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Isolation of yeasts from the vineyard and their using in the production of wine
Ždiniaková, Tereza ; Trachtová, Štěpánka (referee) ; Vránová, Dana (advisor)
The aim of this thesis was the isolation and identification of yests obtained from the wine berries by different molecular methods. The yeasts for identification were collected from the species of Veltlínské zelené white wine from the wineyard from Milotice, district Hodonín. Identification based on analysis DNA by PCR method and followed by RFLP using restriction enzymes HaeIII, HinfI, HhaI a TaqI. To obtain values lenghts of the restriction fragments was used gel electrophoresis. For getting genetic similarity of analyzed yeasts was used BioNumerics software, that processe the results using cluster analysis using Jaccard´s coefficients and the yeasts were identified by comparing the characteristic lenghts with number of articles and educational databases.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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The analysis of DNA isolated from different types of probiotic products using real-time PCR and HRM analysis
Sedláková, Lucie ; Rittich, Bohuslav (referee) ; Trachtová, Štěpánka (advisor)
The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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Identification of lactic acid bacteria in probiotic products (tablets) using amplification methods
Balogová, Petra ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Lactobacillus have been playing a crucial role in the fermentation processes for millenia. Their probiotic effects have been studied deeply and nowadays they are used in the production of fermented products and food adjunkt including probiotic preparations. This thesis was focused on bakteria of genus Lactobacillus in probiotic preparations (tablets). Polymerase chain reaction method (PCR) based on amplification of DNA was used to identify lactobacillus in seven probiotic preparations. As DNA matrices whole DNA recovered from tablets with the help of magnetic microparticles (P(HEMA-co¬-GMA) coated by carboxylic groups was used into a PCR mixture. Genus specific PCR products (250 bp) were detected with the help of gel electrophoresis on agaróse. DNA of genus Lactobacillus was proved in all but one product. In this product only probiotic bacteria were declared presence without family identification was proved. It contained different bakteria then bakteria of Genus Lactobacillus probably.
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Study of Reversible Adsorption of Nucleic Acids on Solid Surfaces
Trachtová, Štěpánka ; Zendulková,, Dagmar (referee) ; Drbohlav,, Jan (referee) ; Rittich, Bohuslav (advisor)
Magnetically driven separation techniques using magnetic solid carriers are one of modern methods to speed up and facilitate the previously used separation and purification procedures. The use of magnetic particles in biology imposes strict requirements on physical, and chemical properties of the particles, including low toxicity, biocompatibility and non-interference with the chemical environment in diagnostics. The aim of this study was to evaluate carboxyl-functionalised magnetic non-porous P(HEMA-co-GMA), P(HEMA-co-EDMA), PGMA, silica-coated lanthanum manganese peroskvite La0.75Sr0.25MnO3 and thermosensitive poly(N-isopropylacrylamide) microspheres – P(NIPAAm) for DNA isolation from different types of complex food and environmental samples containing PCR inhibitors. The solid-phase reversible immobilisation (SPRI) of nucleid acids on microsphere surface and the release of adsorbed DNA were optimised. DNA from real samples (milk products and probiotic food suplements, mouse faeces) was apparently adsorbed on solid particles from the aqueous phase system composed of 16% PEG 6000 and 2M NaCl. The conditions of the subsequent release absorbed DNA to the elution buffer (pH of elution buffer, temperature and time of elution) were optimized. The quality of eluted DNA and the presence of target DNA were examined by PCR and q-PCR using domain-specific Bacteria and genus-specific Lactobacillus primer set. Real-time PCR was used for an estimation of the PCR interference by comparing the amplification efficiencies of purified DNA containing solid nanoparticles with the DNA standards free of any nanoparticles
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Methods for detection of antimicrobial properties of lactic acid bacteria
Vránová, Petra ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Lactic acid bacteria are classified as probiotics producing substances that act against microorganisms. These antimicrobial substances include organic acids, carbon dioxide, hydrogen peroxide and bacteriocins. Currently, the focus is on bacteriocins, which are used in the food and pharmaceutical industries. The aim of this thesis is creating an overview of antimicrobial properties of lactic acid bacteria, methods of their determination and their applications in the food industry. The experimental part deals with identification of lactic acid bacteria present in selected samples of bacterial DNA. In addition, we tested for bacteriocin Gassericin K7A-producing gene. Molecular diagnostic methods such as real-time polymerase chain reaction (qPCR) and conventional polymerase chain reaction were used to distinguish bacterial DNA as well as the bacteriocin.
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Probiotics in food products
Silná, Renata ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms with a positive effect on the consumer when they are added to food in adequate amount. The best known probiotic are lactic acid bacteria and yeast Saccaromyces cerevisiae var. boulardii. The theoretical part of the thesis is focused on using probiotics microorganisms in food. In the experimental part of the thesis were prepared crude lysates from three food products and the presence of bacterial DNA was proved by PCR method.
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Utilization of wastes
Šachlová, Žaneta ; Trachtová, Štěpánka (referee) ; Omelková, Jiřina (advisor)
Bachelor's thesis was focused on recovery from food processing industries, where she described the issue of the types of waste handling, recycling, processing by-products in the food industry, waste prevention. Attention is also given to food from industrial wastes such as sugar industry, production of starch and flour, meat, dairy, production of fats and oils, fruit and vegetable processing, production of malt and beer, wine, alcohol and yeast. There were considered suitable packaging materials in food, different waste treatment. Important microorganisms are set out using in waste treatment. At the conclusion of this study was designed a perfect procedure how to dispose with waste and included legislation on this subject.
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Imunomagnetic separation of lactic acid bacteria using magnetic microparticles functionalised by antibodies
Vaňásek, Jakub ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
Immunomagnetic separation is based on binding of antibody with antigen, where antibody is bound to magnetic particle. In this thesis there were used particles of magnetic pearl cellulose with antiLactobacillus and antiBifidobacterium antibodies. Immunomagnetic separation method was optimalized and verified for its efficiency and specifity with bacterial and yeast cells. This cells were identified by polymerase chain reaction. Efficiency of immunomagnetic separation was verified on probiotic meat product, where Lactobacillus cells were isolated. With DNA from isolated Lactobacillus cells the high resolution melting was performed. The results show presence of several bacterial strains of Lactobacillus species.
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