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DNA isolation from probiotic lactic acid bacteria in food additives
Tvrdíková, Jana ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
In this work the functionalised magnetic particles were tested with streptavidin to selective DNA isolation. The method of selective DNA isolation was tested by using DNA probiotic strain Lactobacillus paracasei subsp. paracasei CCDM 211/06. A test was done on the biotinyl oligonucleotic particles, which was immobilised by containing streptavidin and it was used like a DNA probe for isolation complementary DNA chain by means of DNA/DNA hybridization. The primer R 5´ bio and the biotinyl denatured specific PCR product were tested for species Lb. paracasei as a DNA probe. These following experimental conditions were optimized for selective DNA isolation: temperature and time of hybridization, amount of DNA and the release of DNA from microspheres. Isolation of DNA was verified by PCR with specific generic primers. The specific generic PCR product was amplified in extent 250 bp, which was detected by using electrophoresis in agarose gel. This optimized method was successfully used in selective isolation of DNA Lactobacillus from a complementary sample of supplementary food (BIFI pangamin).
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Preparation of PCR-ready DNA
Čuta, Robert ; Vojtíšková, Marie (referee) ; Rittich, Bohuslav (advisor)
In these days are probiotic lactic acid bacteria (LAB) very often used in food processing industry, such as milk products, cheese and fermented salami production. As well as the food conservation agent. Except of the industry usage of the LAB there are microbiological aspects. Identification of the bacterial species methods based on the isolation and amplification of DNA are very often used last few years. Diploma thesis deal with the bacterial cell of Lactobacillus species lysis and it´s optimalization. At first it was tested the optimal concentration of lysozyme (3mg/ml, 5mg/ml, 10mg/ml) and the exposure times (3, 5 a 10 hours). Another testing was aimed to the use and suitability of washing powder to the bacterial cell of Lactobacillus species lysis. I tested the Amway washing powder optimal concentration (1%, 2%, 3% a 4%). Four of another comercial washing powders were tested too. All these tests were performed at the pure Lactobacillus bacterial culture. To ensure the results I tested the washing powders at the real food matrix (Acidified milk, yogurt mango, yogurt white). All the methods were evaluated at the amplification method PCR with specific primers for the Lactobacillus genus. The DNA isolation was performed with the paramagnetic microsperes P(HEMA-co-GMA) and the amount of the DNA was quantified spectrophotometrically. The PCR products detection was performed with the agarose gel electrophoresis.
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Yeasts colonizing the leaf surfaces and their identification
Bělochová, Kamila ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This diploma thesis is focused on optimalization and employing the PCR-RFLP method, based on the molecular biology principles, for an identification and taxonomy of the yeasts which colonize the leaf surfaces. Simultaneously the yeasts identification techniques based on physiological and morfphological attributes are compared and replaced. PCR-RFLP takes advantage of thermostable polymerases´ ability to amplify the specific segment in the rDNA, which can be split by restriction endonucleases to characteristical polymorphical fragments. Comparing these fragments and restriction´s positions which are for each species unique, demanded results were obtained. They´re summarized in the conclusion part. The theoretical part describes the morphology and cytology of the yeasts, taxonomy as a science, genuses of examined yeasts Cryptococcus, Rhodotorula a Saccharomyces are covered more thoroughly and the method of PCR-RFLP is described in detail.
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Identification of selected genes in lactic acid bacteria
Kristová, Mária ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
Lactic acid bacteria are natural habitants of human gastrointectinal tract. Among the most important are bacteria of genus Lactobacillus and genus Bifidobacterium that contain a lot of probiotic species. Probiotic species are used as food supplements. This work was focused on DNA separation from crude cell lysates of 4 food supplements using magnetic carrier P(HEMA-co-GMA) covered by carboxyl groups. DNA was reversible adsorbed to the carriers in the presence of PEG 6000 (16%) and NaCl (2 M) (final concentrations) and eluted into TE buffer. Lysis of cells from food supplements was performed by lysozyme, SDS and proteinase K. The amount of lysozyme was optimalized. Concentration of separated DNA was measured by spectrophotometric method. The amount of isolated DNA was suitable for PCR. Isolated DNA was used for PCR with universal primers, PCR specific for genus Lactobacillus and genus Bifidobacterium and for 9 different species-specific PCRs: Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/paracasei, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis. Amplicons were detected by agarose gel electrophoresis (1,8%). It was shown that DNA amplification methods are quick and precise for identification of studied species. The results of bacteria identification were compared with data provided by the manufacturer. In all food supplements, bacteria of genus Lactobacillus and Bifidobacterium were detected. However, only some species provided by manufacturer were identified by PCR in each tablet.
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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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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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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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Emanuel Radl, his conception of philosophy and education
Vojtíšková, Michaela ; Pelcová, Naděžda (advisor) ; Blažková, Miloslava (referee)
1 Abstract: Bachelor's thesis Emanuel Rádl, his conception of philosophy and education will follow work and ideas of Czech philosopher and biologist Emanuel Radl. I will study the basic titles of works Consolation of Philosophy and essays devoted to partial topics and to the problem of education in the new Czechoslovak state. The theme of the analysis will be how Rádl thoughts were admitted to the society and how its thinking is valued today. Keywords: philosophy, education, morality, history, justice, verity, human, hero, nation, God
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