Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaça (alembic´s cachaça), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaça production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaça fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Yeast communities in two Atlantic rain Forest fragments in Southeast Brazil

We studied the yeast communities associated with fruits, mushrooms, tree exudates, and flies of the genus Drosophila, in two Atlantic Rain Forest fragments in state of Minas Gerais, Brazil. A total of 456 samples were collected from Rio Doce State Park and 142 from Ecological Station of Universidade Federal de Minas Gerais. From these samples, 608 yeast isolates were obtained, belonging to 71 different species. Among the yeasts isolated from Rio Doce State Park, 17 isolates were recovered from fruits, 12 from mushrooms, 13 from tree exudates, and 299 from Drosophila spp. In the Ecological Station of Universidade Federal de Minas Gerais, 24 isolates were recovered from fruits and 243 from Drosophila spp. Distinct communities of yeast were observed in Drosophila flies, fruits, mushrooms and tree exudates. The highest number of yeast species was recovered from Drosophila flies suggesting that flies are the natural vectors of these microorganisms.

O objetivo deste trabalho foi estudar as comunidades de leveduras associadas a frutos, cogumelos, exudatos de árvores e moscas do gênero Drosophila, em dois fragmentos de Mata Atlântica no Estado de Minas Gerais, Brasil. Foram coletadas 456 amostras no Parque Estadual do Rio Doce e 142 na Estação Ecológica da Universidade Federal de Minas Gerais. Destas amostras foram obtidas 608 isolados de levedura, distribuídas em 71 espécies. Entre os isolados obtidos a partir do Parque Estadual do Rio Doce, 17 foram provenientes de frutos, 12 de cogumelos, 13 de exudatos de árvores e 299 de Drosophila spp. A Estação Ecológica da Universidade Federal de Minas Gerais possibilitou a obtenção de 24 isolados de frutos e 243 de Drosophila spp. Foram observadas comunidades distintas de leveduras associadas a Drosophila, frutos, cogumelos e exudatos de árvores. O maior número de espécies foi obtido em drosófilas, sugerindo que estas moscas são vetores naturais destes microrganismos.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

Probióticos são definidos como microrganismos (bactérias e leveduras) que exercem um efeito benéfico na saúde do hospedeiro quando ingeridos em quantidades adequadas. A seleção desses agentes bioterapêuticos normalmente é feita por testes in vitro simulando o ambiente gastrointestinal que determina a capacidade de sobrevivência no trato digestivo. Neste trabalho, a possibilidade de extrapolação dos dados obtidos nos testes in vitro para as condições in vivo foi estudada utilizando cinco linhagens de Saccharomyces cerevisiae isoladas da floresta Atlântica brasileira. O conteúdo de trealose e a sobrevivência após a exposição a diversos estresses fisiológicos geralmente encontrados no trato gastrointestinal de humanos foram determinados para as cinco linhagens e os resultados comparados com a Saccharomyces boulardii, um probiótico conhecido. Esses resultados foram completados com a capacidade de colonização do trato gastrointestinal de camundongos gnotobióticos pelas leveduras. Pelos resultados obtidos, concluimos que os testes in vitro não são confirmados pelos ensaios in vivo, indicando que essa extrapolação não pode sempre ser feita.

Biological control of Penicillium italicum, P. digitatum and P. expansum by the predacious yeast Saccharomycopsis schoenii on oranges

In this study we evaluated the ability of Saccharomycopsis schoenii Nadson and Krassiln (UWO-PS 80-91) as biocontrol agent against plant pathogenic filamentous fungi P. expansum Link (UFMG 01-2002), P. italicum Wehmer (LCP 61.1199), and P. digitatum (Pers.: Fr.) (LCP 984263, LCP 68175 and LCP 4354). S. schoenii was able to reduce disease severity in oranges inoculated with all fungi. Among the phytopathogens, P. digitatum LCP4354 was the most virulent whereas P. digitatum LCP 68175 was the most susceptible to predation. The yeast was able to survive for 21 days on the fruit surface and did not produce lesions on oranges. Production of antagonistic substances by S. schoenii was not detected using standard techniques. Our results point to the potential use of S. schoenii to control postharvest phytopathogens in fruits.

