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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.
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Production, characterization and application of polymers of malic and glutamic acids
Čangelová, Katarína ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Subject of this bachelor thesis was study of biopolymers production using microorganisms. Polymer of malic acid was produced by Aureobasidium pullulans, a yeast-like fungus. Biosynthesis of polymer of glutamic acid was carried out by Bacillus licheniformis bacterium. Particular polymers, their microbial producers and possible application in different areas are characterised in the theoretical part of the thesis. Through experiments, the effect of various conditions on cell growth and production of these polymers was studied. Cultivation conditions were optimised in terms of polymers production. Subsequently, waste substrates - whey and poultry feathers were used. Microbially-produced polymer of malic acid was hydrolysed with 2 M sulfuric acid. Concentration of malic acid was analysed by IEC with a conductivity detector. Concentration of polymer of glutamic acid was determined by a spectrophotometric method using CTAB.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. 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 to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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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.
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Production, characterization and application of polymers of malic and glutamic acids
Čangelová, Katarína ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Subject of this bachelor thesis was study of biopolymers production using microorganisms. Polymer of malic acid was produced by Aureobasidium pullulans, a yeast-like fungus. Biosynthesis of polymer of glutamic acid was carried out by Bacillus licheniformis bacterium. Particular polymers, their microbial producers and possible application in different areas are characterised in the theoretical part of the thesis. Through experiments, the effect of various conditions on cell growth and production of these polymers was studied. Cultivation conditions were optimised in terms of polymers production. Subsequently, waste substrates - whey and poultry feathers were used. Microbially-produced polymer of malic acid was hydrolysed with 2 M sulfuric acid. Concentration of malic acid was analysed by IEC with a conductivity detector. Concentration of polymer of glutamic acid was determined by a spectrophotometric method using CTAB.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. 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 to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Production of carotenoids and enriched yeast biomass on rape-seed waste substrates
Mikheichyk, Nadzeya ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the group of the most widely used natural pigments with significant biological effects. Recently, microbiological production of carotenoids prior synthetic production is examined. The aim to the study was to analyse the properties of carotenogenic yeasts on waste rapeseed substrate. The main purpose of this thesis was conceived as a comparative study of three strains of carotenogenic yeasts: Rhodotorula glutinis, Sporobolomyces roseus a Cystofilobasidium capitatum. Yeasts were cultivated on different nutrient media, in which selected waste material was used as a nutritional source - rape substrate of two types with different lipid content. For all strains the growth characteristics on rapeseed substrate of the first type with a lower content of lipids were performed. All tested strains were able to use waste substrates as a source of nutrients. Most of strains produced increased amounts of carotenoids, in some cases accompanied with a relatively high production of biomass. As a potentially best producer of enriched biomass yeasts Rhodotorula glutinis and Sporobolomyces roseus were showed. They exhibited the highest production of biomass and metabolites in the media with the ratio glucose:rapeseed cake 3:1 and 1:1. The results showed that rapeseed cake can be used as a potential carbon source for growth of carotenogenic yeasts and production of carotenoids and selected lipids to some limit ratio of waste. Further processing of rapeseeed cake could be a suitable target for following study in related works.
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