Transcriptome profiling brings new insights into the ethanol stress responses of Spathaspora passalidarum.

Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: • Ethanol stress halts Spathaspora passalidarum metabolism and fermentation • Genes encoding nutrient transporters showed downregulation under ethanol stress • Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.

A unique effector secreted by Pseudozyma flocculosa mediates its biocontrol activity.

BACKGROUND: Pseudozyma flocculosa is a highly efficient biocontrol agent (BCA) of powdery mildews whose mode of action remains elusive. It is known to secrete unique effectors during its interaction with powdery mildews but effectors have never been shown to be part of the arsenal of a BCA. Here, we characterize the role of the effector Pf2826 released by Pseudozyma flocculosa during its tripartite interaction with barley and the pathogen fungus Blumeria graminis f. sp. hordei. RESULTS: We utilized CRISPR-Cas9-based genome editing and confirmed that secreted P. flocculosa effector Pf2826 is required for full biocontrol activity. We monitored the localization of the effector Pf2826 with C-terminal mCherry tag and found it localized around the haustoria and on powdery mildew spores. His-tagged Pf2826 recombinant protein was expressed, purified, and used as bait in a pull-down assay from total proteins extracted during the tripartite interaction. Potential interactors were identified by LC-MS/MS analysis after removing unspecific interactions found in the negative controls. A two-way yeast two-hybrid assay validated that Pf2826 interacted with barley pathogenesis-related (PR) proteins HvPR1a and chitinase and with an effector protein from powdery mildew. CONCLUSIONS: In contrast to the usual modes of action of competition, parasitism, and antibiosis ascribed to BCAs, this study shows that effector pf2826 plays a vital role in the biocontrol activity of P. flocculosa by interacting with plant PR proteins and a powdery mildew effector, altering the host-pathogen interaction.

Identification of the main proteins secreted by Kluyveromyces marxianus and their possible roles in antagonistic activity against fungi.

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing.

Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

Phakopsora pachyrhizi triggers the jasmonate signaling pathway during compatible interaction in soybean and GmbZIP89 plays a role of major component in the pathway.

The biotrophic fungus Phakopsora pachyrhizi is currently the major pathogen affecting soybean production worldwide. It has already been suggested for the non-host interaction between P. pachyrhizi and Arabidopsis thaliana that the fungus in early infection induces jasmonic acid (JA) pathway to the detriment of the salicylic acid (SA) pathway as a mechanism to the establishment of infection. In this study, we verified that this mechanism might also be occurring during the compatible interaction in soybean (Glycine max L. Merril). It was demonstrated that P. pachyrhizi triggers a JA pathway during the early and late stages of infection in a susceptible soybean cultivar. The expression of the GmbZIP89 was induced in a biphasic profile, similarly to other JA responsive genes, which indicates a new marker gene for this signaling pathway. Additionally, plants silenced for GmbZIP89 (iGmZIP89) by the virus-induced gene silencing (VIGS) approach present lower severity of infection and higher expression of pathogenesis related protein 1 (PR1). The lower disease severity showed that the iGmbZIP89 plants became more resistant to infection. These data corroborate the hypothesis that the GmbZIP89 may be a resistance negative regulator. In conclusion, we demonstrated that P. pachyrhizi mimics a necrotrophic fungus and activates the JA/ET pathway in soybean. It is possible to suppose that its direct penetration on epidermal cells or fungal effectors may modulate the expression of target genes aiming the activation of the JA pathway and inhibition of SA defense.

Assessment of the phenolic content, mutagenicity and genotoxicity of ethanolic extracts of stem bark and leaves from Strychnos pseudoquina A. St.-hil.

Strychnos pseudoquina is a plant species whose stem bark is used as bitter tonic beverage. The phytochemical analysis, as well as quantification of phenolic constituents and antioxidant activity of ethanolic extracts from S. pseudoquina stem bark, and leaves were conducted. The extracts were tested for mutagenicity (Ames test) and DNA-damaging activity (Plasmid Cleavage test). Leaves recorded the largest amount of flavonoids. The performed high-performance liquid chromatography (HPLC) showed flavonoids such as isorhamnetin and strychnobiflavone (phytochemical markers of the investigated species) in stem barks, but not in leaves. The proanthocyanidin content and antioxidant activity were significantly higher in stem barks than in leaves. Stem bark and leaf extracts presented mutagenic activity against TA98 and TA100 strains with, and without, metabolic activation (S9). The Plasmid Cleavage test did not indicate DNA-damaging activity. Our results suggest that extracts deriving from S. pseudoquina should be used with extreme caution, mainly the stem bark extract, which is widely used in folk medicine.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Antioxidant study indicative of antibacterial and antimutagenic activities of an ellagitannin-rich aqueous extract from the leaves of Miconia latecrenata.

