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Biosynthesis of polyhyroxyalkanoates in non-suflur purple bacteria
Fiala, Tomáš ; Vodička, Juraj (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis focuses on polyhydroxyalkanoates (PHA) and their production using non-sulfur purple bacteria, specifically Rhodospirillum rubrum. The cultivation conditions were optimized especially with respect to the determination of NaCl concentrations for the following evolution experiments. Then this microorganism was repeatedly cultured at a high concentration of NaCl (40 g/L). Passage system was used in this cultivation with 48 h between inoculations under aerobic conditions in the dark. In the samples cultivated in this way, the growth rate was investigated using optical density and the amount of biomass and PHA using gas chromatography with an FID detector. As the last experiment PCR analysis was performed to test the 16S rRNA and phaC genes. The conclusion of the work is the successful adaptation of R. rubrum to a concentration of 40 g/l NaCl. Furthermore, the positive effect of stress stimulus on the increased PHA production was proven.
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Changes in the sensory quality and volatile compounds profile of smear ripened cheese during storage
Šebestová, Adéla ; Zemanová, Jana (referee) ; Vítová, Eva (advisor)
This bachelor thesis deals with the changes in sensory quality, volatile compounds profile and microbial profile of two smear ripened cheese, Olomoucké tvarůžky and Romadur, during their storage for four weeks. The sensory analysis, specifically the assessment of appearance, colour, texture, aroma and taste, showed differences between the two cheeses, which increased during storage (ripening). The greatest differences were observed in the assessment of taste, which improved during ageing for Olomoucké tvarůžky and deteriorated for Romadur. Analysis of volatile compounds by headspace solid-phase microextraction coupled with gas chromatography and mass detection revealed the presence of 57 substances belonging to 9 chemical groups. The composition of the cheese varied in terms of number and content of compounds, with alcohols, ketones and acids being the most abundant. In Romadur, esters predominated quantitatively, and in Olomoucké tvarůžky, sulphur compounds. The total amount of volatile substances in tvarůžky increased during maturation, whereas the opposite was true for Romadur. Microbial analysis showed the presence of a diverse microbiome on the surface of the cheese. There were slight differences between the microbial composition of the cheeses. There was no evidence of change in the microbiome during ripening. The qPCR method was used to identify the microorganisms present.
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Evaluation of the presence of probiotic bacteria in a food supplement using molecular biological methods
Dvornyi, Nikolai ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
Probiotics are beneficial microorganisms that, when administered properly, can provide health benefits to the host, such as aiding digestion and boosting the immune system. Their effectiveness and benefits depend on the specific strain of microorganism. These strains are often from the genera Lactobacillus, Bifidobacterium, and represent natural hosts of the gastrointestinal tract. This bachelor thesis focuses on the identification of probiotic bacteria in a commercially available dietary supplement Linex® Forte using molecular biological methods, mainly polymerase chain reaction (PCR). The aim was to verify the presence of the claimed probiotic cultures of Lactobacillus acidophilus, Bifidobacterium animalis, and to compare the results with the manufacturer's claims. Therefore, two DNA isolation methods were used: phenolchloroform extraction and the commercial purification kit OMNI International. The isolated DNA was then analyzed by domain, genus and species-specific PCR. The results show that the methods used are suitable for the identification of probiotic bacteria and confirm their presence in the selected product.
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Testing of primers for real-time PCR-HRM analysis of fruit products containing one fruit species
Boháčová, Barbora ; Dzurendová, Simona (referee) ; Fialová, Lenka (advisor)
Determining the authenticity of fruit products, which are often counterfeited by substituting part of a more expensive fruit with a cheaper but botanically similar fruit, is a current topic in the food industry. A prime example is the dilution of apricot products with peach puree. This study focuses on testing specific primers AGS18 and PdCass to reveal the true proportion of apricot content in model products mixed with peach puree using PCR analysis followed by HRM analysis. Testing of these primers for authenticity determination revealed limited utility of AGS18 primers due to the formation of small amount or no specific products during reactions with fruit DNA. PdCass primers required PCR condition optimization, but subsequently, specific DNA sequences from fruit leaves could be amplified in sufficient quantities. PCR analysis of DNA from model products with PdCass primers provided specific products in samples with apricot content of 70% or less. HRM analysis of samples and calculation of GCP did not distinguish purees with 0%, 10%, and 30% apricot content. However, purees with 50% and 70% apricot content exhibited significantly different melting curves compared to other samples.
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Utilization of PCR technique for identification of probiotic bacteria in daily hygiene product
Horobets, Yuliia ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Probiotic bacteria have traditionally been used in the food industry, but their applications have now expanded to the prevention and treatment of various diseases. Increasing evidence supports the efficacy of bacteria of the genus Lactobacillus in the oral cavity has led to the application of probiotic strains of this genus in mouthwashes and other personal care products. In this bachelor thesis, DNA was isolated by two methods, then quantified spectrophotometrically and amplified via conventional PCR. The results of the polymerase chain reaction were detected by gel electrophoresis. The presence of probiotic bacteria species Lactobacillus delbrueckii, Lactobacillus plantarum and Lactobacillus pentosus was confirmed.
