|
|
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.
|
|
|
Preparation and characterization of biomaterials with natural UV filtres
Pavelková, Renata ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This presented diploma thesis is focuses on preparation and characterization of biomaterials with natural UV filters. The theoretical part is aimed at effects of sunlight on the skin and how to avoid these effects by using cosmetic products. The materials and methods for extraction of lipid part of the selected vegetable and microorganism species and methods for preparation and characterization of nanoparticles and emulsions were characterized. The practical part deals with extraction of lipid part of selected vegetable and microorganism species. The liposome particles with encapsulated active compounds were prepared. The particle size, polydispersity index, colloidal stability, encapsulation efficiency, SPF, antioxidant capacity, phospholipid content and cytotoxic effect on human keratinocytes were observed. These particles were used for preparation of the resulting emulsions. Their protection level on UV light were set and stability were tested by using analytical centrifugation.
|
|
|
Preparation of encapsulated enzymes for cosmetics application
Bokrová, Jitka ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on testing of an appropriate form of encapsulated enzymes intended for application in cosmetic and pharmaceutical industry. For encapsulation, proteolytic enzymes bromelain, papain and collagenase were used. These enzymes were encapsulated into alginate and chitosan microparticles prepared by an encapsulator and packed into liposomes. Encapsulation effectiveness was evaluated by analysis of total proteins. Particles stability was evaluated in model and real conditions by photometrical analysis of released proteins. Proteolytic activity of released enzymes in model and real conditions were observed too. Alginate and chitosan microparticles prepared by the encapsulator were found as an appropriate form of encapsulated enzymes designed to wound healing. Encapsulation effectiveness of these particles and stability in model conditions were good in comparison with liposomes. Hydrogel and water-oil emulsion were used for analysis of particles stability at real conditions. Hydrogel was found as a good option for preservation of particles as well as proteolytic enzyme activity. Emulsion made particles less stable and proteolytic activity of enzymes decreased rapidly. Encapsulation enables long-term stabilization of biologically active compounds as well as possibility of targeted transport and controlled releasing. Presented diploma thesis suggests possibilities of application encapsulated enzymes in designing more effective formulations for wound healing.
|
|
|
Use of encapsulation techniques for production of food for infants
Hoová, Julie ; Čarnecká, Martina (referee) ; Skoumalová, Petra (advisor)
The Diploma thesis deals with use of selected probiotic strains Lactobacillus acidophilus and Bifidobacterium breve in different forms in food for infants. The theoretical part is focused on describing probiotics, encapsulation methods and intestinal gut microbiota of infants. Further, characterization of individual periods of infant feeding and food for infants were introduced. In experimental part the possibilities of encapsulation and lyophilisation of probiotic cells were observed. Probiotic cells were encapsulated into alginate particles. The encapsulator was used for preparation of particles and the most appropriate particles were prepared by encapsulation nozzle with size of 300 µm. Moreover, probiotics viability was monitored by Flow Cytometry, Fluorescence Microscopy and by cultivation (CFU method). Viability of probiotics was monitored during long-term storage in selected food for infants. The appropriate shelf life of non-lyophilized alginate particles in real food have been set at 1 to 2 months. Lyophilized alginate particles could be stored for more than 3 months. Finally, the stability of the particles and viability of encapsulated and non-encapsulated cells in the gastrointestinal tract conditions were also examined. The viabilities of lyophilized cells and cells encapsulated in lyophilized particles were also compared. From the results obtained, non-encapsulated probiotic bacteria cells are more susceptible to negative effects of digestive juices, the percentage of dead probiotic cells after digestion was approximately 80 %. On the other hand, alginate particles showed cell protection from digestive juices, after incomplete cell releasing from particles the percentage of dead probiotic cells did not exceed 20 %. After adequate rehydration, similar results were gained with lyophilized alginate particles. Lyophilized alginate particles have been determined to be the most suitable application form for infants’ food.
|
|
|
Regulation of polyhydroxybutyrate production in Wautersia eutropha
Grossová, Marie ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
The aim of this work is study of microbial production of biodegradable polymers (polyhydroxyalkanoates; polyhydroxybutyrate PHB). Bacterium Wautersia eutropha was used for laboratory production of PHB. In this bacterium application of exogenous stress factors (osmotic shock, hydrogen peroxide and ethanol) was tested to enhance PHB production. Influence of exogenous stress on bacterial culture depended on stress factor concentration and time of stress application. Substantial influence on PHB yield exhibited changes of biomass production during stress experiments. The main part of this work is focused on study of PHB production in microbial cells exposed to stress in different growth phases. Application of strong stress in early phases of growth led to PHB and biomass decrease. The most suitable seems to be application of stress at the beginning of stationary phase accompanied by increase of PHB formation. Concentration of stress factor could be optimized too. The highest yields were obtained after addition of 0.5 % ethanol into production medium at 60th hour (PHB increase 21 %). These results can form a basis for new fermentation strategy which leads to enhanced effectiveness of PHB production.
|
|
|
Production of carotenoids by red yeasts grown on some waste substrates
Kubáčková, Martina ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the most widespread and abundant classes of natural pigments with utilised in food industry, pharmacy and cosmetics. Presented work was realized as a comparative study of some substrates for cultivation of red yeast Rhodotorula glutinis, which produces carotenoid pigments. Yeasts were cultivated in different media with several waste substrates (for example potato fiber, apple waste, cereals, grains, etc.) as nutrition sources. To some substrates hydrolytic enzymes isolated from the fungi genus Fusarium were added. Majority of waste substrates were acceptable for cultivation. However, the best conditions for production of carotenoid enriched biomass (6,4 g/l of biomass enriched 3,2 mg /g of beta-carotene) exhibited media with hydrolytic fungal enzymes.
|
|
|
Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
|
|
|
Influence of storage conditions on content of biologically active substances in apple fruits.
Ferdová, Jitka ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This study deals with antioxidants in diet and their effects on human organism. Further, it summarizes the agents affect the quality of apples in the course of long-term storage and it outlines the possibility of defence against them. In the experimental part methods of determination of antioxidant enzymes superoxid dismutase (SOD), catalase and polyphenol oxidace (PPO) in apples were introduced. The enzymes were measured in apples tissues in liquid nitrogen after 158 days in normal or modified atmosphere. In Apple the quantitative and qualitative analysis of proteins was realized. Further, some low molecular antioxidants (total phenolics, total flavonoids and vitamin C) as well as total antioxidant status were measured in frozen raw juice. This values were compared with values from apples analyzed immediately after the harvesting. Artificial inoculation with fungi was made in the last part and the fruitfulness was observed.
|
|
|
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.
|
|
|
Production and characteritzation of extracellular hydrolases from selected moulds
Skoumalová, Petra ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.
|
guest ::
RSS feed.