Resumo Os sorvetes à base de frutas são produtos amplamente consumidos em países tropicais e, devido à sua composição, podem ser uma boa fonte de crescimento microbiano, incluindo patógenos oportunistas. Os objetivos deste estudo foram caracterizar as populações de leveduras presentes nos sorvetes brasileiros à base de frutas e investigar sua susceptibilidade aos antifúngicos anfotericina B, fluconazol e itraconazol, e a capacidade dos isolados, que puderam crescer a 37 °C, de aderir em células epiteliais bucais. Duzentos e sessenta e sete isolados de levedura obtidos das amostras de sorvete foram identificados como pertencentes a 29 espécies, com contagens variando de 1,5 a 5,2 log UFC/mL. As espécies predominantes foram Candida intermedia, Torulaspora delbrueckii, C. parapsilosis, Clavispora lusitaniae, Saccharomyces cerevisiae e Pichia kudriavzevii. Pelo menos 16 espécies de leveduras isoladas neste estudo foram relatadas como agentes patogênicos oportunistas. Quarenta e um isolados de leveduras apresentaram resistência ou susceptibilidade dose-dependente a pelo menos um dos antifúngicos testados. Um isolado de C. parapsilosis foi resistente a todos os antifúngicos testados e mostrou capacidade de aderir a células epiteliais bucais. A porcentagem de adesão foi alta principalmente para isolados de P. kudriavzevii, Meyerozyma guilliermondii, C. parapsilosis, S. cerevisiae e Debaromyces hansenii. Os dados sugerem que a presença dessas leveduras oportunistas como contaminantes nos sorvetes pode representar um risco para o consumidor final, especialmente para indivíduos imunocomprometidos que podem consumir esses produtos.

Abstract Fruit-based ice creams are products widely consumed in tropical countries and, because of their composition, can be a good source for microbial growth, including opportunistic pathogens. The aims of this study were to characterize the yeast populations present in Brazilian fruit-based ice creams, and to investigate the antifungal susceptibility to amphotericin B, fluconazole and itraconazole, and the ability of the isolates that were able to grow at 37 ºC to adhere to buccal epithelial cells (BEC). Two hundred and sixty-seven yeast isolates obtained from the ice cream samples were identified as belonging to 29 species, with counts that ranged from 1.5 to 5.2 log CFU/mL. The predominant species were Candida intermedia, Torulaspora delbrueckii, C. parapsilosis, Clavispora lusitaniae, Saccharomyces cerevisiae and Pichia kudriavzevii. At least 16 yeast species isolated in this study have been reported as opportunistic pathogens. Forty-one yeast isolates showed resistance or dose-dependent susceptibility to at least one of the antifungal drugs tested. One isolate of C. parapsilosis was resistant to all the antifungals tested and showed ability to adhere to BEC. The percentage of adhesion to BEC was high mainly for isolates of P. kudriavzevii, Meyerozyma guilliermondii, C. parapsilosis, S. cerevisiae and Debaromyces hansenii. The data suggest that the presence of these opportunistic yeasts as contaminants in ice creams may represent a potential risk to the final consumer, especially to immunocompromised individuals who may consume these products.

ABSTRACT Chicha, a type of beer made mainly with maize or cassava, is a traditional fermented beverage of the Andean region. There have only been a few studies on yeasts associated with chicha fermentation, and the species diversity occurring during the production of this beverage is not known. The objective of this study was to determine the biodiversity of yeasts in chicha, and to characterize the Saccharomyces cerevisiae populations associated with the production of chicha de jora, seven-grain chicha, chicha de yuca, and chicha de morocho in Ecuador. The molecular diversity of S. cerevisiae populations was determined by restriction polymorphism mitochondrial profiles. The beverages were characterized based on their physicochemical parameters. Twenty-six species were identified, and the most prevalent species were S. cerevisiae and Torulaspora delbrueckii. Other yeast species were isolated at low frequencies. Among 121 isolates of S. cerevisiae, 68 different mtDNA molecular profiles were identified. These results showed that chichas are fermented by a high number of different strains of S. cerevisiae. Some other species provided a minor contribution to the fermentation process. The chicha presented generally similar physicochemical parameters to those observed for other traditional fermented beverages, and can be considered as an acid fermented beverage.