Ethnopharmacological relevance; Several plant species of Miconia genus are commonly used in Brazilian folk medicine as anti-inflammatory agents and for the treatment of infectious diseases. Infusions and extracts of Miconia species are also reported as analgesic, antimicrobial, antimalarial, antioxidant, anti-inflammatory, antinociceptive, antimutagenic, and antitumoral. Aim of the study; To determine the phytochemical composition of an aqueous extract of Miconia latecrenata leaves and to evaluate its antioxidant, antibacterial, antimutagenic and antigenotoxic activities. Materials and Methods; The following methods were used for the different effects: I) antioxidant - ß-carotene/linoleic acid, lipid peroxidation, and DPPH• radical scavenging; II) antibacterial - agar well diffusion and MIC methods); III) antimutagenic assays - Ames Test; and IV) antigenotoxic - Plasmid cleavage test. The phytochemical analysis and phenolic quantification were carried out by UPLC-DAD-ESI-MS/MS and colorimetry, respectively. In addition, statistical correlation analysis was performed aiming to evaluate the Pearson correlation between phenolic compounds and biological assays. Results; A high content of tannins was observed and the ellagitannin isomers of 1,2,3,5-tris-galloyl-4,6-HHDP-glucose were identified as the main constituents of the leaves aqueous extract. High antioxidant effect, in different tests, high antibacterial activity to gram-positive and negative strains, as well as high antimutagenic activity were observed. Statistical analysis showed a high Pearson correlation for the tannin content in relation to the results of the antioxidant and antibacterial tests. In general, the antioxidant action of the aqueous extract showed low correlation with the antimutagenic activity. Conclusions; The present results confirmed the expectations regarding the pharmacological profile of M. latecrenata supporting its therapeutic potential in relation to ROS/RNS related disorders. Furthermore, the phenolic compounds of M. latecrenata can act, in turn, minimizing or inhibiting the biological macromolecules damage, especially DNA.

Genome-wide identification and expression analysis of dormancy-associated gene 1/auxin repressed protein (DRM1/ARP) gene family in Glycine max.

Dormancy-Associated gene 1/Auxin Repressed protein (DRM1/ARP) genes are responsive to hormones involved in defense response to biotic stress, such as salicylic acid (SA) and methyl jasmonate (MeJA), as well as to hormones that regulate plant growth and development, including auxins. These characteristics suggest that this gene family may be an important link between the response to pathogens and plant growth and development. In this investigation, the DRM1/ARP genes were identified in the genome of four legume species. The deduced proteins were separated into three distinct groups, according to their sequence conservation. The expression profile of soybean genes from each group was measured in different organs, after treatment with auxin and MeJA and in response to the nematode Meloidogyne javanica. The results demonstrated that this soybean gene family is predominantly expressed in root. The time auxin takes to alter DRM1/ARP expression suggests that these genes can be classified as a late response to auxin. Nevertheless, only the groups 1 and 3 are induced in roots infected by M. javanica and only group 3 is induced by MeJA, which indicates a high level of complexity in expression control mechanisms of DRM1/ARP family in soybean.

Phytochemical characterization and antioxidant, antibacterial and antimutagenic activities of aqueous extract from leaves of Alchornea glandulosa.

Plant extracts exist as a complex matrix which serves as a source of numerous bioactive metabolites. The ultra performance liquid chromatography with diode-array detection-coupled electrospray ionization-mass spectrometry/mass spectrometry technique was used to characterize the aqueous extract from leaves of Alchornea glandulosa (EAG), a species popularly used to treat gastrointestinal problems as an antiulcer agent. Quantification of phenolic derivatives was determined using Folin-Ciocalteu and aluminum trichloride (AlCl3) methods. In addition, antioxidant (2,2-diphenyl-1-picrylhydrazyl [DPPH•] radical scavenging, ß-carotene-linoleic acid, and lipid peroxidation), antibacterial (agar well diffusion method and minimum inhibitory concentration), antimutagenic (Ames test), and antigenotoxic (plasmid cleavage) assays were also performed on this plant extract. The ellagitannin tris-galloyl-hexahydroxydiphenic acid-glucose was identified as the predominant compound along with tannins as majority metabolites. EAG showed high antioxidant activity accompanied by moderate antibacterial activity against Staphylococcus aureus. The highest antimutagenic activity was observed for TA97 strain without metabolic activation (S9) and with metabolic activation, TA100 and TA102 were completely inhibited. In addition, EAG exhibited potential signs of antigenotoxic action. The high antioxidant and antimutagenic activity observed for EAG suggests important therapeutic uses that still need to be verified in future studies.

