeirão Pires e Ibiúna consiste principalmente de precoceno I e II. O óleo essencial das folhas coletadas em Campinas possui apenas traços de precoceno II e uma maior proporção de precoceno I, α-humuleno e (E)-cariofileno quando comparado com os óleos (1) e (2). O crescimento de Aspergillus flavus foi inibido pelos óleos essenciais (1) e (2) em 60 e 64%, respectivamente, enquanto que o óleo (3) foi inativo. Por outro lado, os três óleos essenciais inibiram a esporulação do fungo por mais de 120 dias. Os óleos essenciais de folhas coletadas em locais que mostram semelhanças na latitude, altitude e temperatura média, como Ribeirão Pires e Ibiúna, mostraram composição química e atividade antifúngica semelhante. Isso mostrou a importância dos fatores geo-ecológicos na produção de metabólitos da planta.(AU)

Fungicidal activity and constituents of Ageratum conyzoides essential oil from three regions in São Paulo state, Brazil / Atividade fungicida e constituintes químicos do óleo essencial de Ageratum conyzoides de três regiões do estado de São Paulo, Brasil

The chemical composition and antifungal activity of the Ageratum conyzoides essential oils, obtained from the leaves collected in Ibiúna (1), Ribeirão Pires (2) and Campinas (3) in the São Paulo state, Brazil, were investigated. The essential oils were obtained from A. conyzoides leaves by hydrodestilation and analyzed by GC/MS. The chemical composition of the A. conyzoides oil collected in Ribeirão Pires and Ibiúna consisted mainly of precocene I and II. The essential oil from leaves collected in Campinas had only traces of precocene II and a highest proportion of precocene I, -humulene and (E)-caryophyllene than the oils (1) and (2). The Aspergillus flavus growth was inhibited by essential oils (1) and (2) at 60 and 64%, respectively, and the oil (3) was inactive. On the other hand, the three essential oils inhibited the sporulation of the fungus for more than 120 days. The essential oils of leaves collected in sites that show similarities in the latitude, altitude and average temperatures, as Ribeirão Pires and Ibiúna, showed chemical composition and antifungal activity similar, either, which showed the importance of geo-ecological factors in production of metabolites of the plant.

A composição química e a atividade antifúngica do óleo essencial de folhas de Ageratum conyzoides, coletadas em Ibiúna (1), Ribeirão Pires (2) e Campinas (3) no estado de São Paulo, foram investigadas. Os óleos essenciais foram obtidos a partir de folhas de A. conyzoides por hidrodestilação e analisados por CG/EM. A composição química do óleo essencial de A. conyzoides coletadas em Ribeirão Pires e Ibiúna consiste principalmente de precoceno I e II. O óleo essencial das folhas coletadas em Campinas possui apenas traços de precoceno II e uma maior proporção de precoceno I, -humuleno e (E)-cariofileno quando comparado com os óleos (1) e (2). O crescimento de Aspergillus flavus foi inibido pelos óleos essenciais (1) e (2) em 60 e 64%, respectivamente, enquanto que o óleo (3) foi inativo. Por outro lado, os três óleos essenciais inibiram a esporulação do fungo por mais de 120 dias. Os óleos essenciais de folhas coletadas em locais que mostram semelhanças na latitude, altitude e temperatura média, como Ribeirão Pires e Ibiúna, mostraram composição química e atividade antifúngica semelhante. Isso mostrou a importância dos fatores geo-ecológicos na produ- ção de metabólitos da planta.