Este estudo avaliou a capacidade de levedura Saccharomycopsis schoenii Nadson & Krassiln (UWO-PS 80-91) em controlar o crescimento dos fungos fitopatogênicos Penicillium expansum Link (UFMG 01-2002), P. italicum Wehmer (LCP 61.1199), e P. digitatum (Pers.: Fr.) (LCP 984263, LCP 68175 e LCP 4354). S. schoenii reduziu a severidade da doença em laranjas inoculadas com todos os fitopatógenos testados. Entre estes fitopatógenos, P. digitatum LCP4354 apresentou a maior virulência enquanto que P. digitatum LCP 68175 foi o mais suscetível à predação. A levedura foi capaz de permanecer viável, sem produzir lesões na superfície dos frutos por 21 dias. Outra característica desejável observada foi a ausência de produção de substâncias antagonistas. Sendo assim, este trabalho evidência o potencial de utilização da levedura S. schoenii em protocolos de controle biológico de doenças pós-colheita em laranjas.

Monitoring Saccharomyces cerevisiae populations by mtDNA restriction analysis and other molecular typing methods during spontaneous fermentation for production of the artisanal cachaça

An ecological study on Saccharomyces cerevisiae populations in spontaneous fermentation has been conducted in three vats of a cachaça distillery in Minas Gerais, Brazil. Ninety-seven yeast isolates were collected at the beginning, the middle and at the end of the production period, and were identified by standard methods. Differentiation between the indigenous S. cerevisiae strains isolated was performed by mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR, and PCR fingerprint using an intron splice primer. Analysis of the mtDNA restriction profiles revealed 12 different patterns, 11 corresponding to indigenous yeasts (I to XI) and one (XII) to a commercial strain of the bakery yeast. Pattern II (53.6% of the population) and pattern IV strains were present in all the vats. Pattern IV strain raised from the middle to the end of the period reaching proportions near those of pattern II strain. PCR methods allowed the differentiation of 41 molecular profiles. Both methods showed population fluctuation of S. cerevisiae strains along the period of cachaça production and among different vats of the distillery.

Um estudo ecológico das populações de Saccharomyces cerevisiae em fermentações espontâneas foi conduzido em três dornas de uma destilaria de cachaça em Minas Gerais, Brasil. Noventa e sete isolados foram coletados no início, meio e final do período de produção, e identificados por métodos padrões. A diferenciação entre as linhagens isoladas de S. cerevisiae indígenas foi feita pela analise de restrição do DNA mitocondrial (mtDNA), RAPD-PCR, e PCR por impressão digital do DNA utilizando um iniciador complementar a sítios de processamento de íntron. As análises dos perfis de restrição do mtDNA mostraram a ocorrência de 12 perfis diferentes, sendo 11 correspondentes as leveduras indígenas (I ao XI) e um (XII) a uma linhagem comercial de levedura de panificação. Linhagens com o perfil II (53,6% da população) e o perfil IV estiveram presentes em todas as dornas. A linhagem com perfil IV aumentou do meio para o final do período de fermentação, alcançando proporções próximas a aquelas encontradas para a linhagem com o perfil II. Os métodos baseados em PCR permitiram a diferenciação de 41 perfis moleculares. Ambos os métodos mostraram flutuações populacionais nas linhagens de S. cerevisiae durante o período de produção da cachaça e entre as diferentes dornas da destilaria.