Mutagenic activity and chemical composition of phenolic-rich extracts of leaves from two species of Ficus medicinal plants.

Plant species from the Ficus genus are widely used as food, and in folk medicine as anti-inflammatory, antioxidant and anticancer agents, although some of these species are known to produce adverse effects. The aim of this study was to determine and compare the chemical composition as well as in vitro antioxidant and mutagenic activity of the aqueous extracts of leaves from F. adhatodifolia and F. obtusiuscula. Phytochemical screening using thin-layer chromatography identified 6 classes of secondary metabolites in the extracts. Total phenolic content was estimated by the Folin-Ciocalteau method and the phenolic profile was determined by UPLC-DAD-ESI/MS/MS. Antioxidant activities were evaluated by the DPPH radical assay and by the ß-carotene/linoleic acid system. Mutagenic activity was measured by the Salmonella typhimurium reverse mutation test with 4 strains, in both the presence and absence of metabolic activation. Flavonoids, coumarins, and tannins were detected in both extracts, and 6 major derivatives were identified as flavone compounds. Antioxidant activities were demonstrated for both extracts, while F. obtusiuscula contained higher concentrations of phenolic compounds. Mutagenic activity of the TA97 strain without metabolic activation was observed for both tested extracts, as well as the TA102 strain with metabolic activation. In addition, the extract of F. adhatodifolia was shown to be mutagenic to the TA102 strain without metabolic activation. Evidence indicates that the use of teas obtained from these two plant extracts in folk medicine may raise concerns and needs further investigation as a result of potential pro-oxidant mutagenic effects in the absence or presence of metabolic activation.

Identification of phenolic compounds and biologically related activities from Ocotea odorifera aqueous extract leaves.

Ocotea odorifera (Vell.) Rohwer is popularly used as food and flavoring. The aim of this study was to determine the chemical composition of the aqueous extract from O. odorifera leaves and evaluate the correlation of their phytochemical composition and biological activities. The antioxidant effect was determined by DPPH radical scavenging, ß-carotene-linoleic acid and lipid peroxidation assays; the antibacterial activity was evaluated by the hole plate and MIC techniques and the antimutagenic activity was evaluated by the Ames test. Identification of phytochemicals was performed by LC-ESI/MS and the correlation between the phytochemical composition of the extract and the evaluated activities. The results allowed the identification of 13 phenolic compounds in the extract that exhibited high antioxidant activity and moderate antibacterial and antimutagenic action. Statistical analyses showed correlation of the total phenolic content with biologically related activities. The phytochemical analyses, together with the biological results, support the popular use of O. odorifera.

Euterpe edulis Extract but Not Oil Enhances Antioxidant Defenses and Protects against Nonalcoholic Fatty Liver Disease Induced by a High-Fat Diet in Rats.

We investigated the effects of E. edulis bioproducts (lyophilized pulp [LEE], defatted lyophilized pulp [LDEE], and oil [EO]) on nonalcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) in rats. All products were chemically analyzed. In vivo, 42 rats were equally randomized into seven groups receiving standard diet, HFD alone or combined with EO, LEE, or LDEE. After NAFLD induction, LEE, LDEE, or EO was added to the animals' diet for 4 weeks. LEE was rich in polyunsaturated fatty acids. From LEE degreasing, LDEE presented higher levels of anthocyanins and antioxidant capacity in vitro. Dietary intake of LEE and especially LDEE, but not EO, attenuated diet-induced NAFLD, reducing inflammatory infiltrate, steatosis, and lipid peroxidation in liver tissue. Although both E. edulis bioproducts were not hepatotoxic, only LDEE presented sufficient benefits to treat NAFLD in rats, possibly by its low lipid content and high amount of phenols and anthocyanins.

Avaliação do potencial genotóxico de extratos etanólicos de cascas e folhas de Bathysa cuspidata (A. St-Hil) Hook / Evaluation of the genotoxic potential of ethanolic extracts of stem bark and leaves of Bathysa cuspidata (A. St-Hil) Hook

Bathysa cuspidata (A.St.-Hil.) Hook. is a native tree of the Brazilian Atlantic Forest biome, widely used in Brazilian herbal medicine. Despite its widespread use, there is no report as yet regarding the toxicology of this plant. In this study, the mutagenic and genotoxic effects of ethanolic extracts of B. cuspidata stem bark (SBC) and leaves (LBC) were assessed. The mutagenicity of the extracts was estimated by performing the Ames test on strains TA98 and TA100 of Salmonella typhimurium, in the absence and presence of metabolic activation. The direct action of the extracts on DNA was assessed through plasmid treatment. Phytochemical screening was conducted to compare the secondary metabolite composition of the extracts. No mutagenic activity was found in LBC when tested on strain TA98 or TA100, without or with metabolic activation. However, SBC did show mutagenic activity. Plasmid tests did not indicate genotoxic action for either SBC or LBC. Differences in the composition of secondary metabolites present in the bark and leaves, detected by phytochemical analysis, appear to be a deciding factor in differences in mutagenicity between SBC and LBC. The findings in this study suggest caution in the use of B. cuspidata bark.

