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1.
Curr Microbiol ; 58(1): 58-63, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18941833

RESUMO

In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism (RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.


Assuntos
Actinas/genética , Impressões Digitais de DNA/métodos , Microbiologia de Alimentos , Técnicas de Tipagem Micológica , Reação em Cadeia da Polimerase/métodos , Saccharomyces/classificação , Saccharomyces/genética , Primers do DNA/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Desnaturação de Ácido Nucleico , Filogenia , Temperatura de Transição
2.
Int J Food Microbiol ; 102(2): 173-83, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15992616

RESUMO

A pectinolytic industrial yeast strain of Saccharomyces cerevisiae was generated containing the S. cerevisiae endopolygalacturonase gene (PGU1) constitutively expressed under the control of the 3-phosphoglycerate kinase gene (PGK1) promoter. The new strain contains DNA derived exclusively from yeast and expresses a high polygalacturonic acid hydrolyzing activity. Yeast transformation was carried out by an integrative process targeting a dispensable upstream region of the acetolactate synthase locus (ILV2), which determines sulfometuron methyl resistance. Microvinification assays were performed on white and red musts with the transformed UCLMS-1M strain and with the same strain untransformed. It was found that the changes in the pectic polysaccharide contents did not directly affect the taste or flavor of the wine. From the data reported, it is deduced that the chief advantage of using the modified strain is that it improves the yield of must/wine extraction, while it also positively affects some variables relating to appearance.


Assuntos
DNA Fúngico/genética , Microbiologia Industrial , Poligalacturonase/metabolismo , Saccharomyces cerevisiae/genética , Vinho/microbiologia , Fermentação , Tecnologia de Alimentos , Poligalacturonase/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Transformação Genética
3.
FEMS Microbiol Lett ; 237(2): 261-6, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15321671

RESUMO

A total of 61 S. cerevisiae strains, 60 of them isolated from wine ecosystems, were evaluated for the presence of the gene encoding endopolygalacturonase (PGU1) and for polygalacturonase (PG) activity. Nine strains lack the gene PGU1 and did not exhibit PG activity on plate assays. Of the 52 strains showing an amplified band corresponding to the size of PGU1 gene, only 36 degraded polygalacturonic acid (PGA) and 17 did not degrade it at any of the pH values used. The coding region of the PGU1 gene (ORF YJR153w) was not present in some PG activity negative strains. The S. cerevisiae UCLMS-39 strain was selected for its specific activity at different pHs, temperatures and oenological parameters. The temperature and pH optima were 50 degrees C and 3.5-5.5 respectively and it was only affected by ethanol. The PGU1 gene was cloned and sequenced. The production of a biologically functional endoPG in S. cerevisiae UCLMS-39 brings us a step closer to improving the qualities of outstanding enological yeasts naturally lacking PG activity.


Assuntos
Poligalacturonase/metabolismo , Saccharomyces cerevisiae/enzimologia , Vinho/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Pectinas/metabolismo , Poligalacturonase/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética
4.
Syst Appl Microbiol ; 24(4): 634-8, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11876371

RESUMO

Yeasts isolated from must before and during fermentation at a wine cellar of La Mancha region in Spain were characterised using Polymerase Chain Reaction / Restriction Fragments Lengths Polymorphism and Polymerase Chain Reaction / Temporal Temperature Gradient Gel Electrophoresis. S. cerevisiae strains were differentiated using mtDNA restriction analysis. Direct PCR-TTGE was also used to study biodiversity during wine fermentation, and revealed the variations in the population. It was observed that isolation by conventional plating may afford a skewed view of the strains taking part in wine fermentation.


Assuntos
Saccharomyces/isolamento & purificação , Vinho/microbiologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Eletroforese em Gel de Ágar , Fermentação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Saccharomyces/genética , Espanha
5.
Int J Food Microbiol ; 59(1-2): 29-36, 2000 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-10946836

RESUMO

Enzymatic activity of potential interest in wine-making was studied for 182 non-Saccharomyces yeasts isolated from musts before and at the onset of fermentation at wine cellars operating under the La Mancha Appellation of Origin in Spain. Tests were carried out on plates containing differential substrates appropriate for each case (casein, gelatin, polygalacturonic acid, and arbutin) to determine whether each of the isolates exhibited proteolytic, polygalacturonase, and beta-glucosidase activities. Nearly 80% of the wild yeasts possessed one or more enzymes of biotechnological interest. Once the enzymatic activities of the isolates had been established, 69 of the isolates that exhibited pronounced enzymatic activity and 11 randomly selected isolates that were devoid of any activity were typed using PCR/RFLP, which gave 13 different molecular profiles. The isolates for each of the profiles were then identified by classical methods. The enzyme beta-glucosidase was linked to the species Metschnikowia pulcherrima, and polygalacturonase activity was common in most of the species identified. Proteolytic activity was observed in Pichia membranifaciens and in Metschnikowia pulcherrima. Typing revealed the possibility of intraspecific differences in Pichia membranifaciens, because six different molecular profiles with one or more shared restriction bands were recorded for that species.


Assuntos
Vinho/microbiologia , Leveduras/classificação , Leveduras/enzimologia , Análise por Conglomerados , Meios de Cultura , DNA Fúngico/análise , Endopeptidases/análise , Técnicas de Tipagem Micológica , Poligalacturonase/análise , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Espanha , Leveduras/genética , Leveduras/isolamento & purificação , beta-Glucosidase/análise
6.
Int J Food Microbiol ; 28(3): 369-77, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8652345

RESUMO

Pulsed field electrophoresis is a suitable technique for differentiation of Saccharomyces cerevisiae strains. In this work, karyotype analysis was used to study the ecology of the wild S. Cerevisiae flora in musts in fermentation in the A.C. (Appellation Controlee) area of Valdepeñas (Spain). In order to do this, 392 colonies isolated from different vats in different cellars, where dry yeast was never used to make wines, were submitted to the Countour Homogeneous Electric Field (CHEF) technique. Each of the resulting CHEF profiles was subjected to Cluster Analysis and four main karyotypes were found in this viticultural area.


Assuntos
Fermentação , Frutas/microbiologia , Saccharomyces cerevisiae/classificação , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Ligação Genética , Variação Genética , Cariotipagem , Saccharomyces cerevisiae/genética
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