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Intrinsic fluorescence of bacteria Cupriavidus necator
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.
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Aflatoxins in food and their influence on DNA and cell lines
Šislerová, Lucie ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
Aflatoxins present a great danger due to their high toxicity and carcinogenicity, which is not easily avoided in everyday life. Intoxication with aflatoxins causes a wide range of diseases ranging from mild diseases to organs necrosis or death. Aflatoxins mostly affect the liver, where it degrades and the formation of subsequent metabolites, which are the most toxic to the body. For this reason, their precise determination and understanding of the principle of their effect is very important. In this work, methods for monitoring and closer determination of aflatoxin effects on human cells were calibrated. The methods that were used are: MTT viability assays, fluorescence microscopy and flow cytometry. Next, the amount of aflatoxins present in different foods with different storage conditions was measured. For this analysis were used ELISA assays RIDASCREEN Aflatoxin Total and RIDA Aflatoxin column. Calibrated methods were compared with the methods already used to determine the effect of aflatoxins and the results of the ELISA tests were compared with the limits of aflatoxin levels permitted by the Czech legislation. None of the controlled foods contained above-the-limit concentration of aflatoxins, which in the Czech Republic is set at 4-10 µg/l (varies for different types of food). Foods that were poorly stored but not visibly affected by fungi showed the highest levels of aflatoxins. The LD50 value for aflatoxin B1 was determined to 12,25 µM. The type of cell death caused by aflatoxins was determined by flow cytometry and these data were further confirmed by fluorescence microscopy images.
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Evolutionary engineering of cyanobacteria with respect to PHA accumulation
Vašířová, Kristýna ; Slaninová, Eva (referee) ; Samek, Ota (advisor)
The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.
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Characterization of adsorption properties of probiotic bacteria
Černá, Klára ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of the diploma thesis is to characterize the adsorption properties of probiotic bacteria. Characterization was performed using of designed and optimized spectrophotometric method of determination of adhesion of probiotic bacteria to mucin as the main viscoelastic component of mucus. In order to provide a more detailed description of the adhesion interactions involved in the adhesion of probiotic bacteria to mucin, the viability of selected probiotic bacterial strains Lactobacillus rhamnosus CCM 1825, Lactobacillus plantarum CCM 7039, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190, Lactobacillus acidophilus CCM 4833, Lactobacillus casei CCM 4798, Bifidobacterium breve CCM 7825, Bacillus coagulans CCM 2658 and a potential probiotic strain of Lactobacillus zeae CCM 7069. Moreover, bacterial adhesion to carbohydrates was also determined for all the bacteria tested. The last proposed and optimized technique was dynamic and electrophoretic light scattering characterizing surface properties such as -potential, average size distribution and isoelectric point. From this method, information was obtained on aggregation of bacterial cells and on electrostatic interactions. The combination of these methods was used as a complex tool to characterize adhesion of the tested bacterial cultures as a very specific, sensitive and key parameter of a successful probiotic microorganism that is influence by the multiple effects.
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Connection between stress resistance of bacteria to temperature-mediated stress and PHA accumulating ability
Nováčková, Ivana ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the involvement of polyhydroxyalkanoates (PHA) into the stress response of bacteria, specifically when they are exposed to temperature-mediated stress. The theoretical part focuses on the stress response of bacteria in general, the mechanism of response to temperature-mediated stress and PHA involvement into the stress response. The aim of experimental work was to study the correlation between the amount of PHA in the biomass of the bacteria and their viability after exposure to temperature-mediated stress. PHA producing bacterial strains Cupriavidus necator H16, Burkholderia cepacia and Burkholderia sacchari and non-producing mutant strain Cupriavidus necator PHB4 were used for experimental work. Bacteria accumulated poly(3-hydroxybutyrate) (PHB), the amount of PHB in the biomass was determined by gas chromatography with FID. Viability of live bacterial cells and cells after exposure to temperature-mediated stress was determined by using flow cytometry. It turned out that the content of PHB in biomass assists bacteria to resist the freezing shock. By comparing the viability of bacteria C. necator H16 and C. necator PHB4 after exposure to multiple stresses it also has been observed that PHB increased the resistance of cells against temperature-mediated stress and stress caused by low pH.
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Probiotics and prebiotics - a study of interactions, effects and co-encapsulation
Vrtná, Monika ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on encapsulation probiotics and co-encapsulation with some types of prebiotics. In theoretical part is aimed to probiotics, their general characteristics and application of probiotics in food industry. There are described prebiotics and their classification, there is described principles of encapsulation and encapsulation techniques. Methods, which are used for analysis of particles and encapsulation components were introduced too. The experimental part describes methods of prebiotics characterization by high performance liquid chromatography, thin layer chromatography and spectrophotometric methods. Cultivation of probiotics with prebiotics - hydrolyzed and non-hydrolyzed wad tested. Using flow cytometry cell viability was measured too. Finally probiotics and prebiotics were encapsulated, mainly by encapsulator machine. Long-term stability of particles during 6 week storage was observed. The particles were exposed to effect of artificial intestinal, gastric and bile juices.