Fungicidal activity and constituents of Ageratum conyzoides essential oil from three regions in São Paulo state, Brazil

The chemical composition and antifungal activity of the Ageratum conyzoidesessential oils, obtained from the leaves collected in Ibiúna (1), Ribeirão Pires (2) and Campinas (3) in the São Paulo state, Brazil, were investigated. The essential oils were obtained from A. conyzoidesleaves by hydrodestilation and analyzed by GC/MS. The chemical composition of the A. conyzoides oil collected in Ribeirão Pires and Ibiúna consisted mainly of precocene I and II. The essential oil from leaves collected in Campinas had only traces of precocene II and a highest proportion of precocene I, -humulene and (E)-caryophyllene than the oils (1) and (2). The Aspergillus flavus growth was inhibited by essential oils (1) and (2) at 60 and 64%, respectively, and the oil (3) was inactive. On the other hand, the three essential oils inhibited the sporulation of the fungus for more than 120 days. The essential oils of leaves collected in sites that show similarities in the latitude, altitude and average temperatures, as Ribeirão Pires and Ibiúna, showed chemical composition and antifungal activity similar, either, which showed the importance of geo-ecological factors in production of metabolites of the plant.

A composição química e a atividade antifúngica do óleo essencial de folhas de Ageratum conyzoides, coletadas em Ibiúna (1), Ribeirão Pires (2) e Campinas (3) no estado de São Paulo, foram investigadas. Os óleos essenciais foram obtidos a partir de folhas de A. conyzoides por hidrodestilação e analisados por CG/EM. A composição química do óleo essencial de A. conyzoides coletadas em Ribeirão Pires e Ibiúna consiste principalmente de precoceno I e II. O óleo essencial das folhas coletadas em Campinas possui apenas traços de precoceno II e uma maior proporção de precoceno I, -humuleno e (E)-cariofileno quando comparado com os óleos (1) e (2). O crescimento de Aspergillusflavus foi inibido pelos óleos essenciais (1) e (2) em 60 e 64%, respectivamente, enquanto que o óleo (3) foi inativo. Por outro lado, os três óleos essenciais inibiram a esporulação do fungo por mais de 120 dias. Os óleos essenciais de folhas coletadas em locais que mostram semelhanças na latitude, altitude e temperatura média, como Ribeirão Pires e Ibiúna, mostraram composição química e atividade antifúngica semelhante. Isso mostrou a importância dos fatores geo-ecológicos na produção de metabólitos da planta.

Potential of essential oils for protection of grains contaminated by aflatoxin produced by Aspergillus flavus.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oils of Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) on the mycelial growth and aflatoxin B1 production by Aspergillus flavus have been studied previously in culture medium. The aim of this study was to evaluate aflatoxin B1 production by Aspergillus flavus in real food systems (corn and soybean) treated with Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) essential oils. Samples with 60 g of the grains were treated with different volumes of essential oils, 200, 100, 50, and 10 µL for oregano and 50, 30, 15, and 10 µL for mentrasto. Fungal growth was evaluated by disk diffusion method. Aflatoxin B1 production was evaluated inoculating suspensions of A. flavus containing 1.3 × 10(5) spores/mL in 60 g of grains (corn and soybeans) after adjusting the water activity at 0.94. Aflatoxin was quantified by photodensitometry. Fungal growth and aflatoxin production were inhibited by essential oils, but the mentrasto oil was more effective in soybeans than that of oregano. On the other hand, in corn samples, the oregano essential oil was more effective than that of mentrasto. Chemical compositions of the essential oils were also investigated. The GC/MS oils analysis showed that the main component of mentrasto essential oil is precocene I and of the main component of oregano essential oil is 4-terpineol. The results indicate that both essential oils can become an alternative for the control of aflatoxins in corn and soybeans.

Produção de aflatoxinas e ácido ciclopiazônico por cepas de Aspergillus flavus isoladas de amendoim

Aspergillus flavus is a filamentous fungus that can produce aflatoxins and cyclopiazonic acid, and the presence of these mycotoxins in food and feed can lead to a toxic effect on humans and animals. Strains of A. flavus producing aflatoxin and cyclopiazonic acid are often isolated from peanuts, indicating the the co-occcurrence of these toxins in the natural substrate. In this study, we isolated 47 strains of A. flavus on peanut kernels and hulls during different stages of fruit ripening and its storage. Of the isolated strains, we assessed the potential for aflatoxin and cyclopiazonic acid production, in which 91.5% could produce aflatoxins and 70% produced cyclopiazonic acid; 63.8% produced both toxins and 2.1% produced no toxin. The presence of toxigenic A. flavus strains in peanut samples indicate a potential risk of contamination of these products, if they are exposed to environmental conditions that are favorable to fungal growth and mycotoxin production.

