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Monitoring of wine yeasts population during fermentation of wine cider
Krätschmerová, Kateřina ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This thesis deals with identification of wine yeasts isolated during fermentation process of wine cider and grapes of Sauvignon grape variety, grown in the integrated vineyard. The identification and taxonomic classification is faster and easier due to the progress of molecular methods. In this thesis PCR-RFLP method was used for identification of yeasts. Sequences of DNA specific for each species were analysed. These sequences were amplified by means of PCR method and by using ITS1 and ITS4 primers. In following step, they were put through the restrictiction analysis with five restriction endonucleases. Fragments of DNA were separated by horizontal electrophoresis. The electrophoreograms were evaluated by BioNumerics software and final dendrogram representing genetics similarity of isolated yeasts was created by using UPGMA claster analysis. The basic information about yeasts and their identification by molecular methods are described in the theoretical part of this thesis.
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Identification of wine yeasts by PCR-RFLP method
Šuranská, Hana ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This thesis deals with identification of the wine yeasts by applying the PCR-RFLP method. The identification and characteristic of the yeasts has gone through substantial changes in recent years. There have been introduced new methods of taxonomic classifying based on the molecular methods, which are oriented to easy and fast identification. One of these methods is the PCR-RFLP method. The amplification of the 5•8S-ITS rDNA sequence by the polymerase chain reaction with use of the primers ITS1 and ITS4 leads to the amplification of the specific sequence of DNA. Such multiplied DNA is after repurifying by the ethanol and drying submitted to the restriction analysis. With use of the restriction endonuklases DNA is chopped into the specific segments typical for the particular genus. The chopped fragments can be separated in the electric field in the agarose gel and subsequently evaluated. In this thesis together 63 type yeasts were used. These yeasts were analysed by applying of the seven restriction endonuklases – HaeIII, HhaI, HinfI, HpaII, TaqI, AluI a MseI. The final image of type yeasts splitting was compared to the results of splitting of already identified wine yeasts and these yeasts were subsequently taxonomically classified. Evaluation of genetic similarity was conducted by program BioNumerics and as the results the dendrograms that were created with use of Jaccard‘s coefficients are obtained.
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PRODUCTION OF BETA-GLUCANS AND OTHER POLYSACCHARIDES BY YEAST AND MICROALGAE
Byrtusová, Dana ; Kráčmar, Stanislav (referee) ; Kovalčík, Adriána (referee) ; Márová, Ivana (advisor)
Beta-glukany jsou polysacharidy složeny z monomerů D-glukózy. V dnešní době se -glukany těší zvýšené pozornosti zejména kvůli imunomodulační aktivitě a využitelnosti ve farmaceutickém a potravinařském průmyslu. Saccharomyces cerevisiae je dodnes jediným kvasinkovým zdrojem požívaným v biotechnologické produkci. Avšak některé kvasinky z oddělení Basidiomycetes, které jsou schopny produkce lipidů a karotenoidů, mohou být využity rovněž jako alternativní zdroj -glukanů. Dizertační práce se zabývá možností a optimalizací produkce -glukanů a dalších mikrobiálních sacharidů u karotenogenních kvasinek a mikrořas. Testovány byli zástupci rodů Rhodotorula, Sporobolomyces, Cystofilobasidium a Dioshegia. Z nekarotenogenních kvasinek byly do screeningu zařazeny kvasinky rodu Metschnikowia, askomycetní kvasinky a z mikrořas zástupci zelených a červených řas. Experimentální část cílí rovněž na možnosti koprodukce dalších metabolitů, jako jsou lipidy, pigmenty a extracelulární polymery. První část experimentu se zabývá vlivem čtyř C/N poměrů (10:1, 40:1, 70:1 a 100:1) na produkci biomasy, -glukanů, karotenoidů a lipidů. Ze všech testovaných kmenů, S. cerevisiae CCY 21-4-102, C. infirmominiatum CCY 17-18-4, P. rhodozyma CCY 77-1-1 a R. kratochvilovae CCY 20-2-26 vykazovaly nejvyšší produkci -glukanů a byly proto vybrány k podrobnější optimalizaci, zejména osmotického stresu, teploty a zdroje dusíku v kultivačním médiu. Dodatečně, kmen R. kratochvilovae CCY 20-2-26 je schopný produkce extracelulárních glykolipidů a S. pararoseus CCY 19-9-6 extracelulárních polysacharidů. Následně bylo stanoveno množství -glukanů u dalších dvanácti kmenů S. cerevisiae a rovněž možnost produkce polysacharidů u mikrořas.
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Speciation analysis of selenium in selenized yeast
Motlová, Tereza ; Vitoulová, Eva (referee) ; Diviš, Pavel (advisor)
The aim of the theses was determination of selenium species in yeast Saccharomyces cerevisiae cultivated in medium with added inorganic form of selenium (Sodium Selenite). Concentrations of Sodium Selenite in cultivation medium were 0,1; 1; 10 and 100 mg.l-1. Cultivation was undertaken in fermenting tub for period of 72 hours. Cultivated yeasts were extracted by use of enzymes and subsequently the species of selenium in particular parts of yeasts were determined. In order to determine selenium species, the method of high-performance liquid chromatography in combination with atomic fluorescent spectrometer and technique of hydride generation was used. Having analysed different fractions of the yeasts Saccharomyces cerevisiae it was ascertained that during cultivation the sorption of selenium occurred in form of Se4+ in cell membranes while in cytoplasm no inorganic forms of selenium were found. Furthermore, it was stated that yeasts Saccharomyces cerevisiae are able to metabolically change inorganic forms of selenium to organic forms (selenomethionine), while these forms are present in cytoplasm and they are likely to be bound to proteinic structures of cell membranes. An increase of concentration of Se4+ in cell membranes could be observed as a result of increasing concentration of Sodium Selenite in cultivation medium. In proteinic structures the concentration of organic selenium forms increased only to concentration 10 mg.l-1 of Sodium Selenite in cultivation medium.
