Toxigenic Aspergillus in coffee and cocoa : incidence, mycotoxins production and real-time PCR discrimination of Aspergillus niger

Orientadores: Anderson de Souza Sant'Ana, Marta Hiromi TaniwakiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O uso das técnicas moleculares para identificação de micro-organismos é indispensável em pesquisas cientificas. A dificuldade em caracterizar e identificar fungos do gênero Aspergillus ocorre devido ao fato de algumas espécies estarem intimamente relacionadas entre si, e as diferenças morfológicas entre os indivíduos serem praticamente imperceptíveis. O gênero Aspergillus engloba importantes fungos toxigênicos presentes em alimentos. Desta maneira este trabalho teve como objetivo a caracterização da micobiota toxigênica de cacau e café provenientes dos estados de Espírito Santo, Minas Gerais, São Paulo, Bahia e Pará e o desenvolvimento de uma metodologia para identificação de Aspergillus niger baseada em técnicas moleculares. Foram analisadas 114 amostras, divididas em 64 amostras de café e 50 amostras de cacau. Para o isolamento dos fungos, foi realizado o plaqueamento direto em ágar Dicloran 18% Glicerol (DG18), após desinfecção com hipoclorito de sódio 0,4 %. Um total de 1020 fungos proveniente do café e cacau, foram isolados em meio de cultura Czapek Extrato de levedura (CYA), sendo que, 769 (75,4%) foram identificados como gênero Aspergillus, 120 (11,8%) Eurotium, 52 (5%) hifomicetos, 41 (4%) Penicillium e 24 (2,4%) Fusarium. Dos 429 isolados de cacau, 267 pertenciam à seção Flavi. Em café foram 591 isolados, dos quais 451 foram identificados como A. section Nigri. Na avaliação de produção de micotoxinas, pela técnica de ágar plug associada à cromatografia de camada delgada (TLC), verificou-se que 25 % das cepas de A. section Flavi foram produtoras de aflatoxinas, enquanto que 8% dos 471 isolados de A. section Nigri foram produtores de Ocratoxina A (OTA). Para as análises moleculares foram selecionadas 127 cepas pertencentes à seção Nigri isolados das amostras de cacau e café. Para extração do DNA dois kits foram testados: PowerLyzerTM Power Soil® DNA Isolation Kit da MOBIO e o kit PrepManTM Ultra da Applied Biosystems. O Kit PowerLyzerTM Power Soil® DNA Isolation foi escolhido para efetuar as análises, por apresentar maior reprodutibilidade nos testes. Das 127 cepas analisadas 112 foram identificadas por PCR em tempo real como A.niger/A. welwitschiae. A.niger e A. welwitschiae são espécies irmãs em curso de especiação, o que torna sua identificação bastante difícil, mesmo utilizando técnicas moleculares. Contudo apesar destas dificuldades, a correta identificação através das técnicas moleculares é necessária, tornando-se uma valiosa ferramenta para auxiliar no controle de qualidade e pesquisas em micologia de alimentos, uma vez que estas duas espécies são produtoras de OTAAbstract: Molecular techniques to identify micro-organisms are becoming an indispensable tool in scientific research. The difficulty in characterizing and identifying fungi of the genus Aspergillus is due to the fact that some species are closely related to each other, and morphological differences between them are practically imperceptible. The genus Aspergillus is an important group of toxigenic fungi present in food. The aim of this study was the characterization of cocoa and coffee toxigenic mycobiota from the states of Espírito Santo, Minas Gerais, São Paulo, Bahia and Pará. And the development of a molecular methodology for identification of Aspergillus niger. One hundred and fourteen samples were analyzed, divided into 64 cocoa 50 coffee samples. For isolation of fungi, the samples, waere plated in agar Dichloran Glycerol 18% (DG18) after disinfection with sodium hypochlorite 0,4%. A total of 1020 fungi have been isolated in CYA, of which, 769 (75.4%) were identified as genus Aspergillus, 120 (11.8%) Eurotium, 52 (5%) hyphomycetes, 41 (4%) Penicillium, 24 and (2 ,4%) Fusarium. Of the 429 cocoa isolates, 267 belonged to section A. Flavi. In Coffee 591 fungus were isolated, of which 451 were identified as A. section Nigri. The production of mycotoxins evaluation, showed that 25% of the strains isolated as A. section Flavi were producing aflatoxins, while 8% of 471 A. section Nigri were producing ochratoxin A. For the molecular analysis were selected 160 strains belonging to A. Nigri section. For DNA extraction two kits were tested. The PowerLyzerTM Power Soil® DNA Isolation Kit Mobio and PrepMan Ultra kit from Applied Biosystems. The Mobio Kit was chosen for the analysis due to its reproducibility. Of the 127 strains analyzed, 112 were identified as A.niger/A. welwitschiae. A.niger and A. welwitschiae are close species in the process of speciation, which makes their identification, even with molecular techniques, very dificult. Nevertheless these two species are OTA producers and their identification is a valuable tool for food mycology researchMestradoCiência de AlimentosMestra em Ciência de Alimentos149842/2013-92011/50136-0CNPQFAPES

Genetic diversity, antimicrobial resistance and virulence profile of Salmonella isolated from the peanut supply chain

Thirty-Eight Salmonella isolates recovered from different stages of the peanut supply chain in three Brazilian States (São Paulo, Minas Gerais and Bahia) were subtyped by pulsed-field gel electrophoresis (PFGE) and characterized by phenotypic and genotypic tests for antimicrobial resistance and virulence genes. The isolates were distributed into seven PFGE pulsotypes. All the isolates were resistant to sulfonamide. However, only one isolate from a production site in Minas Gerais had resistance to two types of antimicrobials (sulfonamide and ampicillin). Furthermore, the isolates had intermediary resistance to kanamycin (16/38), streptomycin (14/38) and ceftazidime (12/38). Four isolates had the antimicrobial resistance gene related to phenicols (floR) and 37 related to aminoglycosides (strA). The blashv gene related to β-lactams was detected in isolates recovered from all the production regions. Six virulence genes (invA, sefA, sivH, mgtC, ssaQ and agfA) were observed in all isolates. The sopE gene was detected in 24 isolates, avrA in 12. The gtgB, ipfA and rck genes were not detected. The results showed that the pulsotype 1 was restricted to Minas Gerais whereas the pulsotype 7 was present in São Paulo and Bahia. In addition, most of the isolates were not multidrug resistant2945054COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2016/18724-300

Extrusion of λ‐carrageenan gum : physical properties and in vitro bifidogenic effect

Extrusion was used as a tool for modifying carrageenan gum. Increased extrusion temperature and shear led to a considerable decrease in dissolution time (DT) (7,200 to 30 s) and faster solubilization (30 to 7 s) when coarse particle sizes (> 212 mu m) and hot water were used (p 38%), suggesting in vitro bifidogenic effect. In terms of industrial applications, extruded carrageenan is promising as a food supplement fiber product438COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO CARLOS CHAGAS FILHO DE AMPARO À PESQUISA DO ESTADO DO RIO DE JANEIRO - FAPERJnão temE-26/112.069/201

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-