Identification and characterization of non-saccharomyces spoilage yeasts isolated from Brazilian wines

The industry of fine wines and also locally consumed table wines is emerging in Brazil with an increasing volume and economic impact. Enologists in this region currently lack information about the prevalence and characteristics of spoilage yeasts, which may contaminate and potentially undervalue Brazilian wines. Herein, we analyzed 50 local red wines including 27 fine wines (V. vinifera) and 23 table wines (V. labrusca). Presumptive spoilage yeasts were isolated on differential medium, and classified by RFLP-PCR and sequencing of the ITS1-5.8S-ITS2 and D1/D2 26S rDNA loci. The prevalence of spoilage yeasts in fine wines (11 %) was comparable to that reported in European and US wines, and significantly lower than that observed for local table wines (70 %). The majority of isolates belonged to Brettanomyces bruxelliensis, followed by Pichia guillermondii, and more rarely Candida wickerhamii and Trigonopsis cantarelli. The Brettanomyces isolates varied greatly in off-flavor production, displayed ethanol tolerance (>10 % by volume), tolerated sulfite (≥0.68 mg/l mSO₂), and 39 % of them grew on ethanol as sole carbon source. We discuss the causes and consequences of spoilage yeasts in relation to the Brazilian wine industry.     

Genetic Variability in Hesperozygis ringens Benth. (Lamiaceae), an Endangered Aromatic and Medicinal Plant of Southern Brazil

Hesperozygis ringens Benth. (Lamiaceae) is an aromatic and medicinal plant, characterized by a high concentration of isopulegone; it is endemic to the mountains of southeast Rio Grande do Sul, Brazil, and currently is considered endangered. The present study evaluated the intra- and interpopulation genetic variability of H. ringens by means of RAPD and ISSR molecular markers. The results showed that H. ringens populations are genetically structured, with low gene flow between populations, confirming the fragmentation imposed by anthropic action. Populations from the two areas of occurrence are genetically different. Low intrapopulation variability and heterozygosity were detected, indicating genetic drift and inbreeding. Based on the data, conservation strategies are discussed.     

The fungicide Mancozeb induces metacaspase-dependent apoptotic cell death in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> BY4741

Mancozeb (MZ), a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Toxicologic studies in mammals and mammalian cells indicate that this fungicide can cause neurological and cytological disorders, putatively associated with pro-oxidant and apoptotic effects. Yeast adaptation to sub-inhibitory concentrations of MZ has been correlated with oxidative response, proteins degradation, and energy metabolism, and its main effect on yeast has been attributed to its high reactivity with thiol groups in proteins. Herein, we show that acute MZ treatments on aerobic exponentially growing yeast of wild type (BY4741) and deletion mutant strains, coupled with multiplex flow cytometry analysis, conclusively demonstrated that MZ displays the typical features of pro-oxidant activity on Saccharomyces, elevating mitochondrial ROS, and causing hyper-polarization of mitochondrial membranes leading to apoptosis. A drastic reduction of cellular viability associated with the maintenance of cell membrane integrity, as well as phosphatidyl serine externalization on yeast cells exposed to MZ, also supports an apoptotic mode of action. Moreover, abrogation of the apoptotic response in yca1 deficient mutants indicates that metacaspase-1 is involved in the programmed cell death mechanism induced by MZ in yeast.     

Variation in phenolic compounds,anthocyanins, and color in red wine treated with enzymatic extract of Kluyveromyces marxianus

The effect of the addition of enzymatic extract of Kluyveromyces marxianus NRRL-Y-7571 during the maceration and fermentation steps of Cabernet Sauvignon wine production was evaluated. The results obtained in the analytical determinations of the wines showed levels within the limits established by legislation and similar to values found in other studies. The results show that by adding the enzyme to the red wines these showed color characteristics considered to be superior to those of the control wine and accelerated the extraction of phenolic compounds and anthocyanins. It was observed that by using the commercial enzyme preparation there was an increase of 15 % in polyphenol content compared to the control wine and an increase of 28 % when the crude enzyme extract was used. Anthocyanin content in the wine increased after treatment with the commercial enzyme preparation (10 %) and with the use of the crude enzymatic extract (22 %). Considering all comparison criteria, the K. marxianus enzymatic extract showed results statistically similar or superior to those obtained with the commercial enzyme preparation.     

Essential oil variability within and among populations of Cunila incisa Benth.

To evaluate the intra- and interpopulational variability of volatile compounds in Cunila incisa, 72 samples representing 12 populations, were collected all over the distribution area of the species in Rio Grande do Sul State. The samples were extracted by steam distillation and analyzed using GC and GC–MS. A total of 19 volatile compounds were detected and identified. In average, the main compounds were 1,8-cineole, α-terpineol, p-cymene, sabinene, terpinene-4-ol, and γ-terpinene. Negative correlation was detected between the concentrations of 1,8-cineole and α-terpineol. Significant variation in the concentration of volatile compounds was detected within and among populations. Multivariate analysis of volatile oil composition identified three groups. Most populations exhibited low intra-populational variation indicating foundation effect. Relation between geographic distribution and chemical composition was observed.     

Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest

The Brazilian Atlantic Forest is one of the world's major biodiversity hotspots and is threatened by a severe habitat loss. Yet little is known about the processes that originated its remarkable richness of endemic species. Here we present results of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene of the pitviper, jararaca lancehead (Bothrops jararaca), and two closely related insular species (Bothrops insularis and Bothrops alcatraz), endemic of this region. Phylogenetic and network analyses revealed the existence of two well-supported clades, exhibiting a southern and a northern distribution. The divergence time of these two phylogroups was estimated at 3.8 million years ago, in the Pliocene, a period of intense climatic changes and frequent fragmentation of the tropical rainforest. Our data also suggest that the two groups underwent a large size expansion between 50,000 and 100,000 years ago. However, the southern group showed a more marked signal of population size fluctuation than the northern group, corroborating evidences that southern forests may have suffered a more pronounced reduction in area in the late Pleistocene. The insular species B. alcatraz and B. insularis presented very low diversity, each one sharing haplotypes with mainland individuals placed in different subclades. Despite their marked morphological and behavioural uniqueness, these two insular species seem to have originated very recently and most likely from distinct costal B. jararaca populations, possibly associated with late Pleistocene or Holocene sea level fluctuations.     

Growth of Aeromonas species on increasing concentrations of sodium chloride

The growth of 16 strains of Aeromonas, representing 12 species of the genera, were examined at different salt levels (0-1.71 M NaCl). All the strains grew on media with 0.34 M NaCl, and nine on media with 0.68 M. Two strains, Aer. enteropelogenes and Aer. trota, were able to grow on media with 0.85 M and 1.02 M NaCl, respectively. Comparison of the growth curves of Aer. hydrophila ATCC7966 and Aer. trota ATCC 49657 on four concentrations of NaCl (0.08, 0.34, 0.68 and 1.02 M) confirm the high tolerance of Aer. trota, and indicate that high concentrations of salt increase the lag time and decrease the maximum growth rate. However, both strains were able to grow, slowly, in at least 0.68 M NaCl, a sodium chloride concentration currently used as food preservative.     

RAPD marker polymorphism among commercial winery yeast strains

Random amplified polymorphic DNA (RAPD) analysis was employed to characterize commercial winery yeast strains currently used in Brazilian industries and to examine the relationship between them. Sixteen strains were subjected to RAPD analysis using eight primers, selected from the 40 primers of the kits B and X of Operon Techn. From the total of 93 scorable bands, 39 (41.9%) were polymorphics. Using the accumulated information of several reactions, all strains under investigation were differentiated. Cluster analysis revealed three groups. Most of the strains (11) were clustered in one group. The highest distance (28%) was obtained with the strain Redstar (Universal Food Corp.).     

Rules extraction from neural networks applied to the prediction and recognition of prokaryotic promoters

Promoters are DNA sequences located upstream of the gene region and play a central role in gene expression. Computational techniques show good accuracy in gene prediction but are less successful in predicting promoters, primarily because of the high number of false positives that reflect characteristics of the promoter sequences. Many machine learning methods have been used to address this issue. Neural Networks (NN) have been successfully used in this field because of their ability to recognize imprecise and incomplete patterns characteristic of promoter sequences. In this paper, NN was used to predict and recognize promoter sequences in two data sets: (i) one based on nucleotide sequence information and (ii) another based on stability sequence information. The accuracy was approximately 80% for simulation (i) and 68% for simulation (ii). In the rules extracted, biological consensus motifs were important parts of the NN learning process in both simulations.     

Triplet entropy analysis of hemagglutinin and neuraminidase sequences measures influenza virus phylodynamics

The influenza virus has been a challenge to science due to its ability to withstand new environmental conditions. Taking into account the development of virus sequence databases, computational approaches can be helpful to understand virus behavior over time. Furthermore, they can suggest new directions to deal with influenza. This work presents triplet entropy analysis as a potential phylodynamic tool to quantify nucleotide organization of viral sequences. The application of this measure to segments of hemagglutinin (HA) and neuraminidase (NA) of H1N1 and H3N2 virus subtypes has shown some variability effects along timeline, inferring about virus evolution. Sequences were divided by year and compared for virus subtype (H1N1 and H3N2). The nonparametric Mann–Whitney test was used for comparison between groups. Results show that differentiation in entropy precedes differentiation in GC content for both groups. Considering the HA fragment, both triplet entropy as well as GC concentration show intersection in 2009, year of the recent pandemic. Some conclusions about possible flu evolutionary lines were drawn.