ABSTRACT Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.

RESUMO: Este estudo teve como objetivo conhecer a biodiversidade de leveduras não-Saccharomyces em maçãs com potencial para a melhoria da qualidade aromática da sidra brasileira. As cepas foram isoladas de trinta e cinco (35) mostos de maçã Gala e Fuji de diferentes locais da região Sul do Brasil. Quarenta e cinco (45) cepas foram isoladas e identificadas por análise de PCR. Os resultados indicaram dez (10) espécies: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans e Rhodotorula mucilaginosa. Três desses gêneros (Rhodotorula sp., Lodderomyces sp. e Clavispora sp.) juntos constituíram 71,2% das cepas identificadas. Entre estas cepas, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum e H. guilliermondii foram selecionadas em teste qualitativo devido a produção de aroma frutado, indicando potencial para a produção de compostos aromáticos na sidra.

ABSTRACT: This study aimed to know the biodiversity of non-Saccharomyces yeasts in Brazilian apples must with potential to improve of the aromatic quality of ciders. The strains were isolated from thirty-five (35) Gala and Fuji apple musts from different locations from south region of Brazil. Forty-five (45) strains were isolate and identified by PCR analysis. Results indicated ten (10) species: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans and Rhodotorula mucilaginosa. The genus Rhodotorula sp., Lodderomyces sp. and Clavispora sp. constituted 71.2% of the strains identified. The following strains, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum and H. guilliermondii were selected in qualitative tests due the fruity aroma production by trained team in the aromatic assessment of cider.

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.

The objective of this study was to evaluate the aerobic microbiota of the rumen fluid from Holstein cows and heifers fed different tropical forage in the north of Minas Gerais, Brazil. A total of 30 samples of rumen fluid from cows fed with sorghum silage were collected: 32 from cows fed Brachiaria brizantha, 12 from heifers that received sorghum silage and 11 from calves fed sugar cane foliage. The culture was carried out using the agar Sabouraud medium and the solid C medium, containing microcrystalline cellulose. The isolated mycelial fungi were identified by microculture technique and yeasts by micromorphological and physical-chemical analysis. Specific identification for yeasts was confirmed by ribosomal DNA sequence analysis. The presence of fungal colonies was confirmed on the Sabouraud medium for 100% of the samples. No significant differences were observed comparing the concentrations of mycelia fungi in the rumen fluid from cows fed different forages and for the two categories evaluated, fed sorghum silage. Yeast populations in the rumen fluid from heifers fed sugarcane were higher compared with those receiving sorghum silage. The yeast Pichia kudriavzevii (Candida krusei) was the most frequent and among the mycelial fungi, the genus Aspergillus was the most frequently observed, corresponding to 56% of the samples. Future studies should elucidate the variations in the populations of these microorganisms considering the carbohydrate sources in the tropical forages and the animal categories. The ecological or pathogenic role of these microorganisms should also be considered, aiming at improved productivity and health of cattle.

Enzimas pectinolíticas de origem microbiana são conhecidas por desempenhar um papel importante comercialmente em uma série de processos industriais. Dois tipos de levedura podem ser distinguidos para a produção dessas enzimas. Um grupo inclui aqueles que têm capacidade de produzi-las na ausência de um indutor e outro grupo compreende as leveduras que as produzem na presença de um indutor. O objetivo deste estudo foi investigar a influência de substâncias pécticas, de glicose, do pH e da temperatura na atividade da poligalacturonase de Kluyveromyces marxianus CCMB 322. O cultivo foi em caldo de fermentação contendo diferentes concentrações de glicose e substâncias pécticas. A atividade da poligalacturonase foi determinada pelo método do DNS, e pH e temperatura otimizados pelo delineamento experimental central composto de experimentos. A poligalacturonase secretada por K. marxianus CCMB 322 foi parcialmente constitutiva, apresentando pH e temperatura ótima de 7,36 e 70 °C, respectivamente e reteve 93% de sua atividade original após 50 minutos a 50 °C. A termoestabilidade da enzima poligalacturonase foi estudada em várias temperaturas (50, 60, 70 e 80 °C) em diferentes tempos de incubação (0, 10, 20, 30, 40 e 50 minutos). Este estudo mostrou que a glicose tem influência na regulação da síntese da poligalacturonase.

