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Production of polyhydroxy alkanoates from selected waste substrates
Vlasáková, Terézia ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This bachelor's thesis is focused on study of polyhydroxyalkanoates (PHA) production by bacterial strains Burkholderia sacchari and Burkholderia cepacia. It also deals with influence of various factors on their production. The main studied area was PHA production from substrate prepared from corn stover. In theoretical part a brief description of PHA was given. We also summarized options on their practical applications and possibilities of their bacterial production from waste substrates. In experimental part of work the impact of used precursors on biomass growth, PHA production and incorporation of 3HV monomer into the polymer chain was monitored. Further, a method of corn material hydrolysis was optimized. Bacterial cultures were cultivated on prepared hydrolysates. The best results in cultivation on hydrolysates of corn silage were achieved when the hydrolysis was performed by combination of chemical and enzymatic way. Cultivations of both bacteria were also transferred into the laboratory bioreactor.
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Involvement of polyhydroxyalkanoates into stress response of bacteria
Kučera, Dan ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates (PHA) into stress response of bacteria. The theoretical part of the thesis deals with the possibility of establishing the viability of microorganisms using modern techniques, in particular flow cytometry. Furthermore, the research focused on selected stress factors and PHA involvement in stress response was described. In the experimental part of the work the stress response with regard to the ability to accumulate PHA was assessed. Bacteria Cupravidus necator was used for the experiment. Its ability to accumulate PHA at a later stage of growth increased resistance to ethanol, high temperature and freezing. Conversely, the PHA-producing strain showed lower resistance to the action of inorganic acids and bases. This may be caused by different morphology of PHA-producing cells. One of partial objectives was also to study the possibilities of staining of living cells accumulating PHA using Nile red. The research proved that the dye penetrates into living cells at elevated temperature of 40-45°C. This temperature is not lethal to the cells and the intensity of staining is sufficient to distinguish PHA-producing cells using flow cytometry; that can be applied in the selection of industrial PHA producers.
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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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Hyaluronan ionocomplexes for cosmetic and farmaceutical applications
Cimalová, Jana ; Obruča, Stanislav (referee) ; Pekař, Miloslav (advisor)
This bachelor thesis is focused on the study of a system of cationic surfactant – hyaluronan. Using fluorescent spectroscopy the critical micellar concentration of Carbethopendecinium bromide (Septonex) was determined in three environments – water, 0,15 M NaCl, and buffer PBS, with three fluorescent probes – pyrene, perylene, and nile red. It was found that the physiological environments (buffer and NaCl) reduce the value of CMC about one order. During the experiments with native hyaluronan, precipitate and gel formation were observed. The following part dealt with aggregate behaviour of the new biologically acceptable amphoteric surfactant tetradecylfosfocholine (TPC) in the environment of water and NaCl according to the increasing molar molecular weight of hyaluronan. As a fluorescent probe perylene was used. These measurements showed that the molar molecular weight or a change of ionic force has no effect on the value of CMC and therefore there are no interactions with hyaluronan. The last surfactant to study was 1,2-dihexadecanoyl-3-dimetylammonium-propan (DPTAP), which is water insoluble. DPTAP is soluble in chloroform, but after evaporation of chloroform and after addition of hyaluronan, it precipitated. The results so far, have confirmed that in terms of research of aggregation by fluorescence spectroscopy the new cationic surfactants TPC and DPTAP are not suitable for complexation with hyaluronan.
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Potential use of waste lipid substrates and glycerol to production of carotenoids by yeasts
Kostovová, Iveta ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of this study was use of lipid waste substrates and waste glycerol as carbon sources for red yeasts. Six yeast strains, namely Rhodotorula glutinis, Rhodotorula rubra, Rhodotorula aurantiaca, Cystofilobasidium capitatum, Sporobolomyces roseus and Sporobolomyces shibatanus were used for cultivation. All studied yeast strains were able to utilize glycerol and rapeseed oil as a carbon source. Cultivation in oil medium was in most of strains accompanied by decreased production of carotenoids. Increased production of ergosterol, lycopene and biomass was observed in some strains only. Most of yeast strains cultivated in glycerol medium produced increased amount of biomass as well as carotenoids. The best producer of bi-omass and pigments was yeast strain Sporobolomyces roseus CCY 19-6-4 grown in medium containing glucose and glycerol in a 1:1 ratio.
