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Real-time PCR and it´s use in food processing
Tomanová, Barbora ; Pravečková, Martina (referee) ; Trachtová, Štěpánka (advisor)
Polymerase chain reaction (PCR) is a method abundantly used in molecular diagnostics. PCR in real time or quantitative PCR (qPCR) is one of its modifications and thanks to its advantages it finds still wider utilization nowadays. It finds its use in the food-processing industry too with relatively precise detection, identification, and qualification of both desirable and undesirable components in food, which often brings considerable difficulties and leads to an intensive development of this method. In the experimental part a DNA was isolated from dairy product Bio Via Natur drink for its further processing by means of PCR and gain more detailed information about a bacterial composition using real-time PCR and HRM analysis.
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Testing of lactic acid bacteria Lactobacillus genus for bacteriocins production
Volecová, Veronika ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Antimicrobial substances, or bacteriocins are substances produced by probiotic lactic acid bacteria. They have a positive effect on the gastrointestinal tract of humans and are especially suitable for the food, but also the pharmaceutical industry. The aim of the thesis was the molecular identification of lactic acid bacteria of the Lactobacillus genus, species and their subsequent inclusion PCR method. Using the PCR method were tested also genes responsible for the production of bacteriocins. To confirm the production of bacteriocins has been selected the microbiological method, agarose droplet spot-test. In the present study also included the bioinformatics part to assess the specificity and non-specificity of the primers using in Primer-BLAST program.
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Production of magnetic particles by microorganisms
Chvalkovská, Eva ; Mgr. Martina Mikešová, Ph.D. (roz. Pravečková) (referee) ; Trachtová, Štěpánka (advisor)
This thesis focuses on the magnetic nanoparticles produced by bacteria. These are two types of bacteria. First of them we can find in an industrial environment that produce magnetic nanoparticles using magnetosomes. Magnetosomes are units containing nanocrystals and are surrounded by a lipid layer. They are made up of chains and work as a compass. In this thesis I deal with possibilities of cultivation od these bacteria, acquisition of magnetosomes and subsequent processing. The second type is bacteria living in the human body, they start to produce magnetic nanoparticles after the addition of trigger, such as silver nitrate. Bakteria which can produce silver or ferrous nanoparticles are Bacillus sp, Escherichia coli, Lactobacillus casei ssp. casei and Lactobacillus fermentum. Experimental part of bachelor thesis focused on the production of silver nitrate in concetration of 0,1; 1; 2; 4 mM. The bacteria that have been shown to produce silver nanoparticles are Lactobacillus casei ssp. casei CCM 7088T a Lactobacillus casei ssp. casei CCM 7089 a Lactobacillus rhamnosus CCM 1825T.
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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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