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. Two kinds of yeast can be discerned regarding the production of enzymes. One group includes those which can produce enzymes in the absence of an inducer, and the other group comprises the yeasts that produce enzymes in the presence of an inducer. The objective of this study was to investigate the influence of pectic substances, glucose, pH, and temperature on the polygalacturonase activity by Kluyveromyces marxianus CCMB 322. The yeast was grown in a fermentation broth containing different concentrations of glucose and pectic substances. The polygalacturonase activity was determined by the DNS method, and the pH and temperature were optimized using a central composite experimental design. The polygalacturonase secreted by K. marxianus CCMB 322 was partially constitutive showing optimum pH and temperature of 7.36 and 70 °C, respectively, and maintained approximately 93% of its original activity for 50 minutes at 50 °C. Thermal stability of the polygalacturonase enzyme was studied at different temperatures (50, 60, 70, and 80 °C) and different incubation times (0, 10, 20, 30, 40, and 50 minutes). This study showed that glucose can influence the regulation of the synthesis of polygalacturonase.

Fungos endofíticos representam um grande e ainda pouco explorado componente da biodiversidade. O trabalho avaliou a riqueza e a distribuição endofítica nas folhas de Bauhinia brevipes (Fabaceae). Foram obtidas 1110 colônias que foram agrupadas, por suas características morfológicas em 126 táxons. O número total de táxons por estágio foliar foi: 102 em folhas maduras, 93 em folhas recém-expandidas e 79 em não expandidas. O principal gênero de endofítico encontrado foi Phomopsis, seguido por Dothiorella, Pestalotiopsis e Acremonium. A riqueza e o número de isolados não foram estatisticamente influenciadas pela região foliar. Contudo, alguns táxons foram específicas de um estágio foliar; seis foram isoladas apenas em folhas não expandidas, nove em folhas recentemente expandidas e 17 foram encontradas apenas em folhas maduras. A composição endofítica variou de acordo com as regiões foliares; a similaridade (índice de Jaccard) entre as regiões das folhas de diferentes estágios variou de 0,36 a 0,46, indicando uma alta variação espacial da comunidade de fungos endofíticos dentro da folha. A alta riqueza de endofíticos neste hospedeiro demonstra uma significante contribuição dos fungos para a biodiversidade tropical e a necessidade de pesquisas futuras nesta área.

Endophytic fungi represent large, yet unexplored components of biodiversity. This work evaluated the richness and the distribution of endophytes in the leaves of Bauhinia brevipes (Fabaceae). A total of 1110 colonies were recovered from the samples and grouped by their morphological traits into 126 taxa. The total number of taxa according to leaf development was: 102 in mature leaves, 93 in recently expanded leaves and 79 for unfolded leaves. The major endophyte genera were Phomopsis, followed by Dothiorella, Pestalotiopsis and Acremonium. The richness and the isolate numbers of endophytes were not statistically affected by leaf region. However, some taxa were leaf-age specific; six were isolated only from unfolded leaves, nine from recently expanded leaves and 17 were exclusively found in mature leaves. The composition of endophytes varied with leaf region; the similarities (Jaccard's Index) among the leaf regions of different leaf ages ranged from 0.36 to 0.46, indicating a high spatial variation in the community of endophytic fungi inside the leaves. The high richness of endophytes in this host plant highlights a significant contribution of fungi to tropical biodiversity and the need for further research in this area.

