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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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Nutritional benefit of cricket flour
Šťastná, Martina ; Zemanová, Jana (referee) ; Němcová, Andrea (advisor)
Edible insects have been part of the diet of many countries across the world for several decades. It is becoming increasingly popular in the europian developed countries, mainly because of the content of nutritionally important substances. The theoretical part of the thesis deals with the characterization of edible insects in terms of nutritional benefits, explains the reasons and method of breeding edible insects. For the analysis of nutritionally active substances, flour of house cricket (Acheta domesticus) was used. A. domesticus belongs to the representatives of insects legalized from 1.1. 2018 on the EU market. Of the significantly nutritional substances, proteins, lipids, fatty acids, fiber and selected minerals were analyzed. In this study, optimalization of vertical electrophoresis SDS-PAGE was performed to determine protein fragment sizes. In the second part the influence of acricket flour addition protein bars was investigated by sensory analysis. In the experimental part that was found that cricket flour contains protein, lipids and fatty acids in significant amounts. It also contains important minerals for human health such as magnesium, potassium, iron or zinc. However, the sensory properties of protein bars for consumers were not very attractive.
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Characterization of specific proteins form selected animal products.
Janhuba, Filip ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.
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Impact of elevated carbon dioxide concentration on Rubisco content in Fagus sylvatica and Picea abies
Hamříková, Dominika ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
In this bachelor’s thesis content of Rubisco in beech (Fagus sylvatica) and Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient carbon dioxide concentration (350 mol·mol-1) and elevated carbon dioxide concentration (700 mol·mol-1). The content of Rubisco in exposed and shaded leaves or needles was compared. The content of Rubisco was determined by SDS-PAGE method. A significant decrease in Rubisco content was demonstrated in conditions E versus A in shaded beech leaves. In other cases, the decrease was not so significant. A comparison of exposed and shaded needles also did not show a statistically significant difference, but in shaded beech leaves against exposed leaves Rubisco content was significantly lower, especially in conditions E.
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Protein calculator
Sedlo, Petr ; Maděránková, Denisa (referee) ; Škutková, Helena (advisor)
This work is focussed on studying and analysis of protein qualities while using experimental methods which are next confronted with numerical analysis, which have been introduced by the expansion in computer engineering. Within that task I built up a program for calculation of isoelectric point and molecular mass of an entered chain of amino acids. The program will draw a graph of pH dependance of molecular charge in the wide range of pH values in accordance with the given criteria. We can gain results very quickly and easily in this way, however, these results are just a rough estimate. We´re assumed to be aware of this fact because incorrect use of that program where exact analysis is required would cause errors.
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Analysis of low-molecular proteins in barley by the SDS-PAGE method during malting
Myslivcová, Pavla ; Svoboda, Zdeněk (referee) ; Mikulíková, Renata (advisor)
This diploma thesis deals with the analysis of low molecular weight proteins in barley during malting by SDS-PAGE method. Attention was paid to PR proteins, specifically LTP proteins and thionins, considered to be connected with gushing effect. Barley samples, malt intermediates and malt samples taken in 10 consecutive days to cover the whole malting process were used for the experiment. In total, 5 samplings were used for the analysis. Proteins extracted from the samples were separated by SDS-PAGE using a Tris-tricine buffer system. The protein lines of LTP proteins and thionins were identified in the resulting gels. The relative optical density values of the selected protein bands were obtained to assess the effect of malting on these proteins. A similar pattern of change in the content of mentioned low molecular weight proteins during the malting process was observed. This was confirmed by finding a statistically significant positive correlation between the relative optical density values of LTP proteins and thionins. Furthermore, the relationship between the low molecular weight protein content and the gushing potential and the microbiological contamination of the samples was investigated, but was not confirmed.
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The effect of temperature and drought on the glutenin proteins content in two genotypes of wheat
Vybíral, Lukáš ; Vespalcová, Milena (referee) ; Hrstka, Miroslav (advisor)
This bachelor thesis deals with the influence of water and temperature deficiency on protein content of gliadin fraction and on glutenin/gliadin ratio in two wheat varieties. The varieties selected for the experiment are called Pannonia and RGT Reform. Samples were cultivated at three different temperatures, 26 °C, 32 °C and 38 ° C, and irrigation was checked at the time of flowering. For wet-grown samples, the soil had a moisture content greater than 70 %, and drought-grown specimens grew on soil with a moisture content not exceeding 30 %. After harvest, the grains were ground into flour and glutenins and gliadins were extracted from the individual samples. SDS-PAGE was used for separation and protein quantification was performed by computer densitometry. A significant effect of the variety on individual gluten proteins was found. In the Pannonia variety, it has been found that the interaction of higher temperature and drought has no major effect on glutenin content and glutenin/gliadin ratio. The RGT variety was significantly affected by these conditions, resulting in a decrease in glutenin content and a significant change in ratio. In both varieties, the glutenin content was higher when cultivated at higher humidity but only at two higher cultivation temperatures.
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Analysis of glutenin fraction in winter wheat cultivated in ambient and elevated carbon dioxide concentrations
Čileková, Marta ; Vránová, Dana (referee) ; Hrstka, Miroslav (advisor)
Increasing carbon dioxide concentrations in the atmosphere has influence on plant’s photosynthesis and its metabolits. This bachelor thesis is focused on analysis of glutenin fraction in winter wheat (Triticum aestivum) var. Bohemia grown in ambient (AC) and elevated (EC; 700 mol.mol-1) carbon dioxide concentrations. Plants were cultivated in conditions with nitrogen fertilization (N+; 200 kg.ha-1) and without fertilization (N), in presence of UV-B radiation (UV+) and absence of UV-B radiation (UV), at drought (DRY) and with ambient irrigation (WET). Grain was harvested at 2014. The glutenin proteins were extracted from flour by isopropanol and DTT then separated by SDSPAGE and quantified by computer densitometry. Elevated carbon dioxide concentration decreased glutenin concentration in variant UV+ otherwise differences between AC and EC variants were nonsignificant. The nitrogen fertilization markedly increased glutenin content. Higher concentrations of glutenins were found in variants UV– WET and UV+ DRY. The highest concentrations of glutenins were found in conditions with presence of UV radiation, drought and nitrogen fertilization.
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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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The changes of Rubisco amount in Norway spruce grown at gradient of altitudes
Tomasz, Teresa ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
The study deals with differences of Rubisco enzyme content in Norway spruce needles (Picea abies L.) growing at different altitudes, thus at different concentrations of carbon dioxide. The collection of samples was performed in Jeseníky by Global Change Research Centre, Academy of Sciences of the Czech Republic. The samples of exposed and shaded needles were collected from 39 spruce trees in three altitudes – low (L), medium (M) and high (H). To separate Rubisco enzyme, the SDS-PAGE method was used, the content od Rubisco was determined by computer densitometry. The results of exposed needles confirmed that Rubisco content decreases with increasing altitude, however, in the case of shaded needles, there was no proof of a significant influence of altitudes on Rubisco enzyme amount. At low altitude, Rubisco content was significantly higher in exposed needles in comparison with shaded needles. At medium and high altitude no significant difference was detected between exposed and shaded needles.
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