Enteroxigenic Staphylococcus spp. and other microbial contaminants during production of Canastra cheese, Brazil

Canastra cheese is produced from raw cow's milk, and it is made at the farmhouse level using artisanal procedures and natural starters. The aim of this work was to determine the main hygienic-sanitary indicators and enterotoxigenic staphylococcal strains present during the manufacturing of traditional cheese of Serra da Canastra region, Minas Gerais state, Brazil. Samples from 10 farms were studied, and they included: water employed in the process, raw milk, natural starters, cheese curd before salting and cheese after five days of ripening. All water samples exhibited faecal coliform contamination above the maximum acceptable value recommended by Brazilian standards. Pseudomonas aeruginosa and sulfite-reducing clostridia were also isolated from the water samples. In five samples of raw milk faecal coliform were above the limits allowed by the Brazilian legislation. The counts of Staphylococcus spp. in milk were between 2.0 to 4.9 log.cfu.ml-1. The counts of microbiological indicators were higher in natural starters and curd. High levels of faecal and total coliform, as well as molds, were found in the cheese samples. In all cheeses analyzed Staphylococcus spp. were found in levels above 5.0 log.cfu.g-1. The enterotoxins (SE) most frequently produced by Staphylococcus spp. strains were SEB and SEC. A high number of coagulase negative Staphylococcus strains were also enterotoxin producers. None of the samples contained Salmonella spp. or Listeria spp. These results point out a need for improvements in the production process of the artisanal cheese produced at Serra da Canastra in Brazil.

O queijo Canastra é produzido a partir de leite cru, e é fabricado em fazendas utilizando procedimentos artesanais e culturas naturais de soro como iniciadoras. O objetivo deste trabalho foi determinar os principais indicadores higiênico-sanitário e as linhagens enterotoxigenicas de Staphylococcus presentes durante a fabricação do queijo tradicional da região da Serra da Canastra, Minas Gerais, Brasil. Amostras provenientes de 10 fazendas foram estudadas, e estas incluíram: a água utilizada no processo, o leite cru, o soro iniciador, a coalhada antes da salga, e o queijo após cinco dias de cura. Todas as amostras de água apresentaram contaminação por coliformes fecais acima do valor máximo recomendado pelos padrões brasileiros. Pseudomonas aeruginosa e clostrídios sufito-redutores também foram isolados das amostras de água. Em cinco amostras de leite cru os coliformes fecais apresentaram-se acima dos limites permitidos pela legislação brasileira. As contagens de Staphylococcus spp. no leite variaram de 2.0 a 4.9 log.ufc.ml-1. As contagens dos indicadores microbiológicos foram maiores no soro iniciador do que na massa coagulada. Níveis altos de contaminação por coliformes totais e fecais, como também bolores, foram observados nas amostras de queijo. Em todos os queijos estudados Staphylococcus spp. foram encontrados em níveis acima de 5.0 log.ufc.g-1. As enterotoxinas mais freqüentes produzidas pelas linhagens de Staphylococcus spp. foram B e C. Um número elevado de linhagens de Staphylococcus coagulase-negativa foram também produtores de enterotoxinas. Em nenhuma das amostras foi isolada Salmonella spp. ou Listeria spp. Estes resultados mostram a necessidade de melhorias no processo de produção do queijo artesanal produzido na Serra da Canastra, Brasil.

Uptake of free and complexed silver ions by different strains of Rhodotorula mucilaginosa

Five strains of Rhodotorula mucilaginosa were tested for the ability to accumulate free and complexed silver ions by metabolism-dependent and -independent processes. The ability to take up Ag+ was observed in both live and dead biomass, whereas silver dicyanide [Ag (CN)2-] uptake was strictly glucose dependent. In contrast to Ag (CN)2-, glucose addition inhibited by 16 to 25% the Ag+ uptake rate of living UFMG - Y02, Y27, and Y35 cells, while strains CBS 316 and UFMG-Y01 showed an improved uptake rate of about 115% and 13%, respectively. The Langmuir sorption model was used to evaluate the silver sorption capability of the R. mucilaginosa strains. The calculated q max value suggested that R. mucilaginosa strains UFMG-Y27 had the highest loading capacity. The type strain CBS 316 had the lowest q max but showed the highest affinity for silver ions. The results provided by the Fourier Transform Infra Red analysis (FTIR) suggest that C=O groups represent the main reactive site for silver uptake by the strain UFMG-Y27.