Aspergillus flavus é um fungo filamentoso que pode produzir aflatoxinas e ácido ciclopiazônico, sendo que a presença dessas micotoxinas em alimentos e rações pode levar a um efeito tóxico no homem e em animais. Cepas de A. flavus produtoras de aflatoxinas e ácido ciclopiazônico são frequentemente isoladas do amendoim, indicando a natural co-ocorrência dessas toxinas neste substrato. Neste estudo, foram isoladas 47 cepas de Aspergillus flavus em grãos e cascas de amendoim durante diferentes fases de maturação do fruto e também durante seu armazenamento. Das cepas isoladas, foram avaliados os potenciais para produção de aflatoxinas e ácido ciclopiazônico, em que 91,5% foram produtoras de aflatoxinas e 70% produziram ácido ciclopiazônico, sendo que 63,8% produziram ambas as toxinas e 2,1% não produziu nenhuma. A presença de cepas toxigênicas de A. flavus nas amostras de amendoim analisadas indica um risco potencial da contaminação deste produto, caso seja exposto a condições ambientais favoráveis ao crescimento do fungo e produção de micotoxinas.

Production of aflatoxin and cyclopiazonic acid by Aspergillus flavus strains isolate from peanuts / Produção de aflatoxinas e ácido ciclopiazônico por cepas de Aspergillus flavus isoladas de amendoim

Aspergillus flavus is a filamentous fungus that can produce aflatoxins and cyclopiazonic acid, and the presence of these mycotoxins in food and feed can lead to a toxic effect on humans and animals. Strains of A. flavus producing aflatoxin and cyclopiazonic acid are often isolated from peanuts, indicating the the co-occcurrence of these toxins in the natural substrate. In this study, we isolated 47 strains of A. flavus on peanut kernels and hulls during different stages of fruit ripening and its storage. Of the isolated strains, we assessed the potential for aflatoxin and cyclopiazonic acid production, in which 91.5% could produce aflatoxins and 70% produced cyclopiazonic acid; 63.8% produced both toxins and 2.1% produced no toxin. The presence of toxigenic A. flavus strains in peanut samples indicate a potential risk of contamination of these products, if they are exposed to environmental conditions that are favorable to fungal growth and mycotoxin production. (AU)

Aspergillus flavus é um fungo filamentoso que pode produzir aflatoxinas e لcido ciclopiazônico, sendo que a presença dessas micotoxinas em alimentos e raçُes pode levar a um efeito tَxico no homem e em animais. Cepas de A. flavus produtoras de aflatoxinas e لcido ciclopiazônico sمo frequentemente isoladas do amendoim, indicando a natural co-ocorrência dessas toxinas neste substrato. Neste estudo, foram isoladas 47 cepas de Aspergillus flavus em grمos e cascas de amendoim durante diferentes fases de maturaçمo do fruto e também durante seu armazenamento. Das cepas isoladas, foram avaliados os potenciais para produçمo de aflatoxinas e لcido ciclopiazônico, em que 91,5% foram produtoras de aflatoxinas e 70% produziram لcido ciclopiazônico, sendo que 63,8% produziram ambas as toxinas e 2,1% nمo produziu nenhuma. A presença de cepas toxigênicas de A. flavus nas amostras de amendoim analisadas indica um risco potencial da contaminaçمo deste produto, caso seja exposto a condiçُes ambientais favorلveis ao crescimento do fungo e produçمo de micotoxinas. (AU)

Production of aflatoxin and cyclopiazonic acid by Aspergillus flavus strains isolate from peanuts / Produção de aflatoxinas e ácido ciclopiazônico por cepas de Aspergillus flavus isoladas de amendoim

Aspergillus flavus is a filamentous fungus that can produce aflatoxins and cyclopiazonic acid, and the presence of these mycotoxins in food and feed can lead to a toxic effect on humans and animals. Strains of A. flavus producing aflatoxin and cyclopiazonic acid are often isolated from peanuts, indicating the the co-occcurrence of these toxins in the natural substrate. In this study, we isolated 47 strains of A. flavus on peanut kernels and hulls during different stages of fruit ripening and its storage. Of the isolated strains, we assessed the potential for aflatoxin and cyclopiazonic acid production, in which 91.5% could produce aflatoxins and 70% produced cyclopiazonic acid; 63.8% produced both toxins and 2.1% produced no toxin. The presence of toxigenic A. flavus strains in peanut samples indicate a potential risk of contamination of these products, if they are exposed to environmental conditions that are favorable to fungal growth and mycotoxin production.

