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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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SEC-MALLS study on hyaluronan-albumin interactions
Sereda, Alena ; Kalina, Michal (referee) ; Chytil, Martin (advisor)
This work is focused on interactions of hyaluronan (HA) and bovine serum albumin (BSA). These interactions were studied by SEC-MALLS method, where molar mass, root mean square (rms) radii, hydronamic radius and intrinsic viscosity of particles of the system were defined. Additionally, interactions were examined by measurement of DLS and zeta potential, where hydrodynamic radius and a value of the zeta potential were defined. The interactions were confirmed at any of the used media, but with different efficiency. Furthermore it was found out, that the high ionic strength minimizes the range of the interactions, magnifies the radii of the complexes, their intrinsic viscosity and zeta potential. It was proved that complexes become smaller in their radii and viscosity with growing BSA concentraton.
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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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Influence of nanoparticles on polymer chains in solution.
Bayer, Adam ; Chamradová, Ivana (referee) ; Jančář, Josef (advisor)
High surface area of used nanoparticles and their comparable size with polymer chains, are the most important characteristics of fillers in the preparation of non-Newtonian suspensions. Such structures at different concentrations have major influence on the rheological behavior of polymer solution in solvent. With increasing volume fraction of nanofiller, there is pseudoplastic or dilatant behavior present in the dissolved elastomers. Diploma thesis studies the influence of the concentration of different elastomers (SBR and SEBS copolymers) and nanofiller (hydrophobic nanosilica) in suspension, of rheological properties.
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Preparation of photon-upconversion nanoparticles
Kelarová, Štěpánka ; Křivánková,, Jana (referee) ; Hlaváček,, Antonín (advisor)
The present work aimed to characterize photon-upconversion nanoparticles based on lanthanides. The synthesis of NaGdF4:Yb/Tm@NaGdF4:Eu core-shell nanoparticles using a europium activator was designed based on the available literature. Prepared upconversion nanoparticles showed emission in visible spectral range due to energy migration-mediated upconversion (EMU). The upconversion mechanism was initiated using a 980 nm excitation beam. The optical properties of prepared nanoparticles were examined using fluorescence spectroscopy. This work describes the evolution of optical properties of upconversion nanoparticles in dependency on the europium concentration in the shell in the range of 1560 mol. %. It was proved that decrease of europium concentration from 60 mol. % to 15 mol. % led to the increased intensity of emitted radiation. Thus, the efficiency of the upconversion process can be improved by the appropriate choice of europium concentration. The present study compares the upconversion efficiency of prepared NaGdF4:Yb/Tm@NaGdF4:Eu nanoparticles with standard NaYF4:Yb, Er nanoparticles using erbium activator.
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Antimicrobial peptides and their application into foods
Janhuba, Filip ; Petrik, Siniša (referee) ; Márová, Ivana (advisor)
The presented thesis is focused on a study of antimicrobial peptides and their potential applications into foods. Two types of antimicrobial peptides were studied in detail - bacteriocin nisin and antimicrobial enzyme lysozyme. In theoretical part an overview of antimicrobial peptides used in food industry was introduced. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial peptides concentration. Commonly used spectrophotometric methods for quantitative analysis of total proteins as well as high performance liquid chromatography were tested. To determine the molecular weight and purity a modified SDS-PAGE with the addition of urea and tricine buffer was performed. Further, encapsulation of nisin into liposome was tested. Encapsulated particles were prepared by three different procedures: by ultrasonication, ethanol injection and TLE. Particles stability, distribution and size were studied by dynamic light scattering. Stability of encapsulated peptides in model conditions and model foods was studied too. Finally, antibacterial properties of the selected peptides using test system Bacillus subtilis were studied. Nisin already showed antimicrobial effect at low concentrations - about 1 µg/ml. The inhibitory effect of nisin was more evident in liquid cultures.
