MICOTOXINAS EM ALIMENTOS

As micotoxinas são metabólitos tóxicos produzidos por algumas espécies de fungos filamentosos e podem contaminar os alimentos destinados ao consumo humano e animal. As micotoxinas que serão discutidas nesta revisão são: aflatoxinas (AFLA), ocratoxina A (OTA), desoxinivalenol (DON), zearalenona (ZON), fumonisinas (FUMO), toxina T–2 e patulina (PAT). Serão apresentados os aspectos relevantes relacionados às micotoxinas como a natureza química, fatores que governam a produção de toxinas, a presença das micotoxinas mais comuns em alimentos, a metodologia para análise de micotoxinas e finalmente as formas de prevenção de micotoxinas

Evaluation of coffe bean mycobiota and fungal metabolites on beverage quality

Orientador: Neura BragagnoloTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O café passa por vários processos até chegar a ser consumido como bebida e vários fatores contribuem para a sua qualidade final, dentre eles a população microbiana presente. A contaminação dos grãos pelos microrgranismos é diversificada, envolvendo a participação de bactérias, bolores e leveduras, com a predominância de um ou outro grupo, dependendo da etapa de processamento dos grãos. Existem evidências, ainda não conclusivas de que vários fungos presentes no café podem produzir uma série de compostos que podem vir a prejudicar a qualidade da bebida. Esta pesquisa teve como objetivos analisar a micobiota dos grãos obtidos em diferentes etapas da cadeia produtiva do café; investigar a produção dos compostos voláteis produzidos pelos isolados e o impacto dos mesmos na qualidade da bebida e; avaliar sensorialmente a bebida, correlacionando com os fungos presentes. A micobiota de 41 amostras de grãos de café cru, de duas regiões produtoras do Brasil, Cerrado Mineiro/MG e Piraju/SP foram analisadas. As amostras foram coletados do pé (cereja), do solo (varreção), do terreiro (maduro, seco e passas no pé e verde) e da tulha (estocagem) e comparados dois tipos de preparo dos grãos: secagem natural e cereja descascado. As amostras de Minas Gerais apresentaram baixa infecção fúngica, as principais espécies isoladas foram Eurotium spp. e Fusarium spp. Em relação aos cafés da região de Piraju, houve uma grande diversidade de espécies isoladas, dentre àquelas mais predominantes foram Penicillium brevicompactum, Aspergillus foetidus, Penicillium crustosum e Fusarium spp. Cafés varreção e bóia (seco e passas no pé) caracterizaram-se pela alta incidência de Aspergillus foetidus, apresentando infecção superior a 16% por esta espécie e avaliação sensorial negativa. A foetidus produziu compostos voláteis, como 2-butenal, dimetilbisulfeto no meio de cultura e 1-octen 3-ol quando inoculado no café cru. Estes metabólitos são caracterizados pelo aroma desagradável de terra, mofo, estragado e pungente e foram relacionados como alguns dos compostos responsáveis pelas características negativas na análise sensorial da bebida. Foi constatado também que a presença de algumas espécies fúngicas nos grãos, mesmo em alta percentagem de infecção, não implicou necessariamente na redução da qualidade sensorial da bebida. Amostras com alta freqüência de Penicillium brevicompactum apresentaram avaliação final positiva. Esta espécie destacou-se pela produção de vários compostos voláteis com características positivas como aldeídos (2-octenal, decanal e undecanal) com aroma cítrico e herbal, e cetonas (2-nonanona, 3-nonen-2 ona, 2-undecanona e 2 pentadecanona) de aroma frutal e floral. Portanto, metabólitos produzidos durante o desenvolvimento de espécies fúngicas podem estar relacionados à introdução de características sensoriais de sabor ao café, tanto desejáveis quanto indesejáveisAbstract: Coffee goes through several processes until consumed as a beverage and many factors contribute to its final quality, including the presence of the microbial population. The coffee beans contamination by microorganisms is diversity envolving bacteria, moulds and yeast, with predominance of one or another and is dependent of the coffee beans processing stages. There is inconclusive evidence that many fungi present in coffee can produce several volatile metabolites that can damage beverage quality. This research had the objectives to analyze the mycobiota ot the coffee beans obtained on the different stages of coffee production chain; investigate the production of volatile compounds produced by the isolates and the impact of them on the beverage quality and carry out sensory evaluation of beverage in relation to the fungal species. The mycobiota of forty-one samples of raw coffee beans from two Brazilian production areas, Cerrado Mineiro/MG and Piraju/SP were analyzed. Samples were collected from the tree (cherry beans), from the soil (¿varreção¿), from the drying yard (ripe, dry, over-ripe and immature) and drying storage (¿tulha¿) and two kinds of bean separation were compared: natural and pulped. The Minas Gerais samples had low fungal infection with the main species being Eurotium spp and Fusarium spp. In relation to the Piraju samples there was a considerable diversity of isolated species and the following were among the most predominant: Penicillium brevicompactum, Aspergillus foetidus, Penicillium crustosum and Fusarium spp. Coffee beans collected from the soil along with the over-ripe ones were a highly incidence of Aspegillus foetidus with a percent infection above 16% and a negative sensorial evaluation. Aspergillus foetidus produced volatile compounds such as 2-butenal, dimethyl dissulfite in the culture medium and 1-octen-3-ol when inoculated on the raw coffee. These metabolites were characterized by as unpleasant aroma of soil, musty, rotten and pungency and they were related as one of the responsible compounds for the negative characteristcs of the sensorial analyses.In this work the presence of some fungal species were found in the beans wich even at high levels of infection, did not necessarily result in a decrease of the sensorial evaluation. Samples with a high percentage of Penicillium brevicompactum infection had a positive final evaluation. This specie stood out from the rest due to the production of many volatile compounds with positive characteristics such as aldehydes (2-octenal, decanal and undecanal) showing citric and herbal aromas and cetones (2-nonanone, 3-nonen-2-one, 2-undecanonc and 2-pentadecanone) showing frutal and floral aroma. Therefore, metabolites produced during fungal growth can be related to the insertion of sensorial properties of flavour on coffee, both positive or negativeDoutoradoDoutor em Ciência de Alimento

