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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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DNA microextraction from plant vegetable matrix
Cesnak, Filip ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.
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Identification of lactic acid bacteria in fermented dairy products using amplification methods
Tycová, Martina ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.
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Separation and purification of nucleic acids with the use of magnetic microparticles
Vlachová, Jana ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Application of magnetic microparticles for nucleic acid isolation from real samples is a modern and fast separation procedure. This method is suitable for application in laboratory practice. The aim of the bachelor work was isolation of DNA from a dairy product (Activia sweet white) in the PCR-ready quality. DNA isolation was carried out by magnetic microparticles P(HEMA co GMA) in the presence of 2 M NaCl and 16% PEG 6000 or 8% PEG 6000. According to the declaration of the producer the presence of DNA from probiotic bacteria of the genera Lactobacillus and Bifidobacterium was confirmed by PCR.
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Preparation and purification of plasmid DNAs
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of therapeutic methods in medicine, like are DNA vaccines and gene therapy increases demand for new processes for the isolation and purification highly pure plasmid DNA. Most often used methods of purification plasmid DNA are chromatographic methods. In experimental part of this thesis was performed isolation of plasmid pUC-19 DNA plasmid via alkalyne lysis. And purification of plasmid was performed by liquid chromatography and agarose gel electrophoresis.
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Identification of bacteria of Lactobacillus acidophilus species in probiotic products
Sznapková, Veronika ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria (LAB) are an important part of fermented dairy products, pharmaceuticals and food supplements. At present, rapid and accurate identification of bacteria is carried out using molecular biological methods based on DNA amplification. The aim of the thesis was to identify by non-cultivation bacteria of genus Lactobacillus and bacteria of species Lactobacillus acidophilus in complex matrices at total of seven different food supplements. Total DNA was isolated from crude cell lysates using magnetic carrier P(HEMA-co-GMA). Amplificability of DNA was verified by PCR using primers specific for the domain Bacteria. In next step isolated DNA was amplified using primers specific for the genus Lactobacillus and species Lactobacillus acidophilus to demonstrate the presence of this bacterial genus and species declared by the producers. The results of bacteria identification obtained by PCR were compared with declared specification given by the producers.
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Analysis of the composition of selected probiotic products by PCR-HRM
Tomanová, Barbora ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
This work was focused on the detection of probiotic bacteria in four different probiotic products (probiotic cream, probiotic tampons, oral probiotics and soy beverages with probiotics). The viability of the bacteria contained in the products was verified. Complex matrices of the products were used to isolate DNA in a quality suitable for the PCR method, followed by identification of the declared bacterial genus and species. Amplification was achieved with conventional PCR and real-time PCR, genus- and species-specific primers were used. Bacteria, of the genus Lactobacillus and Bacillus and bacterial species Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus gasseri, were proven to be within the products. Subsequently, the DNA from mixed bacterial species in the probiotic tampon were distinguished using PCR-HRM. Five sets of primers were used to test this. Two sets of primers (primers P1V1, P2V1 and V1F-HRM, V1R-HRM) were evaluated as the most suitable for resolution.
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Magnetic carriers and their practical use
Chlopková, Barbora ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The theoretical part summarizes the current knowledge for practical use of magnetic carriers in molecular diagnostics. It includes both already used methods and methods with potential for the future. In the experimental part was tested by use of magnetic media for isolation of DNA from a dairy product and a bacterial culture. It was confirmed that the magnetic carrier DNA was isolated in quantity and quality suitable for carrying out polymerase chain reaction.
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Isolation, Identification and Characterisation of Microbial Communities of Wine and Selected Foods
Šuranská, Hana ; Španová, Alena (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
Proposed dissertation thesis deals with wine and artisanal cheeses microbiology. The first part is focused on identification of yeasts isolated from grapes and musts during production of white and red wines. The grape varieties were grown under the integrated and organic farming on Moravian vineyard. Yeasts were identified by ITS-PCR-RFLP method (amplifying internal transcribed spacer ITS: ITS1, ITS2 and 5.8S rDNA) and unknown species were subjected to partial sequencing of ITS rDNA region. In total, 524 isolates were divided into 14 different species belonging to six genus were identified from. The first stages of fermentation process were characterised by predominance of non-saccharomyces species especially H. uvarum. Due to increased ethanol concentration strains of S. cerevisiae prevailed in the later phases of the process. Further, partial aim of this study was to isolate and to apply selected autochthonous S. cerevisiae strains as starter culture during controlled industrial wine fermentation process. Genus Saccharomyces was distinguished from other non-saccharomyces species by ITS-PCR-RFLP. Further, in order to distinguish Saccharomyces genus at the species and the strain level, several molecular methods were applied including PCR-fingerprinting (rep- and RAPD-PCR), species-specific primers (multiplex and touchdown PCR), LSU-DGGE and interdelta PCR. Species-specific primers enabled us to distinguish some species of the Saccharomyces sensu stricto complex. Furthermore, interdelta PCR seems to be useful tool for S. cerevisiae strains identification. Among 120 isolated autochthonous strains belonging to Saccharomyces genus, 45 different strains were identified. Based on its sufficient technological properties (osmo- and ethanol tolerance, low H2S production etc.), S. cerevisiae 1-09 strain isolated from grape berries coming from moravian vineyard was chosen. Strain S. cerevisiae 1-09 was tested in small amount of must and after that also during industrial fermentation of red and white wine production. Based on the results of chemical and sensorial analysis, the strain seems to be suitable for application as the starter culture for winemaking process. The final part of this thesis is focused on quantification and identification of the yeasts isolated from artisanal cheeses and their by-products coming from Western Balkan Countries. Isolated species were identified by ITS-PCR-RFLP, partial sequencing and by physiological tests. Among the 20 yeast species found, D. hansenii, C. zeylanoides and Y. lipolytica were found to be predominant. Moreover, we developed culture-independent, semi-quantitative technique based on construction of ITS-clone library from metagenomic DNA to investigate complex fungal communities associated with artisanal cheeses and their by-products. Novel technique is based on direct extraction of total DNA from the sample. This was compared with culture-dependent ITS-PCR-RFLP and culture-independent LSU-DGGE methods. The results highlighted the discrepancies among these methods. Finally, the divergences among applied methods were confirmed by correlation analysis and by indices of general biodiversity and dominance of species. ITS-clone library approach combines the advantages of cultivation-based analysis and LSU-DGGE with semi-quantification of fungal species without the requirement of their cultivation. This study might open new perspectives in direct and complex analysis of yeasts and moulds in food matrices.
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Recombinant probiotics
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Theoretical part of this thesis focuses on present state and research of recombinant probiotics and their use in food industry and health care. It also concentrates on their beneficial effects on the health of individuals. The experimental part focuses on the identification of specific bacterial strain in a probiotics food supplements. The DNA was isolated from these products by use of magnetic microparticles and obtained DNA was subsequently analysed through polymerase chain reaction.
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