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Yeasts and wine
Palíková, Petra ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with isolation and identification wine yeasts from grapes and must. For analysis was used white wine Sauvignon that was grown and producing after needs ecological agriculture. Remove samples were processed in laboratory and by the help of dilution method were obtained pure culture isolated yeasts. In the following step, by the application of commercial kit UltraCleanTM Microbial DNA Isolation Kit we were able to isolated individual DNA that it was used to the next analysis. Isolated DNA was amplification by PCR method with ITS1 and ITS4 primers. PCR products were detected on agarose gel. Amplification samples were chopped five restriction endonucleases: HaeIII, HinfI, TaqaI, AluI and MseI. Chopped DNA was detected by the same way as PCR products and it was compared with restriction patterns of collection yeasts. In the next step it was compared genetic similarity of isolated yeasts by using BioNumerics software. As a criterion it was used Pearson coefficients and UPGMA clastering analysis. The result is dedrogram of genetics similarity isolated yeasts.
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Effect of culture medium on the identification of yeasts of the genus Cryptococcus using mass spectrometry
Jurnečková, Alena ; Vadkertiová, Renata (referee) ; Stratilová, Eva (advisor)
Cryptococccus genus is known for its difficult identification and taxonomical classification in area of clinical microbiology. For this bachelor thesis, total 22 yeast strains of the Cryptococcus genus were chosen. The part of strains was firstly analyzed by D1/D2 domain LSU rRNA gene analysis. Three types of culture medium – YPD, potato dextrose agar and Sabouraud’s medium were selected for cultivation. Samples were prepared according to standardized method of Bruker Daltonik company, Institute of Chemistry of SAS and combination of these two methods. Identification was done by MALDI-TOF mass spectrometry. Obtained spectra were compared using corresponding software and evaluated on the basis of specific algorithm. The most advantageous culture medium for cultivation and biotyping with the largest percentage score was YPD (Yeasts pepton dextrose). On the other hand, the least advantageous culture medium was Sabouraud’s agar, which reached the smallest percentage success due to parameters of Bruker Daltonik algorithm. The most succesfull method of sample preparation and application was the combined method with YPD as a culture medium. The results of complete analysis are dendrograms for each medium showing the genetic similarity of yeast strains. The dendrogram shows categorization of the single strains into appropriate groups. Cr. flavescens (CCY 17-3-28, CCY 17-3-30) and Cr. laurentii (CCY 17-3-2) strains were correctly integrated into phylogenetic group Cr. laurentii I (branch in the dendrogram with designation A). Cr. flavus (CCY 17-3-5) doesn’t belong to this group, although it shows similarity with Cr. flavescens. The strains Cr. carnescens (CCY 17-3-13) and Cr. victoriae (CCY 17-3-26) belong to the phylogenetic group Cr. laurentii II (designated as B). Cr. magnus (CCY 17-4-39, CCY 17-4-40) strains show similarity with these strains, but doesn’t belong to the phylogenetic group II. The strains Cr. gastricus (CCY 17-5-1) and Cr. diffluens (non-attached) form a branch designated as C. Cr. aerius (CCY 17-4-9) strain, which was also put into this group, was proposed to sequence analysis, because its spectrum indicates that it should be rather a Cr. diffluens strain. The group D contains Bulleromyces albus (CCY 17-3-35, CCY 17-3-36) and Cr. saitoi (CCY 17-3-18, CCY 17-4-2) strains. The sequenced Cr. albidus (CCY 17-4-1), non-sequenced Cr. diffluens (CCY 17-4-13) and Cr. terreus (CCY 17-8-1) form the group E. The strain CCY 17-4-13 was proposed to sequence analysis because of occurrence of the Cr. diffluens sequenced strain in the group C. The sequenced Cr. aerius (CCY 17-25-1) is also part of this group, but it represents a separate branch. The last group is named as F and consists of Cr. macerans (CCY 17-19-3) and control strains Cr. neoformans var. neoformans (CCY 17-1-4, CCY 17-1-5).
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Optimalization of PCR-RFLP method for taxonomy of yeasts
Olivová, Radana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deal with optimalization method PCR – RFLP for taxonomy enlistment of yeasts. Conventional identification methods for yeasts are time-consuming. Molecular biological method based on PCR are instrumental towards fast and precise identification as compared to conventional phenotypic methods. In this thesis molecular biological method PCR – RFLP was used for identification and enlistment of yeasts. This metod follow repeating spacers of ribozomal DNA of yeast, characteristic for each species and strain. By the help of PCR were amplified specific partitions of DNA. These fragments of DNA were split by restriction endonucleases and identified by horizontal electroforesis. In background of this thesis there are information about yeasts, their taxonomy and molecular biological methods.
