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Analysis of autenthicity of food products with fruit component
Prachárová, Adriana ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.
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The application of magnetic particles for DNA isolation from selected vegetable products
Akwari, Michala ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.
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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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Microbiological analytical methods suitable for dairy industry
Vlasák, Jaroslav ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the thesis is focused on probiotics in dairy products. Thesis deals with molecular genetic methods, which are used to analyze DNA. Especially are discussed methods used for isolation bacterial cells belonging to genus Lactobacillus. The polymerase chain reaction (PCR) is the basic technique at present time. Short chain of oligonucleotide primers are used to amplification specific parts of DNA molecule chain. The practical part of the thesis is focused on the DNA from pure bacterial culture and probiotic dairy product. DNA was isolated using methods of phenol-chloroform extraction and magnetic separation. For magnetic separation was used magnetics particles covered with carbonyl functional groups. Quality of the DNA was confirmed by PCR amplification. Primers specific for domain Bacteria and genus Lactobacillus and Bifidobacterium was used.
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Selective isolation of of the genus Lactobacillus bacteria from foods
Novotná, Eva ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria of genus Lactobacillus play an important role in the digestive tract of human. They are used in food processing and they are the part of food supplements. Lactic acid bacteria of the genus Lactobacillus can be identificated by polymerase chain reaction (PCR). Bacterial DNA was isolated from cell lysates of 4 synbiotic food suplements by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. Magnetic particles with immobilized antibodies against Lactobacillus bacteria were used in the next part of thesis. These particles were used for isolation target cells from products with their identification by genus specific PCR.
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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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The use of magnetic microparticles for DNA isolation
Jelínek, Zdeněk ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.
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Probiotics and prebiotics in food and other products
Langová, Denisa ; Havlíková, Šárka (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of this thesis focuses on present state and research of probiotics and prebiotics, their use for microflora modulation of host and their beneficial effects on the health of individuals. Furthermore thesis deals with efficiency of probiotics strains, which depends on the food matrix and other various factors. The experimental part focuses on the identification of chosen bacterial strain, which is contained in probiotics product. It is realized due the isolation of bacterial DNA by phenol extraction and by use of magnetic particles and subsequent analysis of obtained DNA by polymerase chain reaction (PCR).
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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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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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