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Influence of production environment on the occurrence of contaminating Penicillium in smear ripened cheese
Veselá, Vendula ; Brázda, Václav (referee) ; Vítová, Eva (advisor)
The topic of this thesis is the monitoring of the environment in a cheese production plant specializing in cheese ripened under a smear rind. The aim is to evaluate the occurrence of Penicillium mold as the main contaminant microorganism, depending on the sampling location and production time. Air samples were collected throughout the production process using the sedimentation method at 21 designated locations. The total number of microorganisms, selected groups of microorganisms, and the target microorganism (Penicillium mold) were monitored in the collected samples using cultivation (according to ČSN standards), microscopic, and molecular diagnostic techniques. The aim was to evaluate the occurrence of Penicillium citrinum, chrysogenum and commune species in dependence on the sampling location and production time. As part of the production screening, the broader microflora was first evaluated, followed by monitoring for contaminating molds. Using macroscopic and microscopic methods, 36 morphologically distinct colonies were selected. Microscopic analysis confirmed the presence of bacteria, yeasts and molds of the genera Penicillium, Cladosporium and Aspergillus. In order to identify the microorganisms present, an analysis using the polymerase chain reaction (PCR) method was performed, preceded by DNA isolation using the NucleoSpin Microbial DNA kit and phenol extraction. Based on real-time PCR results, the occurrence of contaminating Penicillium and Cladosporium molds was detected even in the early stages of production. Molds from these genera are found throughout the entire facility except for the single room that serves as a curd cooler before forming. Aspergillus mold, in the form of yellow mold with white mycelium on the edge of the colony, was detected only once in the drying room where cheese ripening occurs, and confirmed by microscopic observation. The genus Cladosporium was found in five morphologically distinct forms in the production environment. In addition, DNA was isolated from 11 morphologically distinct bacteria. Real-time PCR identified this DNA as belonging to coryneforms, with four of them belonging to the genus Brevibacterium. The genus Penicillium was represented by 13 morphologically distinct colonies, real-time PCR confirmed the presence of Penicillium commune and Penicillium chrysogenum. In addition to the mentioned molds, 11 morphologically distinct bacteria were detected, from which DNA was isolated. Real-time PCR identified this DNA as belonging to coryneforms, with 4 of them belonging to the genus Brevibacterium. The results of this work completely map the contamination of the production environment of the plant, and based on these results, methods for environmental sanitation were proposed to ensure the decontamination of the production process.
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Detection of pathogens in joint infections, including total endoprostheses using the BioFire method (Biomérieux)
ŠTĚTINOVÁ, Lucie
This bachelor thesis deals with the detection of pathogens in joint infections including total joint replacements by the BioFire method (Biomérieux, France). Compared to conventional methods, the Biofire Joint Infection Panel (BioMérieux, France) offers simultaneous detection of up to 39 pathogens and antimicrobial resistance markers in about one hour of time.This method is a multiplex PCR system for rapid and comprehensive testing. It is an innovative method that, thanks to its speed and comprehensiveness, will allow clinical microbiologists and physicians to make timely decisions on appropriate antibiotic therapy. Early de-escalation of this antibiotic therapy will also be enabled through the timely and targeted deployment of the appropriate ATB. The theoretical part of the thesis discusses the major infections of joints and joint replacements, introduces the reader to the most important causative agents of these infections, and a section is also devoted to microbiological diagnosis. The practical part describes the classical multiplex PCR, the FilmArray multiplex PCR system and with it the BioFire Joint Infection Panel, which has been used in the analysis of synovial fluid in patients with suspected septic arthritis or periprosthetic infection. Data from the analysis are also evaluated in the practical part. The data are from March 2021 to June 2022 and are presented in the practical part in the form of tables and graphs. The conclusion of the paper summarizes the relevance of the method for routine diagnosis and answers the stated research questions.
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Molecular genetic diagnostics of solitary fibrous tumors
ZÍTOVÁ, Lucie
The aim of this work is to provide an expert review of solitary fibrous tumors (SFT). In the practical part of the work, main aim is to create a comprehensive view of the molecular diagnosis of SFT. These tumors exhibit a broad spectrum of biological behavior from benign to malignant. The prognosis is generally uncertain. Promising marker of aggressive biological behavior is a mutation in the promoter region of the TERT gene, which causes the reactivation of telomerase reverse transcriptase. In this context, the key hypothesis of the work is the question of whether it is possible to determine the biological behavior of the tumor based on sequencing analysis of the promoter region of the TERT gene. A set of 33 patients of the University Hospital in Motol was analyzed and the data was statistically processed.
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PCR testing for selected diseases of bees
GREGOROVÁ, Sabina
Nosema ceranae is a species of microsporidia that negatively affects Apis mellifera. It is most commonly transmitted by coprophagy, or eating faeces when filling faecal sacs. It is a worldwide pathogen that is considered to be one of the causes of CCD (colony collapse disorder). The lesser known Crithidia mellificae belongs to the class Trypanosomatidae, which acts similarly like N. ceranae. Both of these pathogens target the guts of honey bees. As a result, infected bees weaken and the colony dies. The aim of this bachelor thesis was the practical examination of selected Apis mellifera samples from the beehive of the University of South Bohemia Faculty of Agriculture and Technology using the PCR method. In the theoretical part I describe Nosema ceranae and Crithidii mellificae and their effect on honey bees. Furthermore, the route of transmission of these pathogens and the possible treatment of infected honey bee colonies are mentioned. In the practical part, which I did in the laboratory at the Department of Plant Production of the Faculty of Agriculture and Technology, the procedure of DNA isolation, PCR and gel electrophoresis is described in detail. The evaluation of the results is also presented in this part.
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