Aspergillus flavus é um fungo filamentoso que pode produzir aflatoxinas e لcido ciclopiazônico, sendo que a presença dessas micotoxinas em alimentos e raçُes pode levar a um efeito tَxico no homem e em animais. Cepas de A. flavus produtoras de aflatoxinas e لcido ciclopiazônico sمo frequentemente isoladas do amendoim, indicando a natural co-ocorrência dessas toxinas neste substrato. Neste estudo, foram isoladas 47 cepas de Aspergillus flavus em grمos e cascas de amendoim durante diferentes fases de maturaçمo do fruto e também durante seu armazenamento. Das cepas isoladas, foram avaliados os potenciais para produçمo de aflatoxinas e لcido ciclopiazônico, em que 91,5% foram produtoras de aflatoxinas e 70% produziram لcido ciclopiazônico, sendo que 63,8% produziram ambas as toxinas e 2,1% nمo produziu nenhuma. A presença de cepas toxigênicas de A. flavus nas amostras de amendoim analisadas indica um risco potencial da contaminaçمo deste produto, caso seja exposto a condiçُes ambientais favorلveis ao crescimento do fungo e produçمo de micotoxinas.

Mycotoxins detection by chromatography.

Mycotoxins are metabolites and toxic substances produced by certain filamentous fungi that frequently contaminate food and agriculture commodities and it may cause disease in animals or humans. The toxigenic fungi are responsible for mycotoxin production in food that belongs to mainly three genera: Aspergillus, Penicillium and Fusarium. The contamination of food by mycotoxins is difficult to control in addition, causing economic impacts on public health, so their identification and quantifications are very necessary. Various analytical methods are developed for the detection and quantification of mycotoxins in order to control the residual contents of these toxins. Among them there is a widely used chromatography. This paper reports some chromatographic methods for the detection and quantification of mycotoxins described in patents and scientific articles.

Evaluation of antifungal activity of Pittosporum undulatum L. essential oil against Aspergillus flavus and aflatoxin production / Avaliação da atividade antifúngica do óleo essencial de Pittosporum undulatum L. em Aspergillus flavus e produção de aflatoxina

The presence of mycotoxins as a result of fungal attack can occur before, after and during the harvest and storage operations on agricultural crops and food commodities. Considering the inhibitory property of essential plant oils on the mycelial development of fungi and the importance of Aspergillus flavus, the main producer of aflatoxins, this research was designed to evaluate the toxicity of essential oil from Pittosporum undulatum against A. flavus. The essential oils were obtained from P. undulatum leaves, collected in different months and analyzed by GC/MS. The oils were rich in hydrocarbon, monoterpenes and sesquiterpenes and it was observed a significant variation on the chemical composition of the essential oil of leaves at different months. Besides, the essential oils were tested against fungal growth and the results showed different spectrum of inhibition on A. flavus. However, the essential oils inhibited the aflatoxin B1 production.

A presença de micotoxinas como resultado do ataque fúngico pode ocorrer antes, após e durante a colheita e também no armazenamento de grãos e alimentos. Considerando as propriedades inibitórias dos óleos essenciais de plantas no desenvolvimento do micélio dos fungos e a importâncias do Aspergillus flavus, principal produtor de aflatoxinas, relatou-se neste trabalho, a atividade tóxica do óleo essencial do Pittosporum undulatum em cultura de A. flavus. Os óleos essenciais de P. undulatum foram obtidos a partir de folhas coletadas em diferentes meses e analisado por CG/EM. Os óleos se mostraram ricos em hidrocarbonetos, monoterpenos e sesquiterpenos e foi observada uma significante variação na composição química destes óleos nos diferentes meses de coleta. Os óleos essenciais mostraram diferentes espectros de inibição do crescimento de A. flavus, porém todos foram capazes de inibir a produção de aflatoxina B1.

