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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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Lactobacillus DNA analysis using real-time PCR and HRM analysis
Aksamitová, Dagmar ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
The rapid and accurate identification of the bacterium of the genus Lactobacillus, which are an important part of the normal gastrointestinal microflora and fermented dairy products are currently mainly used amplification methods. The aim of the study was to analyze the possibility of resolution of selected bacterial strains of the genus Lactobacillus, using the metod of polymerase chain reaction in the real time combined with high resolution melting curve analysis (qPCR HRM). It was tested five primers designed for qPCR-HRM analysis of lactic acid bacteria. The specificity of the primers was also verified simultaneously using bioinformatic analysis. On the basis of analysis of the DNA were selected as the most appropriate primers P1V1/P2V1, V3F/V3R and V6F/V6R. The suitability of the primers V3F/V3R and V6F/V6R was verified on a complex sample of food supplement from which the DNA was isolated using magnetic particles. The presence of bacteria of the genus Lactobacillus was performed using high resoluting melting analysis curves. The obtained results were in agreement with the information given by the manufacturer.
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Isolation of DNA from probitic products using solid carriers
Bonczek, Ondřej ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Microbial DNA was isolated from lysed cells of Lactobacillus genus in probiotic products. Reversible adsorption DNA on the surface of carboxyl coated nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) magnetic particles and silicagel coated manganase Perovskite nanoparticles. DNA was adsorbed on the surface of the particles in the presence of 16 % poly(ethylenglycol) (PEG 6000) and 2 M sodium chloride (NaCl) concentrations. The adsorbed DNA was released from particles by low ionic strength TE buffer (pH= 8.0). The quality of isolated DNA was checked by spectrofotometric measurement and PCR amplification. DNA samples isolated using magnetic particles and phenol extraction method (control method) were PCR-ready. The DNA isolated from lysed cells of probiotic products was quantificated in real-time qPCR.
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Use of PCR for species identification and searching of selected genes of lactobacilli
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.
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Study of growth and metabolic properties of selected microorganisms
Hudečková, Helena ; Šupinová, Petra (referee) ; Babák, Libor (advisor)
The aim of this Bachelor thesis was study of the growth and production of selected metabolites of microorganisms, namely Lactobacillus casei and Saccharomyces cerevisiae. Microorganisms were cultivated in Erlenmeyer flasks by using the recommended media for their growth. The growth curves were determined from the values of optical density and concentration of biomass. Samples were then analyzed by HPLC for determination of the content of glucose and ethanol in S. cerevisiae and the content of glucose and lactic acid by L. casei.
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Identification of probiotic species of genus Lactobacillus
Vystavělová, Růžena ; Trachtová, Štěpánka (referee) ; Rittich, Bohuslav (advisor)
Germs of the Lactobacillus genus form part of the micro flora, the composition of which exercises the influence on the state of health of its host. In view of the fact that at some types of Lactobacillus there were proved clinical effects (e. g. strengthening of the immune system, prevention of diarrheic illnesses and so on), the Lactobacilli have been often used for making fermented dairy products and they form part of food additives because of their probiotic effect. Germs of the Lactobacillus genus and germs of different types of Lactobacillus can be identified by means of PCR based on amplifying specific DNA fragments. The complete bacillary DNA Lactobacillus rhamnosus was separated by the phenolic extraction method. The presence of germs of the Lactobacillus genus was proved by the generic-specific PCR method; the presence of Lactobacillus rhamnosus germs was proved by the species-specific PCR method.
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Study of aerobic cultivation conditions with select strain of Lactobacillus
Šupinová, Petra ; Burdychová, Radka (referee) ; Babák, Libor (advisor)
The aim of this study was focused on the study of conditions of growth of strains Lbc. paracasei subsp. paracasei CCDM 211, Lbc. paracasei CCDM 212, Lbc. paracasei subsp. paracasei CCDM 213 and Lbc. salivarius CCDM 216 in media with different amount of carbon-source (glucose, lactose and whey). Next part of the experiment was dealed with study of conditions of bacteria growth at stress conditions (lower pH). The purity od bacterial culture was verified with help of streaking. Purity DNA isolated from bacteria was tested using agarose gel electrophoresis, DNA concentration was estimated spectrophotometricaly. The presence of bacteria of genus Lactobacillus was proved using polymerase chain reaction (PCR) with genus specific primers.
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Identification of probiotic lactobacili in dairy products
Dofková, Květoslava ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The genus Lactobacillus is part of intestinal microbiota which constitution plays important role in health and well-being of the host. Probiotic properties of lactobacili have been studied in lot of studies and clinical effects (immune enhancement, prevention of diarrhoeal disease etc.) of some probiotic species have been documented. Therefore bacteria of genus Lactobacillus are used in the production of fermented dairy products and probiotic preparations. PCR method based on amplification of 16S – 23S rRNA intergenic spacer regions was developed for identification of Lactobacillus species presented in dairy products. Whole DNA obtained by phenol extraction from dairy product Actimel Natur was used in this work as template for PCR. Purified DNA was amplificated by genus-specific PCR. Bacteria of genus Lactobacilllus were detected due to the detection of specific PCR product. Using species-specific PCR probiotic species Lactobacillus casei was detected in the Actimel Natur in quantity that approximately corresponded to declared amounts.
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Production and application of antimicrobial substances produced by lactic acid bacteria
Utíkalová, Kristýna ; Dzurická, Lucia (referee) ; Márová, Ivana (advisor)
The bachelor’s thesis deals with the production of antimicrobial substances by selected lactic acid bacteria of the genera Lactobacillus, Pediococcus and Bifidobacterium, in which the production of bacteriocins was assumed based on the conducted research. Bacteriocins are peptide-based antimicrobial substances with the potential to be widely used primarily as biopreservatives in the food industry. The theoretical part of the bachelor’s thesis focuses on antimicrobial substances produced by probiotic bacteria with a further concentration on bacteriocins produced by selected bacterial species and their current research and application in the industry. In the experimental part, the antimicrobial activity of prepared isolates from probiotic bacteria was tested on selected gram-negative and gram-positive bacteria. Specifically, this was done by the agar diffusion method, the broth dilution method and the cell viability test using resazurin. The amount of lactic acid and acetic acid in the individual isolates was determined using high performance reverse-phase liquid chromatography, which made it possible to further discuss their influence on the antimicrobial activity of the isolates. Strong antimicrobial activity against indicator bacteria was found in all isolates. For some isolates, it was higher than the antimicrobial activity of lactic acid itself together with its concentration which is found in the isolate according to the measurements. That is a significant stimulus for further research of these probiotic isolates, especially the L. acidophilus and L. plantarum isolates. For example, it was found that the tested isolate from L. acidophilus showed an inhibitory activity against S. epidermidis of 90,76 % and 81,62 % against E. coli at a concentration of 20,47 mg/ml. The isolate from L. acidophilus was subsequently applied to plant packaging with the potential of usage in the food industry, and the inhibitory activity of this isolate against contaminating microorganisms was observed.
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Use of PCR for species identification and searching of selected genes of lactobacilli
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.
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