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Monitoring of the influence of using indigenous yeasts for wine production in the conditions of winery
Beníčková, Romana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with identification of yeasts by applying the RFLP-PCR method. Objective of the thesis was to identify the yeasts present in wine from Grüner Veltliner during fermentation. Identification was made by amplification of 5,8S-ITS sequences of DNA by the polymerase chain reaction with primers ITS1 and ITS4. Amplified DNA was submitted to the restriction analysis by restriction endonuclease HaeIII, HinfI and HhaI. By restriction analysis with a specific enzyme, the amplified DNA is chopped into the specific fragments which are characteristic for given kind of yeasts. In the analysed wine, the dominance of autochthonal yeast Saccharomyces cerevisiae was confirmed throughout fermentation. The other identified yeasts in the wine were of kind Pichia. The second part of the thesis was to expand the database by characterization of 28 type-yeasts, using RFLP-PCR analysis. To compare the genetic similarity, program BioNumerics was used, which processed the results of UPGMA cluster analysis using Jaccard´s coefficients.
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Isolation and characterization of autochthonous yeasts from interspecific varieties of grapes
Dlapalová, Kristýna ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
The aim of this thesis is the isolation and identification of yeasts obtained from the wine berries and the characterization of the collection yeast by using processes of PCR - RFLP. The type yeasts were obtained from the collection of yeasts of CCY in Bratislava, yeasts from the wine berries were collected from the species of Hibernal wine from the wineries of Štěpán Maňák. Identification of individual yeast is then based on analysis of the DNA segment in the area of 5,8S - ITS using primers ITS1 and ITS4. The restriction analysis was performed using restriction endonucleases HaeIII, HinfI, HhaI a TaqI(a). Restriction analysis is used to chopp the DNA to specific sections that are characteristic for each microorganism. For the assesment of the genetic similarity analyzed yeasts the BioNumerics software has been used. BioNumerics processes the results using cluster analysis using Jaccard´s coefficients.
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Taxonomy of yeasts of the genus Saccharomyces
Augustová, Kamila ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.
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Isolation and characterization of autochthonous yeasts from interspecific varieties of grapes
Dlapalová, Kristýna ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
The aim of this thesis is the isolation and identification of yeasts obtained from the wine berries and the characterization of the collection yeast by using processes of PCR - RFLP. The type yeasts were obtained from the collection of yeasts of CCY in Bratislava, yeasts from the wine berries were collected from the species of Hibernal wine from the wineries of Štěpán Maňák. Identification of individual yeast is then based on analysis of the DNA segment in the area of 5,8S - ITS using primers ITS1 and ITS4. The restriction analysis was performed using restriction endonucleases HaeIII, HinfI, HhaI a TaqI(a). Restriction analysis is used to chopp the DNA to specific sections that are characteristic for each microorganism. For the assesment of the genetic similarity analyzed yeasts the BioNumerics software has been used. BioNumerics processes the results using cluster analysis using Jaccard´s coefficients.
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Monitoring of the influence of using indigenous yeasts for wine production in the conditions of winery
Beníčková, Romana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with identification of yeasts by applying the RFLP-PCR method. Objective of the thesis was to identify the yeasts present in wine from Grüner Veltliner during fermentation. Identification was made by amplification of 5,8S-ITS sequences of DNA by the polymerase chain reaction with primers ITS1 and ITS4. Amplified DNA was submitted to the restriction analysis by restriction endonuclease HaeIII, HinfI and HhaI. By restriction analysis with a specific enzyme, the amplified DNA is chopped into the specific fragments which are characteristic for given kind of yeasts. In the analysed wine, the dominance of autochthonal yeast Saccharomyces cerevisiae was confirmed throughout fermentation. The other identified yeasts in the wine were of kind Pichia. The second part of the thesis was to expand the database by characterization of 28 type-yeasts, using RFLP-PCR analysis. To compare the genetic similarity, program BioNumerics was used, which processed the results of UPGMA cluster analysis using Jaccard´s coefficients.
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Taxonomy of yeasts of the genus Saccharomyces
Augustová, Kamila ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.
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Yeasts and wine
Palíková, Petra ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with isolation and identification wine yeasts from grapes and must. For analysis was used white wine Sauvignon that was grown and producing after needs ecological agriculture. Remove samples were processed in laboratory and by the help of dilution method were obtained pure culture isolated yeasts. In the following step, by the application of commercial kit UltraCleanTM Microbial DNA Isolation Kit we were able to isolated individual DNA that it was used to the next analysis. Isolated DNA was amplification by PCR method with ITS1 and ITS4 primers. PCR products were detected on agarose gel. Amplification samples were chopped five restriction endonucleases: HaeIII, HinfI, TaqaI, AluI and MseI. Chopped DNA was detected by the same way as PCR products and it was compared with restriction patterns of collection yeasts. In the next step it was compared genetic similarity of isolated yeasts by using BioNumerics software. As a criterion it was used Pearson coefficients and UPGMA clastering analysis. The result is dedrogram of genetics similarity isolated yeasts.
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