Fungos toxigenicos e micotoxinas em frutas secas e produção de ocratoxina A em uvas passas em condições de abuso

Orientadores: Hilary Castle de Menezes, Marta Hiromi TaniwakiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Resumo: o objetivo do presente trabalho foi avaliar a qualidade das frutas secas comercializadas no Brasil, verificando quais os fungos mais comuns e sua capacidade para produção de ocratoxina A e aflatoxina, bem como analisar a presença destas toxinas nas frutas. No total foram avaliadas 119 amostras de frutas secas: uvas passas escuras (24 amostras), uvas passas claras (19), ameixas (21), figos (19), damascos (14) e tâmaras (22). As frutas secas eram provenientes da Argentina, Tunísia, Turquia, Chile, Espanha, Irã e Estados Unidos. Um total de 528 cepas de A. niger, A. carbonarius, A. ochraceus e A. fIavus foram isoladas e testadas quanto à produção de ocratoxina A para as três primeiras, e aflatoxinas para a última. A. niger foi a espécie fúngica predominante com 486 cepas isoladas e 24% foram produtoras de ocratoxina A. A porcentagem de infecção por essa espécie variou de O a 90% em amostras de uva passa escura, 0 a 86% em tâmaras, 0 a 60% em ameixa, 0 a 38% em figos e 0 a 8% em uva passa clara, com médias de 22, 8,6, 8,4, 4,5 e 0,5% respectivamente. Foram isoladas 38 cepas de A. ochraceus de tâmaras, uva passa escura e ameixa, com médias de porcentagem de infecção de 1,1, 0,8 e 0,5% respectivamente e 37% das cepas foram produtoras de ocratoxina A. A. fIavus foi isolado apenas de 1amostra de uva passa clara (9 cepas) e figo (1 cepa) e 100% foram positivas para aflatoxinas B1 e B2. As amostras de damasco seco apresentaram ausência de contaminação por qualquer fungo e por ocratoxina A. Verificou-se que as amostras de uvas passas escuras e figos apresentaram os maiores níveis de contaminação de ocratoxina A, com médias de 4,73 e 4,10'mu'g/kg,respectivamente. As tâmaras e ameixas apresentaram nível médio de contaminação por ocratoxina A menor que o limite de detecção (0,10'mu'g/kg).Não houve presença de aflatoxinas em todas as amostras de uva passa escura e apenas duas amostras de uva passa clara, estavam contaminadas com aflatoxina B1, com teores de 0,45 e 0,48'mu'g/kg. Nas amostras de figo, a presença de aflatoxinas foi mais freqüente. Foi encontrada presença de aflatoxinas em 55% das amostras. Com exceção de uma amostra, todas as demais apresentaram teores menores que 10'mu'g/kg. Numa a amostra o teor de aflatoxinas B1 e B2 excedeu 1500'mu'g/kg. A produção de ocratoxina A em frutas secas submetidas às condições externas de abuso de temperatura e umidade relativa, foi verificada. Amostras de uva passa escura mantida na própria embalagem de comercialização, naturalmente contaminada com fungos (A. niger), mas sem ocratoxina A, foram incubadas à temperatura de 30°C e umidade relativa maior que 90% durante 75 dias. A cada 15 dias foram retiradas amostras para análises de ocratoxina A, umidade e atividade de água. A porcentagem de infecção fúngica também foi calculada. O teor máximo de ocratoxina A produzido nestas condições foi de 30'mu'g/kg após 30 dias de incubaçãoAbstract: The objective of this study was to evaluate the quality of dried fruitsold in Brazil, verifying what the most common fungi were and their capacity to produce ochratoxin A and aflatoxin, as well as, the presence of these toxins in the fruit. A total of 119 samples of dried fruit were analyzed: black sultanas (24 samples), white sultanas (19), plums (21), figs (19), apricots (14) and dates (22). The dried fruits were from Argentina, Tunisia, Turkey, Chile, Spain, Iran and the United States. A total of 529 strains of A. niger, A. carbonarius, A. ochraceus and A. fIavus were isolated and tested as to the production of ochratoxin A for the first three, and aflatoxins for the last. A. niger was the predominant fungus with 486 isolated strains and 24% were ochratoxin A producers. The percentage infection for these species varied trom 0 to 90% in black sultanas, 0 to 86% in dates, 0 to 60% in plums, 0 to 38% in figs and 0 to 8% in white sultanas, with average percentage infection of 22, 8.6, 8.4, 4.5 and 0.5% respectively. A total of 38 strains of A. ochraceus were isolated from dates, black sultanas and plums, with infection percentage averages of 1.1, 0.8 and 0.5% respectively and 37% of the strains were ochratoxin A producers. A. fIavus was isolated only from 1 sample of white sultanas (9 strains) and figs (1 strain) and 100% were positive for aflatoxin B1and B2. The apricot samples presented no contamination by any fungi ar ochratoxin A. It was verified that the black sultana and fig samples presented the highest levels of ochratoxin A, with averages of 4.73 and 4.10'mu'g/kg, respectively. The dates and plums presented average levels of ochratoxin A contamination lower than the detection limit (0,10'mu'g/kg).No aflatoxin was found in any of the samples of black sultanas and only two white sultana samples were contaminated with aflatoxin B1 (0.45 and 0.48'mu'g/kg). In the fig samples, the presence of aflatoxin was more common, found in 55% of these samples. With the exception of 1 sample, all of them presented contents lower than 10'mu'g/kg. In one sample the contents of aflatoxins B1 and B2 exceeded 1500'mu'g/kg.The production of ochratoxin A in dried fruit submitted to extreme external conditions of temperature and relative humidity, was verified. Black sultana samples in their original packaging, naturally contaminated with fungi (black aspergilli), but without ochratoxin A, were incubated at 30°C and at relative humidity of more than 90% for 75 days. Every 15 days, samples were taken for the analysis of ochratoxin A, moisture content and water activity. The percentage infection by fungi was also calculated. The maximum level of ochratoxin A produced under these conditions was 30'mu'g/kg after 30 days of incubationMestradoMestre em Tecnologia de Alimento

Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within beta-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.CAPESCAPESCNPqCNPqFAPESPFAPES

Data on the presence or absence of genes encoding essential proteins for ochratoxin and fumonisin biosynthesis in Aspergillus niger and Aspergillus welwitschiae

We present the multiplex PCR data for the presence/absence of genes involved in OTA and FB2 biosynthesis in Aspergillus niger/Aspergillus welwitschiae strains isolated from different food substrates in Brazil. Among the 175 strains analyzed, four mPCR profiles were found: Profile 1 (17%) highlights strains harboring in their genome the pks, radH and the fum8 genes. Profile 2 (3.5%) highlights strains harboring genes involved in OTA biosynthesis i.e. radH and pks. Profile 3 (51.5%) highlights strains harboring the fum8 gene. Profile 4 (28%) highlights strains not carrying the genes studied herein. This research content is supplemental to our original research article, “Prospecting for the incidence of genes involved in ochratoxin and fumonisin biosynthesis in Brazilian strains of A. niger and A. welwitschiae” [1]. Keywords: Ochratoxin, Fumonisin, High-resolution capillary electrophoresi

Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within beta-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.CAPESCAPESCNPqCNPqFAPESPFAPES

Real-time PCR-based method for rapid detection of aspergillus nigerand aspergillus welwitschiae isolated from coffee

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species1488792CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP#305649/2014-0; #302763/2014-7; #310970/2015-6#33003017027P1#2011/50136-

Aspergillus section <i>Flavi</i> diversity and the role of <i>A. novoparasiticus</i> in aflatoxin contamination in the sugarcane production chain

The presence of Aspergillus section Flavi and aflatoxins in sugarcane as well as in by-products, such as molasses, sugar, yeast cream and dried yeast, collected from different fields and processing plants in Sao Paulo state, were investigated throughout the sugarcane production chain. A total of 246 samples was collected and analyzed and 226 isolates of Aspergillus section Flavi were isolated. Aspergillus section Flavi strains were found in sugarcane juice, milled sugarcane, stalk, soil and dried yeast samples. Among the isolates of Aspergillus section Flavi submitted to polyphasic identification (n = 57), Aspergillus novoparasiticus and Aspergillus arathidicola were predominantly found. A significant proportion of the isolates (84.5%) were found to have morphological and physiological characteristics of A. novoparasiticus. Most samples, with the exception of sugar, showed some aflatoxin contamination. The highest level was in dried yeast with an average of 2.55 mu g/kg and maximum value of 10.19 mu g/kg. This is the first report of contamination of sugarcane by A. novoparasiticus2931723CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/10073-