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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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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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The monitoring of the lactic acid bacteria in the Moravian wines
Valicová, Markéta ; Španová, Alena (referee) ; Omelková, Jiřina (advisor)
The aim of this Master Degree Thesis was to monitor the total number of lactic acid bacteria occurring in grape must during wine production. The study was performed on the red wine grape variety Cabernet Moravia from organic vineyard and on the white wine grape variety Sauvignon from both organic and integrated vineyards. The isolation of pure cultures of lactic acid bacteria from mixed cultures and subsequently their identification by genus and species-specific PCR was also subject of the thesis. The experimental results show that the number of viable cells of lactic acid bacteria is influenced not only by the wine grape variety, whether it is a variety of red or white wine grape, but also by the way of wine growing. The method of wine growing also had an impact on the species representation of lactic acid bacteria in each variety.
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Use of DGGE to analysis and identification of selected microorganisms
Jankeje, Kristína ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.
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Identification of lactic acid bacteria in hard cheeses using amplification methods
Herzogová, Jitka ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.
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Probiotics and their use in food industry
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic bacteria are defined as live microorganisms, which when consumed in the determining quantities, have healthy and beneficial effects. Most of probiotics belongs to the genera Lactobacillus and Bifidobacterium. These and other genera of microorganisms are successfully used in industry, including food industry at present. Probiotics are used primarily in dairy products and food additives in food idustry. Probiotic bacteria, like other organisms, can be to identifie by PCR method that allows amplifying specific regions of DNA. Polymerase chain reaction was performed after DNA isolation from bacterial cultures of three strains using phenol extraction method. PCR specific for the domain Bacteria and genus-specific PCR were used for the confirmation of the presence of bacteria of the genus Lactobacillus.
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Characterization of selected yeast strains from food
Ostrihoňová, Katarína ; Vítová, Eva (referee) ; Vránová, Dana (advisor)
This bachelor´s thesis is focused on identification of yeasts of the cheeses by PCR-RFLP method and verifying the lipolytic activity of the yeast. In the theoretical part are processed basic information about yeast, their possible positive and negative effects on the quality of cheeses, the technology of production of cheeses, lipolysis and proteolysis in the cheese and of course of PCR-RFLP (The polymerase chain reaction-restriction fragment length polymorphism). Experimental section shows the isolation of DNA, identification of DNA by PCR made by amplification 5,8S-ITS sections of DNA using primers ITS1 and ITS4. The amplified DNA was purified by ethanol and then was subjected to restriction analysis with the enzymes HaeIII, HinfI, HhaI, TaqI. Then there is listed detection of the PCR product and the restriction fragments by gel electrophoresis. Lengths of the fragments obtained after electrophoresis will be used to identify yeast species isolated from cheeses. In the second part of the thesis in the experimental part we have dealt with evidence of lipolytic activity of the yeast by test on Spirit blue agar.
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DNA analysis of nonpathogenic clostridia isolated from cheeses
Chroboková, Maria ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is a molecular method which allows in vitro replication of nucleic acids. It allows the identification and quantification of microorganisms or to prove specific gene sequentions in different matrices of biological origin. Some nonpathogenic species of genus Clostridium cause damages of cheeses, so their identification and quantification is very important in cheesemaking. In this thesis, specific primers for genus Clostridium were tested. Bacterial DNA from culture collection strains and from strains isolated from damaged cheeses were used. Genus-specific region for Clostridium was amplified using specific primers. The PCR products (619 bp) were detected using electrophoresis in 1,8% agarose gel. Genus-specific character of primers was confirmed. DNA of Lactobacillus was used for negative control.
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Detection of phytoplasmas using DNA-microarrays
MARKOVÁ, Jaroslava
The aim of this thesis was to optimize the method of detection of phytoplasmas using DNA-microarray. It consisted of testing an appropriate method of genetic material isolation, development and optimization of PCR to amplify different groups of phytoplasmas, optimization of detection of DNA at a microarray, and sequence analysis of phytoplasma in order to design more suitable probes. PCR was first optimized for collection isolates, then also for natural samples. All 16Sr groups from the collection were sequenced and phytoplasmas were detected in them by hybridization. Phytoplasmas were detected also in natural samples: oilseed rape (species Brassica napus), red clover (Trifolium pretense), purple coneflower (Echinacea purpurea), and apple tree (Malus domestica). Using the DNA from insect vectors, only sample 202/6 from the group 16Sr-XII was positive. The sequence of red clover and oilseed rape correspond with the database samples in the group 16Sr-I "Aster yellows".
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Detection of phytoplasmas using PCR amplification of selected fragments from the phytoplasma genome
MARKOVÁ, Jaroslava
Phytoplasmas are bacterial pathogens of plants. They can cause devastating losses of yield of diverse crops. It is necessary to find reliable detection technique for detection of phytoplasmas disease. The easiest and the cheapest technique is just PCR (polymerase chain reaction) which is used to selective amplification of selected parts of the molecule DNA. The aim of this bachelor thesis is to find of optimal primers to amplification of selected fragments of phytoplasmas genome in one-step PCR and use this technique in detection of phytoplasmas. A collection of phytoplasmas from professor Bertaccini with 37 samples was used for this study. It was tested 16 natural samples by optimized primers. Phytoplasma was detected in strawberry root. Research was supported by the Czech Science Foundation, Grant No. GP 522/09/P545.
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