Evaluation of fungal burden and aflatoxin presence in packed medicinal plants treated by gamma radiation.

This study was developed to evaluate the fungal burden, toxigenic molds, and mycotoxin contamination and to verify the effects of gamma radiation in four kinds of medicinal plants stored before and after 30 days of irradiation treatment. Eighty samples of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia, and Cassia angustifolia) purchased from drugstores, wholesale, and open-air markets in São Paulo city, Brazil, were analyzed. The samples were treated using a (60)Co gamma ray source (Gammacell) with doses of 5 and 10 kGy. Nonirradiated samples were used as controls of fungal isolates. For enumeration of fungi on medicinal plants, serial dilutions of the samples were plated in duplicate onto dichloran 18% glycerol agar. The control samples revealed a high burden of molds, including toxigenic fungi. The process of gamma radiation was effective in reducing the number of CFU per gram in all irradiated samples of medicinal plants after 30 days of storage, using a dose of 10 kGy and maintaining samples in a protective package. No aflatoxins were detected. Gamma radiation treatment can be used as an effective method for preventing fungal deterioration of medicinal plants subject to long-term storage.

Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus.

Aflatoxin B(1) (AFB(1)) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oil of Ageratum conyzoides, on the mycelial growth and aflatoxin B(1) production by Aspergillus flavus were studied. Cultures were incubated in yeast extract-sucrose (YES) broth for days at 25 degrees C at the following different concentrations of the essential oil (from 0.0 to 30mug/mL). The essential oil inhibited fungal growth to different extents depending on the concentration, and completely inhibited aflatoxin production at concentrations above 0.10microg/mL. The analysis of the oil by GC/MS showed that its main components are precocene II (46.35%), precocene I (42.78%), cumarine (5.01%) and Trans-caryophyllene (3.02%). Comparison by transmission electron microscopy of the fungal cells, control and those incubated with different concentrations of essential oil, showed ultra-structural changes which were concentration dependent of the essential oil of A. conyzoides. Such ultra-structural changes were more evident in the endomembrane system, affecting mainly the mitochondria. Degradation was also observed in both surrounding fibrils. The ability to inhibit aflatoxin production as a new biological activity of A.conyzoides L. indicates that it may be considered as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis.

Avaliação da micoflora e ocorrência de micotoxinas em cascas de amendoim em diferentes estágios de maturação da vagem / Evaluation of mycoflora and occurrence of mycotoxins in peanut hulls at different pod maturation stages

As cascas de amendoim (Arachis hypogaea L.) são de grande importância para confecção de cama de frangos, de gado de leite e como fonte de fibras para ruminantes, portanto a elucidação dos mecanismos de contaminação por fungos toxigênicos e por micotoxinas em amendoim é imprescindível, especialmente para que medidas preventivas possam ser tomadas. Realizou-se, este trabalho, em Junqueirópolis, Estado de São Paulo, Brasil. Os principais fungos isolados nas cascas de amendoim foram Fusarium ssp. (78,75 por cento), Rhizopus ssp. (14,1 por cento) e A. flavus (11,75 por cento). No solo foram isolados Penicillium spp., Fusarium spp. e Aspergillus flavus, entre outros. Aflatoxinas foram detectadas em amostras de cascas de amendoim a partir do estágio de granação em concentrações que variaram de 5,42 μg/kg a 218,52 μg/kg. Ácido ciclopiazônico e fumonisinas B1 e B2 não foram detectadas. A presença de A. flavus e aflatoxinas nas amostras, revela a importância de um controle das cascas de amendoim antes de sua utilização. Boas práticas agrícolas são indicadas para região, uma vez que a contaminação das vagens ocorreu antes da colheita.