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Study of carotenogenic yeasts doring growth by using advanced instrumental techniques
Vaněk, Martin ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
This work is dealing with application of advanced fluorescence techniques for gaining knowledge about culture development during fermentation of red yeasts. Flow cytometry was used for auto-fluorescence measurement a carotenoids quantitation. It was resolved that while carotenoids are stored mainly in membranes the technique was feasible. If red yeast starts to accumulate carotenoids into lipid bodies mainly throughout the course of stationary phase, then the method starts to fail. Flow cytometric method using cell size measurement and light scatter for lipid quantitation was proved as applicable, too. However, it works only if cells are not starved. Individual calibration for each species is needed for elimination inter-species variations of intracellular structures. Fluorescence lifetime imaging microscopy was also used for studying of red yeast. Inherent ability to resolve different fluorescent species of the same molecule, which arise due to different molecular environment, helps with quantitation of cellular lipidic structures changes through the course of fermentation. Increase in the levels of carotenoids and/or rigidity of membranes was found as mechanism of protection during metabolic shifts, when intracellular content is vulnerable to damage.
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Probiotics and prebiotics and their co-encapsulation
Šnajdarová, Karolína ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on study of probiotic cultures and options of coencapsulation with specific substrates called prebiotics. Lactobacillus acidophilus and Bifidobacterium breve were used as encapsulated microorganisms. As the specific substrates inulin, psyllium, apple fiber and hemp fiber were tested. Prebiotics was added to probiotics both in untreated form and in hydrolyzed form. The stability and viability of the cells have been studied by microscopy for the period of 6 weeks. Particles were stored in the enviroment of 2.5% citric acid. As the best substrate for cell growth the hemp fiber was found. The hemp fiber exhibited the highest increase of cells count. During observation of long-term stability in the model digestive conditions, particles with hemp fiber were destroyed in instestinal juices. For that reason, particles with hemp fiber could be used in food supplements with targeted transport. The bachalor thesis is also focused on characterisation of prebiotics components. The highest amount of total sugars in the group of non-hydrolyzed prebiotics exhibited psyllium. The highest content of reducing sugars both in the groups of non-hydrolyzed and hydrolyzed prebiotic had hemp fiber. By the method of thin-layer chromatography was found out that the main components of hydrolyzed prebiotics are glucose and fructose. Further, some disaccharides and oligosaccharides were obtained by TLC analysis. For characterisation of prebiotics by HPLC the column Rezex exhibited better sensitivity. HPLC analysis confirmed that the main components of prebiotics are glucose and fructose, some more complex carbohydrates were found too.
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Use of microscopy and flow cytometry to molecular characterization of carotenogenic yeasts.
Vacková, Hana ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The aim of presented bachelor thesis is to optimize the methods for study of yeasts using flow cytometry and fluorescence microscopy. The theoretical part deals with the structure of yeast cells, principles and application of flow cytometry and fluorescent microscopy. Use of microscopy and flow cytometry in study of pigmented microorganismswas reviewed too. In the experimental part methods optimization was performed on yeasts Cystofilibasidium capitatum, Rhodotorula glutinis, Sporobolomyce sroseus, Sporobolomyces shibatanus. Further, the growth curve and the viability of yeasts under the influence of stress factors (freezing and the heat shock 60 C) was measured. Cystofilobasidium capitatum has shown to be more resistant to stress than Rhodotorula glutinis. It was found that there is the possibility of using methylene blue instead of propidium iodide in yeast cell analysis by flow cytometry.
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Use of selected fluorescence techniques to study of yeasts and yeast metabolites
Mikheichyk, Nadzeya ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The scope of thesis was the optimization of methods for the study of yeast and their metabolites using flow cytometry and fluorescence microscopy. Red yeasts are characterized by overproduction of carotenoids and lipids, which are used in food, pharmaceutical and feed industries. Currently, intensive research is being carried on to find appropriate microbiological alternatives for synthesis of these substances. Present thesis is focused on selected yeast genera: Rhodotorula, Sporobolomyces, Cystofilobasidium and strain Phaffia rhodozyma. Yeasts were cultivated on different nutrient media, in which glucose was used as a nutritional source, and also on glycerol and whey as waste material. In two strains - Cystofilobasidium macerans and Rhodotorula mucilaginosa growth characteristics were determined on a synthetic glucose production medium. All studied strains were able to use waste substrates as a source of nutrients. Some of the strains displayed increased production of carotenoids, and, additionally, in some cases also relatively high production of lipids. In classical cultivation in lipid and glucose medium supplemented with vitamins the best production characteristics displayed Rhodotorula glutinisstrain. In glycerol medium the highest amount of carotenoids and lipidic substances produced Sporobolomyces shibatanus strain. Strain Sporobolomyces roseus showed the best production characteristics on whey as the main source of carbon. The results show use of whey and glycerol seems like appropriate option for potential carbon source to cultivate carotenogenic yeasts and production of carotenoids and selected lipidic substances as products with higher added value. Further optimization of nutrient medium on the given substrates is needed for higher production of selected metabolites. Fluorescence microscopy and flow cytometry have proved to be suitable options for determination of the observed metabolites in the cells, their amount and viability.
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