Quatro linhagens de Rhodotorula mucilaginosa e uma linhagem padrão dessa mesma espécie foram investigadas quanto a habilidade de acumular íons de prata livres (Ag+) e complexados [Ag (CN)2-], através de processos dependentes e independentes do metabolismo. A habilidade de acumular Ag+ foi observada em ambas as células, vivas e mortas. Contudo, o acúmulo de Ag (CN)2- foi um processo estritamente dependente de energia. Durante os estudos que avaliaram a dependência de uma fonte energética para o acumulo de íons de prata livres (Ag+) foi observado que a adição de glicose resultou na diminuição do acúmulo de Ag+ por três linhagens (UFMG -- Y02, 27 and 35). Ao passo que as linhagens CBS 316 e UFMG -- Y01 apresentaram aumento do acúmulo desse metal. A analise dos resultados de biossorção através do modelo de Langmuir, sugerem que a linhagem UFMG -- Y27 possui a maior capacidade de acúmulo de prata (carga). Enquanto que a linhagem padrão CBS 316 apresentou a menor capacidade de carga e a maior afinidade por ions de prata. Analises de infra vermelho da biomassa fúngica, com e sem prata, mostraram que os grupamentos C=O foram os principais sítios de ligação desse metal pela linhagem UFMG -- Y27.

Trehalose accumulation, invertase activity and physiological characteristics of yeasts isolated from 24 h fermentative cycles during the production of artisanal Brazilian cachaça

Trehalose accumulation, invertase activity and physiological characteristics of 86 yeast isolates from short fermentative cycles during the production of cachaça in three artisanal distilleries of the State of Minas Gerais were studied. Among these isolates, 70% were able to grow at temperatures between 40 and 42ºC. Only Saccharomyces cerevisiae isolates were able to grow over 40ºC. Lower temperatures ( 40ºC) favoured the growth of yeasts such as Candida parapsilosis-like, C. maltosa-like, Kloeckera japonica, S. exiguus and C. bombicola-like. The isolates from all three distilleries were ethanol tolerant, produced invertase, and accumulate trehalose in the presence of glucose. The strains isolated from distillery A presented more resistance to ethanol (around 84.2% of the strains were able to grow in the presence of 12% ethanol) when compared to the ones from distilleries C and B (9.5% and no strain, respectively). The strains of S. cerevisiae isolated from the three distilleries presented a higher capacity to produce invertase and accumulate trehalose in the presence of glucose. Based on the results of thermal and ethanol stress experiments, it was possible to identify strong relationship between intracellular trehalose accumulation and cell viability. The increase in cell viability was even more pronounced when the strains were subjected to a pre-treatment at sublethal temperatures.

As características fisiológicas, acúmulo de trealose e produção de invertase de 86 leveduras isoladas do ciclo fermentativo durante a produção artesanal de cachaça em três destilarias no Estado de Minas Gerais foram estudadas. Destes isolados 70% foram capazes de crescer acima de 40 e 42ºC. Somente os isolados de Saccharomyces cerevisiae foram capazes de crescer acima de 40ºC. Baixas temperaturas ( 40ºC) favoreceram o crescimento de leveduras como Candida parapsilosis-like, C. maltosa-like, Kloeckera japonica, S. exiguus and C. bombicola-like. Os isolados das três destilarias foram tolerantes ao etanol, produziram invertase e acumularam trealose na presença de glicose. As linhagens isoladas da destilaria A apresentaram maior resistência ao etanol (cerca de 84.2% das linhagens foram capazes de crescer na presença de 12% de etanol) quando comparadas àquelas das destilarias B e C (nenhuma linhagem e 9,5%, respectivamente). As linhagens de S. cerevisiae isoladas das três destilarias apresentaram uma alta capacidade para produzir invertase e acumular trealose na presença de glicose. Com base nos resultados dos experimentos de estresse térmico e de etanol, foi possível observar uma forte relação entre acúmulo de trealose intracelular e viabilidade celular. O aumento da viabilidade celular foi mais pronunciado quando as linhagens foram submetidas a um pré-tratamento em temperaturas sub-letais.