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Characterization of active substances in several kinds of beer
Benešová, Pavla ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of this bachelor thesis is to analyse biologically active compounds, especially of phenolic character, in 10 kinds of alcohol-free beer in comparison with alcoholic beer. In theoretical part basic steps of brewery technology are described including alcohol-free beer production. Characteristic compounds occurring especially in Czech beer e.g. proteins, phenolics, bitter substances and also methods for their analysis are discussed as well. Microfluidic electrophoresis (Experion, BioRad) was used to beer protein analysis. Phenolic compounds, flavonoids, technological characteristics and antioxidative activity were measured by spectophotometry. Individual phenolics of selected beers were identified and quantified by LC/ESI-MS analysis too. In alcohol-free beers similar concentrations of biologically active compounds were found in comparison with alcoholic beers, especially in the case of beer phenolics.
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Controlled prodution of pullulan by yeast-like organism Aureobasidium pulluans
Skoumalová, Petra ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
Bachelor's thesis is focused on study of influence of exogenous stress factors on biomass and pullulan production by microorganism Aureobasidium pullulans. As a part of this work an overview of stress factors, pullulan producers, its structure, function and technological use was introduced. In the experimental part growth characteristics of Aureobasidium pullulans and pullulan production during growth in optimum conditions and under stress were analyzed. The reduced availability of oxygen resulted in a decrease of biomass production accompanied by increased pullulan production. Chemical stress induced by NaCl significantly affected mainly biomass production. The highest production of pullulan was found at 15 g / l of NaCl. Ethanol stress exhibited growth inhibition and at higher concentration also lack of pullulan production. Peroxid stress exhibited no effect on pullulan production. Short-time exposure to low heavy metal concentration (Se(IV), Cr(III)) influenced pullulan production more positively than long-term effect.
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Analysis of mycotoxins in barley and malt
Čuta, Robert ; Obruča, Stanislav (referee) ; Běláková, Sylvie (advisor)
This bachelor´s thesis deals with the mycotoxines present in cereals, especially in barley and malt. Teoretical part summarizes the knowledge of mycotoxines and their occurrence. The possibilities of mycotoxines determination are held forth in this thesis. In the experimental part an ultrasonic extraction metod of deoxynivalenol was optimized. For the deoxynivalenol determination was used a method of high-performance liquid chromatography with a mass detector (HPLC – MS/MS). This method was used for deoxynivalenol determination in barley and malt produced from this barley. The bachelor´s thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.
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Thermophilic Enzymes
Kuchtová, Ludmila ; Obruča, Stanislav (referee) ; Babák, Libor (advisor)
Thermophilic enzymes are produced by organisms living at temperatures between 60 and 80°C, in some cases even higher, and are active and stabile at these temperatures. This work deals with thermophilic and hyperthermophilic organisms, their habitats, ways of adaptation to environment with considerably higher temperatures than optimal temperature for most organisms, and recent findings and opinions about possible factors enhancing protein thermostability. Properties and behaviour of thermophilic enzymes and their current applications (i.e. PCR, starch industry) are summarized. Advantages and disadvantages of using of these enzymes in comparison to currently used mesophilic enzymes are compared. Possible applications of thermozymes in various industrial branches, where they could replace some environment polluting procedures, simplify technology and be economically important, are introduced.
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Degradation of biomaterials in model physiological conditions
Mikulíková, Zuzana ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of presented work was to study degradation of selected modified protein materials and optimization of methods usable for protein concentration changes in samples that undergo proteolytic degradation. In first part overview of biomaterials used in medicine applications, biocompatibility and of collagen materials and their applications was given. In experimental part some methods for proteolysis determination were tested using standards of albumin, collagen and selected amino acids. As a part of this work proteolysis of selected modified collagen materials was observed. First, following methods for total protein analysis were tested: ninhydrin method, biuret method, Hartree-Lowry, UV-VIS spectrophotometry and TNBS method (trinitro-benzen-sulphonic acid). For collagen proteolysis determination two of these methods were optimized: biuret method (peptide group detection) and TNBS (primary amino group detection). These two methods were applied on enzyme degradation of three collagen samples: soluble collagen standard, collagen standard type I (insoluble) and modified collagen material for clinical application. In soluble and modified collagens collagenase effect exhibited decrease of peptide bounds and, simultaneously, increase of primary amino groups according to enzyme concentration and enzyme:substrate ratio. In insoluble collagen analysis was strongly influenced by structure and conformation changes of material during degradation.
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