Peanut (Arachis hypogaea L.) hulls are very important because they are used as litter to poultry and dairy cattle and as fiber source to cattle. The elucidation of the peanuts contamination mechanisms by toxigenic fungi and their mycotoxins is vital, specially for prevention measurements. The peanuts total mycoflora and mycotoxin contamination were analyzed in plants sampled in Junqueirópolis, in São Paulo State (Brazil) at different stages of the pod maturity. The prevalent mycoflora in peanut hulls were Fusarium spp., Rhizopus spp. and Aspergillus flavus. In soil under the peanut crop, the genus Penicillium spp., Fusarium spp. and A. flavus were detected. Aflatoxins were detected in peanut hull samples since filling pod stage in concentrations from 5.42 μg/kg to 218.52 μg/kg. Cyclopiazonic acid and fumonisins were not detected. The A. flavus presence and the detection of aflatoxins indicate the importance of quality control of peanut hulls before their utilization, and the adoption of agricultural practices showed to avoid the contamination since the peanut pods contamination happened before the harvest.

Study of the antitumor potential of Bidens pilosa (Asteraceae) used in Brazilian folk medicine.

AIM OF THE STUDY: Bidens pilosa (L.) (Asteraceae) is a medicinal plant traditionally used in Brazil for treating conditions that can be related to cancer. Therefore the present study was carried out to evaluate the antitumor activity of extracts obtained from the aerial parts of this plant species. MATERIALS AND METHODS: The crude hydroalcoholic extract (HAE) (water:alcohol, 6:4) and solvent fractions (chloroform=CHCl3,ethyl acetate=EtOAc, methanol=MeOH) were assessed for cytotoxicity assay by the brine shrimp and hemolytic, MTT and NRU assays. The antiproliferative potential of the crude extract and fractions was investigated in vivo using the Ehrlich ascites carcinoma (EAC) in isogenic Balb/c mice that were administered intraperitoneally 150 and 300 mg/kg body weight per day for nine days beginning 24 h after tumor inoculation. RESULTS: In in vitro cytotoxicity using Ehrlich ascites carcinoma cell line assay CHCl3 extract proved to be more toxic than the crude HAE with an IC(50) of 97+/-7.2 and 83+/-5.2 microg/mL to NRU and MTT, respectively. Histomorphological evaluations indicated that the treatment with CHCl3 and HAE extracts significantly reduced (P<0.05) body weight, abdominal circumference, tumor volume, packed cell volume and viable cell count, when compared to EAC control group. Furthermore, nonviable tumor cell count increased significantly (P<0.01) only under treatment with CHCl3 or HAE, and this was accompanied by a marked percentage increase in life span (54.2 and 41.7%, respectively). Biochemical assays revealed that CHCl3 and HAE extracts were also able to decrease serum LDH activity (39.5 and 30.6%) and GSH concentration (94.6 and 50.7%) in ascitic fluid, respectively. CONCLUSION: The chloroform fraction showed the best and methanolic the worst antitumor activity.

Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest.

The total mycobiota and the mycotoxin contamination of peanuts were analyzed in plants collected at different stages of the pod maturity sampled in Junqueirópolis, at São Paulo State (Brazil). The prevalent peanut mycobiota were Fusarium spp. and Aspergillus flavus, present in 26% and 17% respectively of the samples analyzed. In soil, the genus Penicillium and Fusarium were most frequently detected, and A. flavus was detected in 8% of the samples. The screening of mycotoxins indicated that aflatoxins and cyclopiazonic acid were present the highest incidence, being detected in 32% of the samples, in concentrations, respectively, from 4.20 microg/kg to 198.84 microg/kg and from 260 microg/kg to 600 microg/kg. Fumonisins were not detected by HPLC. All data were correlated with the occurrence of wind-dispersed fungi and the environmental and soil conditions. Results indicate that good management of the agricultural environment may offer a way to reduce mycotoxins and the toxigenic fungal contamination in peanuts preharvest because the pods are exposed to different environmental conditions during their formation until harvest, and the optimal conditions for mycotoxin production and fungal growth are frequently found in the crop fields.