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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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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 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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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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The identification of Saccharomyces yeasts during fermentation of white grape must.
Zdeňková, Michaela ; Vojtíšková, Marie (referee) ; Vránová, Dana (advisor)
This thesis is concerned with the identification of the yeasts belonging to Saccharomyces species, which is participating in the particular fermentation phases of the white wine. The rapidity that we are able to identify the yeast strains is the significant factor for appreciation the fermantation proces quality as well as final product quality – the quality of wine. The molecular biology method developing is gradually limiting the using of traditional identification methods mainly because of their time intensity. In this thesis was used molecular method PCR-RFLP as an implement to quick and accurate identification of particular strains of yeasts. The specific DNA section was amplified by the help of PCR and consequently amenabled to the restrict analysis. The restrict endonukleas fissiled DNA to fragments specified for the certain strain of yeast. The fragments were detected by horizontal electrophoresis. To compare the fragments with type yeast fragments made us possible to identify the trast strain and its taxonomy classification. The literature search contains the basic information about the yeasts and their using, the information about wine making as well as the PCR-RFLP method principle.
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