Duas linhagens de Saccharomyces cerevisiae foram testadas como iniciadoras em uma destilaria de cachaça. Foram utilizadas as linhagens de S. cerevisiae UFMG-A829, isolada de fermentação de cachaça, e S. cerevisiae K1-V1116, de origem vinícola. A permanência de cada linhagem durante a fermentação foi determinada por RAPD (Random Amplified Polymorphic DNA)-PCR, utilizando o iniciador M13. As duas linhagens predominaram nas dornas de fermentação por aproximadamente 30 dias. Leveduras não-Saccharomyces e S. cerevisiae indígenas foram isoladas em menor proporção durante o experimento. As linhagens de S. cerevisiae indígenas apresentaram perfis moleculares distintos em relação às linhagens iniciadoras. As duas linhagens foram promissoras para serem utilizadas como iniciadoras do processo fermentativo para a produção da cachaça.

Two Saccharomyces cerevisiae strains were tested as the starter yeasts in a traditional cachaça distillery. The strains used were S. cerevisiae UFMG-A829, isolated from a cachaça fermentation process, and S. cerevisiae K1-V1116, obtained from the wine industry. The permanence of each strain in the fermentation must was determined by RAPD (Random Amplified Polymorphic DNA)-PCR, with primer M13. Both yeast strains were prevalent in the vats for approximately 30 days. Indigenous non-Saccharomyces and indigenous S. cerevisiae strains were isolated in lower counts during the fermentation period. Indigenous S. cerevisiae strains were molecularly distinct when compared to the starter yeasts. The two yeasts appeared promising starter yeasts in the fermentation process to produce traditional cachaça.

O objetivo deste trabalho foi estudar a freqüência de infecções intramamárias ocasionadas por leveduras a partir de amostras de leite (n=1710) coletadas em 40 rebanhos leiteiros do Estado de Minas Gerais. Cinqüenta e seis estirpes de leveduras do gênero Candida e uma linhagem de Trichosporon loubieri foram isoladas. Candida albicans foi a espécie dominante (28,1% das cepas), seguida por Candida parapsilosis (19,3%), Candida catenulata (14,0%), Candida glabrata (14,0%) e Candida tropicalis (8,8%). Infecções mistas foram detectadas em 29,8% das vacas levedura-positivas. Amostras positivas para leveduras foram predominantemente obtidas (84%) de vacas com mastite subclínica. A baixa taxa de isolamento de leveduras sugere que estes microrganismos não são relevantes para mastite bovina na região estudada.

The objective of this study was to report the frequency of intramammary infections by yeasts, in Minas Gerais State, from milk samples (n = 1710) collected in 40 dairy herds. Fifty six yeast strains of the genus Candida and one strain of Trichosporon loubieri were isolated. Candida albicans was the dominant species (28.1% of the strains), followed by Candida parapsilosis (19.3%), Candida catenulata (14.0%), Candida glabrata (14.0%), Candida tropicalis (8.8%). Mixed infections were detected in 29.8% of yeast-positive cows. The yeast infection was more frequent (84%) in cows with subclinical mastitis. The low rate of isolation of yeasts suggests that these microorganisms are not relevant to bovine mastitis in the studied region.

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

A concentração de fósforo total foi utilizada para determinar o grau de trofia das principais sub-bacias do trecho médio da bacia do Rio Doce, em Minas Gerais, e variáveis físicas, químicas e microbiológicas da água foram analisadas nos períodos de chuva e seca durante os anos de 2000 e 2001. O estudo visou determinar mudanças na qualidade da água causadas pela sazonalidade e por diferentes atividades antrópicas. As áreas estudadas variaram de oligotróficas a eutróficas. As variáveis que apresentaram as maiores correlações positivas com a densidades de coliformes fecais foram as concentrações de nitrogênio e fósforo total, além disso, bactérias heterotróficas mostrou-se capaz de diferenciar os ambientes. Estes resultados sugerem a inclusão do grau de trofia e da caracterização das atividades antrópicas na bacia, como ferramentas importantes para a proposição de medidas de recuperação e conservação de trechos de rios sujeitos a impactos antrópicos.