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Synthesis of SiO2/Au core/shell nanoparticles for enhancement of OCT signal
Kantorová, Martina ; Kolář, Radim (referee) ; Drbohlavová, Jana (advisor)
The aim of this master thesis is synthesis of SiO2/Au core/shell nanoparticles. These nanoparticles should improve the signal of optical coherence tomography (OCT). The procedure of nanoparticle synthesis is described in the work. Synthetized nanoparticles were analyzed by scanning electron microscopy (SEM), Ultraviolet-Visible spectroscopy (UV-Vis), Near-infrared spectroscopy (NIRS), Fourier transform-infrared spectroscopy (FT-IR) and Dynamic light scattering (DLS). Then, the synthetized nanoparticles were tested for enhancement of OCT signal. For this testing were also used commercial nanoparticles with the same structure and similar size.
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Characterization of organic nanoparticles with encapsulated antimicrobial peptides
Vejrostová, Petra ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on characterization of particles containing encapsulated antimicrobial enzyme lysozyme. The theoretical part deals with characterization of antimicrobial peptides and their description. Further part of review was focused on lysozyme, the selected representative used in this thesis, its structure, mechanism of action and possible usage. In the experimental part the Hartree-Lowry method was used for lysozyme detection, determination of encapsulation efficiency and for detecting the amount of lysozyme released after incubation in model physiological environment and in model foods. In process of encapsulation the highest amount of lysozyme was packed into 1% chitosan particles, manually prepared alginate particles and into liposomes. During study of stability of particles in model foods as the least stable manually prepared chitosan particles were found. The released lysozyme exhibited changes probably caused by its degradation. The highest influence on particles proved 3% acetic acid. During studying the stability of the particles in artificial digestive fluids as the most unstable manually prepared chitosan particles were found, while alginate particles were the most stable. The thesis also deals with changes in antimicrobial activity of encapsulated lysozyme in prepared particles and after its application into the model environments. A gram-positive bacteria Bacillus subtilis was used in order to test the antimicrobial activity. Antimicrobial tests showed that after encapsulation antimicrobial activity of lysozyme was substantially decreased in most samples. Size and stability of prepared particles was tested using dynamic light scattering.
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Study of hyaluronan-albumin systems using light scattering methods and calorimetry
Sereda, Alena ; Hermannová, Martina (referee) ; Chytil, Martin (advisor)
This thesis, which is a continuation of the previous Bachelor thesis, is dedicated to the study of polyelectrolyte complexes between hyaluronan (HA) and bovine serum albumin (BSA). Interactions between HA and BSA and a formation of complexes were studied by SEC-MALLS method, where a molar mass, a root mean square (rms) radius, a hydronamic radius and an intrinsic viscosity of particles of the system were defined. Furthermore the interactions were studied by calorimetric measurement ITC, where thermodynamic character of complex formation was determined. Additionally, dynamic and electrophoretic light scattering methods (DLS and ELS) were applied, where hydrodynamic radius and a value of the zeta potential were defined. Also the effect of higher temperature on the character and complex formation was examined by SEC-MALLS and ITC measurements. The interactions were confirmed at any of the used media, but with different efficiency. It was also proved that complexes become smaller in their radii and viscosity with growing BSA concentration. Furthermore it was proved, that the high ionic strength hinders surface charges of HA and BSA molecules and minimizes their mutual interactions. At the higher ionic strength the radii of the complexes, their intrinsic viscosity and zeta potential are increased. The higher temperature has only minimal effect on the formation of the complexes.
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Dynamic light scattering and electroanalytical investigation of hyaluronan-amino acids systems
Muchová, Johana ; Kalina, Michal (referee) ; Chytil, Martin (advisor)
The bachelor thesis deals with interactions between low-molecular and high-molecular weight hyaluronic acid (HA) and protonized amphiphilic amino acids Lysine and Arginine. The interactions were observed in the area of low aminoacids concentrations with in the range 0–15 mmoldm-3. The interactions occur between the carboxyl groups of HA and the amino group of the protonated amino acids. Proving these interactions would allow us to physically modify HA and further more, use such a system as a carrier of pharmaceuticals. The resistence towards the ionic strength at the concentration of 0,015 and 0,15 moldm-3 NaCl was investigated. Previous results show that the system with unprotonated amino acids at low concetrations of electrolyte in the solution is rather disturbing. Therefore, the interactions were reinforced by the aminoacids protonation using HCl. The amino acids were protonated not only completely, but also partly because of negative influence of chloride anions. To study the interactions pH-metry, conductance and Dynamic Light Scattering were used.
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