Bathysa cuspidata (A.St.-Hil.) Hook. é uma árvore nativa do bioma Mata Atlântica largamente utilizada na medicina popular brasileira. Apesar do amplo uso, ainda não existe relato sobre a toxicologia dos compostos obtidos dessa espécie vegetal. O presente estudo avaliou efeitos mutagênicos e genotóxicos de extratos etanólicos de B. cuspidata obtidos das cascas do caule (SBC) e das folhas (LBC). A mutagenicidade dos extratos foi avaliada utilizando o teste de Ames nas linhagens TA98 e TA100 de Salmonella typhimurium, na ausência e presença de ativação metabólica. Os efeitos dos extratos diretamente sobre o DNA foram avaliados por clivagem plasmidial. Análises fitoquímicas foram realizadas para comparar a composição de metabólitos secundários dos extratos. Não foi encontrada atividade mutagênica para LBC quando avaliados utilizando as cepas TA98 e TA100, sem ou com ativação metabólica. No entanto, SBC apresentou atividade mutagênica. Clivagem plasmidial não indicou ação genotóxica tanto para SBC quanto para LBC. Diferenças na composição dos metabólitos secundários presentes nos cascas e folhas, detectadas pelas análises fitoquímicas, devem ser determinantes para a variação do efeito mutagênico entre SBC e LBC. Os resultados sugerem cautela no emprego de cascas de B. cuspidata.

Staphylococcus aureus of bovine origin: genetic diversity, prevalence and the expression of adhesin-encoding genes.

Staphylococcus aureus is a well-armed pathogen that is a leading cause of bovine mastitis. Attempts to define a set of bacterial proteins that are crucial for infection have failed. The identification of these proteins is important to define biomarkers that can be used for diagnostic purposes and to identify potential vaccine targets. In this study, seven genes that encode virulence factors were analyzed in 85 bacterial isolates that were derived from animals with bovine mastitis. The clfB, spa, sdrCDE and fnBP genes were detected in 91.8%, 85.9%, 85.9% and 63.5% of the isolates, respectively. At least one gene was present in all of the strains, while the most prevalent combination was clfB and sdrCDE (82.4%). The genetic diversity of the isolates was high and allowed for clustering into more than 40 groups, with each group containing bacteria collected from different locations. The gene expression of the four most prevalent adhesins was examined in nine genetically distinct strains. No common pattern of expression was observed for the genes, suggesting that the capacity of S. aureus to cause infection may rely on differential expression of the virulence factors in different isolates. Our results conclude that using only one antigen is unlikely to provide effective protection against bovine mastitis and suggest that a combination of at least three adhesins may be more suitable for developing preventive therapies. We also conclude that the characterization of isolates distributed worldwide is necessary to improve our understanding of pathogenesis in the natural populations of S. aureus.

The mutagenic, DNA-damaging and antioxidative properties of bark and leaf extracts from Coutarea hexandra (Jacq.) K. Schum.

Coutarea hexandra is a species commonly known in Brazil as quina, and its bark is used in folk medicine. In this study, we assess the mutagenic and DNA-damaging effects of ethanol extracts from C. hexandra stem bark (SCH) and leaves (LCH) by employing the Ames test on the TA98 and TA100 strains of Salmonella typhimurium in addition to a plasmid treatment test. Furthermore, we performed a phytochemical analysis by TLC and HPLC, a quantification of the phenolic constituents and an assessment of the antioxidative activity. SCH and LCH showed mutagenic action in the Ames test for TA98 strains after metabolic activation. LCH also showed mutagenicity for the TA100 strain after metabolic activation. The findings from the plasmid treatment test did not indicate any DNA-damaging activity for either of the extracts with the tested dosages. SCH showed greater flavonoid content and greater antioxidative potential in relation to LCH. This study suggests that caution is advisable in the use of this plant. However, in vivo studies should be conducted to confirm these data.

Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations.

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5',5″-trifluor-D: ,L: -leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. "Cachaça" produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in "cachaça" production.

Protective effect of ions against cell death induced by acid stress in Saccharomyces.

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na(+) in yeast viability in acidic conditions was tested using S. cerevisiae Na(+)-ATPases (ena1-4), Na(+)/H(+) antiporter (nha1Delta) and Na(+)/H(+) antiporter prevacuolar (nhx1Delta) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.

Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production.

Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5',5"-trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.