Microbial dynamics in industrial-scale wine fermentation employing Hanseniaspora uvarum ß-glucosidase-producer strain.

The use of non-Saccharomyces yeast strains in winemaking is becoming a common trend. In fact, consumers are demanding new and healthier styles of wine. On the other hand, these strains are a challenge for the starting process due to winery-resident strains, especially with regard to industrial-scale fermentations. Current assay focuses on the scale-up of the laboratorial inoculum inside the winery environment to ferment 15,000 and 25,000 L of Vitis labrusca Bordô must, using a Hanseniaspora uvarum ß-glucosidase-producer strain as starter culture. This scale-up could confirm the viability of using non-Saccharomyces yeast, as it presented promising results on a laboratory scale. The non-Saccharomyces strain was selected in a previous study since it proved to increase resveratrol concentration in lab scale winemaking. The yeast diversity was followed by the plate culturing method. Species identification and strain typing were determined by ITS-RFLP and PCR-fingerprinting, respectively. Physical and chemical analyses and resveratrol quantification were performed in the elaborated wines.

MALDI-TOF MS Supplementary database for species identification employing the yeast diversity encountered on southern Brazil grapes.

The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application.

Screening of whole yeast free-cells and optimization of pH and temperature for fructooligosaccharides production / Seleção de células inteiras de levedura e otimização dos parâmetros pH e temperatura para a produção de fruto-oligossacarídeos

Fructooligosaccharides are catalyzed by ß­fructofuranosidase enzyme, produced by many microorganisms. However, in order to achieve a more profitable, low time -consuming process with lower cost, researchers have sought alternatives. This study aimed to select and identify yeasts able to produce fructooligosaccharides and evaluate the influence of pH and temperature on their synthesis. Yeast suspensions, solutions of 500 g L-1 sucrose and three values of pH (4.5, 5.5, and 6.5) and temperature (40, 50, and 60ºC) were tested. Yeast species were identified by molecular techniques. Among 141 yeast isolates from grapes, 65 were able to synthesize fructooligosaccharides. The maximum concentration of fructooligosaccharides was 4.8% (w v-1), and Saccharomyces cerevisiae 222 produced 1-kestose and nystose.

Fruto-oligossacarídeos são catalisados pelas enzimas ß­fructofuranosidase, produzida por muitos micro -organismos. No entanto, para obter processos mais rentáveis, de menor custo e tempo, pesquisadores têm procurado alternativas. Este trabalho tem objetivo de selecionar e identificar leveduras capazes de produzir fruto-oligossacarídeos e avaliar a influência do pH e da temperatura na sua síntese. Suspensões de leveduras, soluções de sacarose de 500 g L- 1 e três valores de pH (4,5, 5,5 e 6,5) e de temperaturas de (40, 50 e 60ºC) foram utilizados. As espécies de leveduras foram identificadas por técnicas moleculares. De 141 isolados de leveduras de uvas, 65 foram capazes de sintetizar fruto-oligossacarídeos. A concentração máxima de fruto-oligossacarídeos foi de 4,8% (p v-1), e a levedura Saccharomyces cerevisiae 222 produziu 1-cestose e nistose.

Impact of Fungicide Residues on Polymerase Chain Reaction and on Yeast Metabolism

ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin "in natura" consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR) has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL).Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.

ATP-dependent potassium channels and type 2 diabetes mellitus.

Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

Composition of amino acids and bioactive amines in common wines of Brazil / Composição de aminoácidos e aminas bioativas em vinhos comuns brasileiros

Since most consumed wines in Brazil are common wines and since their representativeness is not accounted for in scientific research, current study quantifies bioactive amines and their precursors in Brazilian sweet and dry common wines, correlates the formation of amines with physical and chemical parameters and clusters studied areas by their amine and amino acid contents. Forty-seven wine samples varying in type, color and origin were analyzed simultaneously for seventeen amino acids, ammonium ion and five bioactive amines by reversed-phase high performance liquid chromatography and ultraviolet detection after the derivation phase. Physical and chemical analyses comprised titratable acidity, pH, organic acids, sugar and alcohol contents. Sweet wines had lower concentrations of amino acids and bioactive amines. Dry white wines had higher amino acid contents when compared to those in dry red wines. Since multivariate data analysis confirmed similarities between the studied regions, their unity as potential viniculture area was reinforced. Amine levels in Brazilian common wines were reported for the first time and results reinforced the importance of bioactive amines quantification and the use of suitable vinification practices to reduce their formation.

Considerando que os vinhos mais consumidos no Brasil são vinhos comuns e que sua representatividade não é demonstrada na pesquisa científica, o presente estudo tem por objetivo quantificar as aminas bioativas e seus precursores em vinhos comuns brasileiros suaves e secos, correlacionar a formação das aminas com parâmetros físico-químicos e agrupar as áreas estudadas por meio do seu conteúdo em aminas e aminoácidos. Foram analisadas 47 amostras de vinhos que variam quanto ao tipo, cor e origem; foram analisados, simultaneamente, 17 aminoácidos, íon amônio e cinco aminas bioativas por cromatografia líquida de fase reversa depois de uma etapa de derivatização. Análises físico-químicas incluíram acidez total, pH, ácidos orgânicos, açúcar e teor alcoólico. Os vinhos doces tiveram menor concentração de aminoácidos e aminas bioativas. Os vinhos brancos secos apresentaram maior conteúdo em aminoácidos quando comparados com os apresentados nos vinhos tintos secos. Uma vez que a análise multivariada confirmou similaridades entre as regiões estudadas, sua unidade como uma área potencial para a vinicultura foi reforçada. Os níveis de aminas em vinhos comuns brasileiros foram reportados pela primeira vez e os resultados reforçam a importância da quantificação de aminas bioativas e o uso de práticas apropriadas de vinificação para reduzir a sua formação.

Evaluation of MALDI-TOF mass spectrometry for identification of environmental yeasts and development of supplementary database.

Yeast identification using traditional methods which employ morphological, physiological, and biochemical characteristics can be considered a hard task as it requires experienced microbiologists and a rigorous control in culture conditions that could implicate in different outcomes. Considering clinical or industrial applications, the fast and accurate identification of microorganisms is a crescent demand. Hence, molecular biology approaches has been extensively used and, more recently, protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved to be an even more efficient tool for taxonomic purposes. Nonetheless, concerning to mass spectrometry, data available for the differentiation of yeast species for industrial purpose is limited and reference databases commercially available comprise almost exclusively clinical microorganisms. In this context, studies focusing on environmental isolates are required to extend the existing databases. The development of a supplementary database and the assessment of a commercial database for taxonomic identifications of environmental yeast are the aims of this study. We challenge MALDI-TOF MS to create protein profiles for 845 yeast strains isolated from grape must and 67.7 % of the strains were successfully identified according to previously available manufacturer database. The remaining 32.3 % strains were not identified due to the absence of a reference spectrum. After matching the correct taxon for these strains by using molecular biology approaches, the spectra concerning the missing species were added in a supplementary database. This new library was able to accurately predict unidentified species at first instance by MALDI-TOF MS, proving it is a powerful tool for the identification of environmental yeasts.

Morphological and molecular identification of filamentous fungi isolated from cosmetic powders

Seven fungi were isolated from 50 samples of cosmetic powders. Morphological analyses and ribosomal DNA Internal Transcribed Spacers sequencing were performed which allowed the discrimination of the isolated fungi as Aspergillus fumigatus, Penicillium sp., and Cladosporium sp. which could have, among their species, potentially pathogenic microorganisms.