Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 x 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

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 percent 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 percent 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 percent of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75 percent of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9 percent 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.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaça (alembicïs 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.

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 percent 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 por cento 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.

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 percent 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 percent of the strains were able to grow in the presence of 12 percent ethanol) when compared to the ones from distilleries C and B (9.5 percent 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