Total phosphorus concentration was used to define the trophic state of the main sub-basins of the middle Rio Doce, in Minas Gerais State (southeast Brazil) and physical, chemical, and microbiological variables of water were analyzed during 2000 to 2001. The study evaluated changes in water quality caused by seasonality and human activities. Water temperature, conductivity, pH, total alkalinity, dissolved oxygen, and concentrations of NH4-N, NO2-N, NO3-N, total-N, PO4-P, total-P, DOC, and chlorophyll-a were measured in seven rivers stretches (sampling stations). Total yeasts, faecal and total coliforms, and heterotrophic bacteria were also determined. The studied areas were considered to be under oligotrophic to eutrophic conditions. The variables that presented highest positive correlation with faecal coliforms were total-P and total-N, and heterotrophic bacteria density was identified as a good parameter to differentiate the ecosystems. These results suggested the inclusion of the trophic level and the distinct activities within a watershed as important elements when proposing conservation and restoration areas.

A total of 103 isolates of basidiomycetes, representing 84 species from different Brazilian ecosystems, were evaluated for their antifungal and antibacterial activity in a panel of pathogenic and non-pathogenic microorganisms. Tissue plugs of the fruiting bodies were cultivated in liquid media and the whole culture extracted with ethyl acetate. Crude extracts from Agaricus cf. nigrecentulus, Agrocybe perfecta, Climacodon pulcherrimus, Gloeoporus thelephoroides, Hexagonia hydnoides, Irpex lacteus, Leucoagaricus cf. cinereus, Marasmius cf. bellus, Marasmius sp., Nothopanus hygrophanus, Oudemansiella canarii, Pycnoporus sanguineus, Phellinus sp., and Tyromyces duracinus presented significant activity against one or more of the target microorganisms. Eight isolates were active only against bacteria while three inhibited exclusively the growth of fungi. Two extracts presented wide antimicrobial spectrum and were active against both fungi and bacteria. Differences in the bioactivity of extracts obtained from isolates from the same species were observed.

O uso alternativo de cacau (Theobroma cacao) para produção de vinho foi testado. A polpa de cacau foi obtida da Fazenda Formosa, Itacaré, Brasil. As amostras de polpa foram diluídas, homogeneizadas e inoculadas em meio de Sabouraud dextrose e incubadas a 28°C por 5-8 dias. Colônias selecionadas foram testadas quanto à habilidade de fermentar a polpa de cacau e divididas em fermentadoras, não-fermentadoras e fermentadoras lentas. As amostras fermentadoras foram identificadas e testadas para produção de vinho de cacau em escala piloto. A maioria das amostras fermentadoras pertencem ao gênero Brettanomyces, com exceção de duas amostras de Kloeckera apis. O vinho obtido apresentou coloração fraca e clara, tornando a clarificação desnecessária, além de sabor doce e agradável. A predominância de espécies de Brettanomyces na polpa de cacau poderia indicar sua importância ecológica neste ambiente e sugere uma participação ativa dessas leveduras nos processos de deterioração da polpa processada do cacau.

The alternative use of cocoa (Theobroma cacao) for wine production was tested. The pulp samples, obtained from Formosa farm, Itacaré, Brazil, were diluted, homogenized and inoculated on Sabouraud dextrose agar medium (SDA) and incubated at 28º C for 5-8 days. Selected colonies were tested for the ability to ferment cocoa pulp and divided into fermentative, non-fermentative and weak/late fermentative species. Isolates characterized as fermentative were further tested in a small-scale wine production plant and identified. Species from the genus Brettanomyces constituted the main fermentative yeasts, with the exception of two Kloeckera apis samples. The final wine product was normally pale or clear, making clarification unnecessary, and with a sweet or dry pleasant flavor. The predominance of Brettanomyces species in cocoa pulp indicated its ecological importance in this environment and pointed to an active role of Brettanomyces in the deterioration process of the processed cocoa pulp.