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Monitoring of the influence of indigenous culture of yeasts on the fermentation process of making wine
Michálek, Petr ; Molnárová,, Jana (referee) ; Vránová, Dana (advisor)
This thesis deals with the identification of wine yeasts isolated from the grape must using PCR-RFLP method. The yeasts were isolated from Pinot Noir grape variety must. Grapes were grown and produced in accordance with the requirements placed on organic and integrated farming. Samples were processed in the laboratory, where pure cultures of individual yeast were obtained. A commercial kit was used for yeast DNA isolation. Obtained DNA was used for further analysis. Using the polymerase chain reaction and the primers ITS1 and ITS4 a specific segment of 5.8S rDNA-ITS region was amplified. The PCR products were then detected by electrophoresis in an agarose gel, and after a subsequent purification, three restriction enzymes: HaeIII, HinfI and HhaI were subjected to restriction analysis. The DNA was digested to fragments specific for yeast species and they were detected by agarose electrophoresis. Similarity of these isolates was compared using BioNumerics program and the result is dendrogram of genetic similarity of isolated yeast. The basic chemical analysis of samples must was also performed.
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Monitoring of the influence of commercial culture of yeasts on fermentation wine making process
Šerý, Filip ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This diploma thesis focuses on isolation and taxonomic classification of yeast species isolated during the red wine (Pinot noir) fermentation. Grapes were grown under organic and integrated farming in South Moravia wine region, Czech Republic. Processing was controlled – for inoculation was used strain Saccharomyces cerevisiae BS6. Polymerase chain reaction followed by restriction fragment lenght polymorphism of PCR-amplified fragments (PCR-RFLP) was used for yeast species identification. For DNA analysis we used coding region of 5.8S ITS rDNA which was amplified using ITS1-ITS4 primers. Amplicon was digested by three restriction endonucleases - HaeIII, HinfI and HhaI. Isolates were divided into eleven groups using UPGMA cluster analysis (software BioNumerics). We identified following yeast species: Candida valida, Candida vini, Issatchenkia occidentalis, Pichia fermentans, Saccharomyces cerevisiae and Zygosaccharomyces bailii. We were not able to identify some yeast species. Differences between organic and integrated farming were demonstrated with varying composition of yeast species.
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Influence of grape growing methods on yeasts community
Jiříková, Ivana ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This diploma thesis has analyzed the effect of organic wine-growing on the wine yeasts population. The wine yeasts were isolated from the Pinot Noir variety. They were identified by the molecular biological method PCR-RFLP. The theoretical research compiles basic information on yeasts, knowledge about the red wine production as well as information on molecular biological methods. The experimental part utilizes the 5,8S-ITS rDNA specific segment for analysis. The segment was amplified using the ITS1 and ITS4 primers and subjected to restriction analysis. The restriction analysis has used these restriction endonucleases - HaeIII, HinfI, Taq?I, AluI and MseI. The BioNumerics software was then used to compare genetic similarity between the isolated yeasts and these were taxonomically classified.
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Raw materials and microorganisms to distilled beverage production
Jechová, Iva ; Vitoulová, Eva (referee) ; Babák, Libor (advisor)
The production of distilled ranks among established fermentative production. Than the first – quality spirits is became from raw material, it is obliged to change ranks processes. The important from the processes are alcoholic fermentation, which proceeds owing to mikrobiological cultures, distillation and subsequent rectification and at not least ranks aging distilled, for which is necessary selected appropriate container. For all processes the granted conditions are appointed, under which the processes would ocur, so resulting distilled achieved the first – quality and the best sensory character. Therefore this work does one’s best map these single processes and it is largely intent on fermentative processes.
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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 changes of yeasts population in the production of red wine
Ducháč, Petr ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
The aim of this diploma thesis is the identification of yeasts isolated during grape must fermentation. The must was obtained from Pinot Noir varieties grown in an integrated and organic production. The partner of this thesis was a winery Holánek. In the theoretical part of the work was the emphasis on information about the determinants quality of wine, yeasts and PCR-RFLP method. Physiological properties of yeasts were described and also the principles of the polymerase chain reaction (PCR) were explained. In the experimental part of the thesis was applied molecular biology method PCR-RFLP for identification of yeasts. The specific segment of DNA was amplified (5, 8S-ITS rDNA sequencing) with the help of ITS1 and ITS4 primers. The incurred amplicons were digested by applying restriction endonucleases: HaeIII, HinfI and TaqI. Subsequently the restriction fragments were analysed by using of electrophoresis. The yeasts were identified and classified by taxonomy on the level of genera and species.
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