|
|
Molecular identification of probiotic bacteria in milk products form commercial yoghurt cultures
Horňan, Samuel ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Lactic acid bacteria are considered as an important group of bacteria with probiotic effects, which are being widely used in the food industry or pharmacology. Identification and characterization of important probiotic strains play an essential role in the validation of probiotic products for commercial purposes. Their identification using molecular-biology techniques (most commonly PCR method) is one of the standard tools in commercial operations and services. The aim of this bachelor thesis is a literature review of probiotics and probiotic strains as well as a summary of current knowledge about the use of molecular biology techniques for identification of these bacteria with probiotic properties in dairy products. The experimental part of this work verifies the presence of probiotic bacteria declared on selected commercial dairy products using the polymerase chain reaction (PCR) method.
|
|
|
Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
|
|
|
The application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
|
|
|
Authenticity of natural plant component in cosmetics products
Kubalová, Michaela ; Fialová, Lenka (referee) ; Němcová, Andrea (advisor)
The purpose of this thesis was to study the authenticity of selected natural ingredients in cosmetic products. These were specifically cosmetic products that contained citruses, mint or lavender. Commercially available isolation kits were used for DNA isolation. The presence of plant origin DNA was verified by PCR method using primers specific for the ITS2 region of plants. The presence of limonene, a significant allergen contained in said plants, was determined in the samples by PCR method using primers for limonene synthase. At the same time, its presence was verified by HPLC method. In addition, two primers were tested for lavender and monitored for their efficacy, with no significant difference in the usage.
|
|
|
Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
|
|
|
Detection of probiotic bacteria in diary food products using PCR technique
Klaška, Dominik ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
In the bachelor thesis, DNA was isolated from commercially available white yogurt. The isolated DNA, which was gained by two different methods, was performer analyse by a spectrophotometer. Both methods provided sufficiently concentrated and high-quality DNA for further analysis by PCR. Precisely defined sections of isolated DNA were amplified using specific primers. The presence of the bacterium domain was detected, and in the case of genus specific amplification, the presence of bacteria of the genus Lactobacillus was detected too, by gel electrophoresis.
|
|
|
Determination of Staphylococcus aureus and Bacillus cereus in confectionery products
Vdolečková, Monika ; Necidová,, Lenka (referee) ; Šárka,, Bursová (advisor)
This bachelor thesis focuses in the theoretical part on the description of bacteria Bacillus cereus and Staphylococcus aureus, as bacteria that can contaminate raw materials of either plant or animal origin. Both of these bacteria can be found in soil, water, but we can also encounter them on our skin. This fact can aid contamination in the manufacture of confectionery products. In the experimental part, samples of Christmas sweets were processed. The presence of the above mentioned bacteria was monitored in Christmas sweets and genes encoding toxin production were the presence of bacteria was detected.
|
|
|
Biofilm formation in probiotic cultures and its application in pharmacy
Ryšávka, Petr ; Obruča, Stanislav (referee) ; Vorlová, Lenka (referee) ; Márová, Ivana (advisor)
The work was comprehensively focused on the development of adhesive forms of probiotics in the form of a biofilm on combined carriers with a prebiotic component. The second part dealed with the influence of food on the multiplication and survival of selected types of probiotic bacteria. Subsequently, the effect of individualized probiotic supplements on changes in the human intestinal microbiome was monitored. Suitable adherent probiotic strains for biofilm formation were selected and tested. Methods have been introduced and different variants of carriers for culturing and binding bacteria have been tested. In vitro experiments verified the stability of biofilm stucture and its resistance to low pH, bile and antibiotics in comparison with the planktonic cell form. The antimicrobial effect of probiotic strains in the form of a biofilm was studied. The cultivation of the multispecies biofilm on the combined carrier was optimized and the stability of the biofilm and the final viability of probiotic bacteria were confirmed. Furthermore, the influence of various foods and beverages on the viability of probiotic bacteria was evaluated with emphasis on the simulation of passage through the gastrointestinal tract. Both models, solutions with standardised concentrations of alcohol, sugar, salts, proteins or different pH and different types of real foods and beverages were tested. The effect of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and on probiotic capsules containing a mixed culture of probiotic microorganisms. The survival of probiotics in various food matrices in the simulated gastrointestinal tract was quantitatively different. We managed to define foods suitable for supporting the multiplication of probiotic bacteria. A separate part of the work was focused on the targeted modulation of the intestinal microbiome by individualized probiotics that were prepared on the basis of molecular biological analyzes of the intestinal microbiome aimed at detecting the percentage of lactobacilli, bifidobacteria and phylum Firmicutes and Bacteroidetes. Personalized probiotic supplementation confirmed the positive effect of this approach on microbiome changes, especially on the increase of the content of lactobacilli, bifidobacteria and the overall diversity of the microbiome.
|
|
|
Characterization of selected yeast strains from the environment
Bečková, Alena ; Omelková, Jiřina (referee) ; Vránová, Dana (advisor)
The aim of this bachelor thesis deals with identification type-yeasts, which were obtained from the Culture Collection SAV in Bratislava. The purpose is extension of yeast database in laboratory of Faculty of Chemistry, which is used by identification yeasts in samples taken from food or environment. Identification of yeasts was implemented by method PCR-RFLP. Obtained segment is located in area 5,8S-ITS and is defined by using primers ITS1 and ITS4. Restriction analysis was carried out by restriction endonucleasis HaeIII, HinfI, HhaI and TaqI. Chosen species were also tested for presence of extracellular lipasis. In this part of characterisation was used SpiritBlue Agar. The presence of blue colour is lost by activity of lipasis.
|
|
|
Characterization of carotenogenic yeasts using molecular techniques
Kostovová, Iveta ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
The aim of this master’s thesis was focused on characterization of carotenogenic yeast using molecular techniques. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method, which approved differences of S. roseus in all analyzed rDNA sequencies compared to the other analyzed carotenogenic yeasts. Parameters of PFGE and isolation procedure of the intact DNA were optimized for caryotypic yeast characterization. At all, nine of carotenogenic yeast strains Rhodotorula, Sporobolomy-ces, Cystofilobasidium a Phaffia were analyzed by this techniques. Further part of this thesis was focused to application of molecular methods to analysis of region D1/D2 of large ribosomal subunit in mutant carotenogenic yeast strains. Mutant strains were pre-viously adapted to waste substrates - pasta and glycerol, and stability of their production properties was verified.
|
guest ::
