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Influencing of molecular mass of hyaluronic acid by fermentation process
Wikarská, Monika ; Jílková, Jana (referee) ; Velebný, Vladimír (advisor)
The biological function of hyaluronic acid (HA) – the polysaccharide, which consists of repeating units of N-actylglucosamine and glucuronic acid – is extremely broad and often contradictory, depending on its molecular weight (MW). The bacterial production provides HA of high molecular weight and purity. The objective of this thesis is to control the molecular weight of produced HA by the addition of hyaluronidases during the process of fermentation, resulting in reduction of viscosity of the medium followed by increase of yield. Hyaluronic acid was prepared by batch fermentation of the microorganism Streptococcus equi subsp. zooepidemicus. During the process of fermentation, the enzyme hyaluronate 4-glycanohydrolase (BTH) at various concentrations and different times of cultivation was added to bioreactors. The HA yield, the molecular weight and the polydispersity by the SEC-MALLS were monitored, together with the residual glucose concentration and the dynamic viscosity of the production medium. The formation of low and medium molecular weight HA was achieved. The addition of 10 U (BTH) / g (HA) had no significant effect on the molecular weight and therefore the HA function, but the corresponding drop of viscosity was noteworthy for the simplification of the product purification. Potential increase in HA yield was not confirmed because of the very low concentration of residual glucose and the method of precipitating low and medium MW HA.
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Study on the Supramolecular Structure of Humic Acids
Širůček, David ; Pekař, Miloslav (referee) ; Kalina, Michal (advisor)
This bachelor thesis is focused on the study of supramolecular structure of humic acids. This issue was studied on a number of HA samples which differed in source matrix (lignite, leonardite, soil, peat and compost). For these purposes, methods of size exclusion chromatography, dynamic and electrophoretic light scattering and UV-VIS spektrometry were used. In order to better understand the formation of the supramolecular structure of HA, the ways of its stabilization and how it was influenced by the change in pH, individual HA samples were studied in three pH-varying mediums (3,5; 7 and 12). The results obtained on a wide range of used humic acids are in accordance with the supramolecular theory of the structure of humic acids in aqueous solutions which describes them as freely-bound supramolecular associates of relatively small molecules having both polar and nonpolar parts and can be formed into more complex supramolecular aggregates depending on the conditions in the solution (pH, content of low molecular weight ions, etc.).
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Control the molecular weight of hyaluronic acid (HA) by adjusting the specific growth rate of culture Streptococcus equi subsp. zooepidemicus
Osičková, Jana ; Pepeliaev,, Stanislav (referee) ; Franke,, Lukáš (advisor)
This master thesis focuses on the effect of cultivation parameters on hyaluronic acid synthesis during its biotechnological production. The cultivation parameters were temperature of the cultivation, aeration, agitation, carbon source and addition of phosphatidylcholine. Changes in molecular weight and yield of hyaluronic acid, growth of biomass and medium viscosity were observed. From the obtained data we learned, that the specific growth rate greatly impacts final characteristics of hyaluronic acid. Under suboptimal temperatures a high molecular weight polymer was synthesised along with comparable yields from the control cultivation. High temperatures (40 °C) caused a dip in molecular weight. The next cultivation parameters were aeration and agitation. The highest molecular weights were obtained in cultivations with high agitation rates and intensive aeration, specifically 1 vvm/800 rpm and 2 vvm/800 rpm. Agitation had a bigger influence on molecular weight than aeration. When the carbon source was changed from sucrose to glucose, the lowest molecular weight and yield were obtained. Addition of phosphatidylcholine with concentration 160 mg/l had a positive effect on the cultivation, where the molecular weight grew and biomass growth was higher.
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Automatic improvements of images from 1D gel electrophoresis
Kovář, Martin ; Vítek, Martin (referee) ; Škutková, Helena (advisor)
In this bachelor’s thesis are explained the basic principles of electrophoresis and its modalities with focusing on 1D gel electrophoresis. It describes analysis of an electrophoreogram and causes of its possible distortion, and states specifications of applications of the method in microbiology, genomics and proteomics. The practical part presents development, optimization and outputs of a programme for automatic electrophoreogram analysis, which was created in Matlab environment. The analysis contains lane and band detection and computation of samples’ molecular weight. The ending of the thesis is constituted by evaluating efficiency of detection and accuracy of weight computation.
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Direct Synthesis of High-Molecular Polymers of Lactic Acid
Mikulík, David ; Kupka, Vojtěch (referee) ; Petrůj, Jaroslav (advisor)
This master thesis deals with the direct synthesis of polymers from lactic acid. The theoretical part focuses on both natural and synthetic ways of production the lactic acid monomer, their advantages and properties. Furthermore, the theoretical part focuses on the synthesis of poly(lactic acid) (PLA) from lactide, and direct polycondensation from lactic acid discussing about influences of catalysts, co-catalysts as well as chain extenders. The experimental part focuses on the synthesis of PLA polymers and co-polymers wherein investigates suitable catalysts, reaction medium for azeotropic dehydration and co-catalyst influence on products. Thermal and analytical analysis of PLA polymers are mentioned at the end of the experimental part.
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CHARACTERIZATION OF METALLOCENE-MADE POLYPROPYLENE WITH NARROW DISTRIBUTION OF MOLECULAR WEIGHT
Fojtlová, Lucie ; Poláček, Petr (referee) ; Bálková, Radka (advisor)
Metallocene based polypropylene (mPP) with very narrow distribution of molecular weight was peroxide-degraded to materials of four different molecular weights including the original mPP labeled MET1–MET3 and MET0, respectively. Double bonds formed after peroxide-degradation was proved on material surfaces by FTIR-ATR (attenuated total reflection of Fourier-transformed infrared spectroscopy). The decreasing molecular weight led to gradual decrease of the tensile strength, tensile modulus as well as the strain and to the decrease of the temperature of thermal decomposition. Confocal laser scanning microscopy (CLSM) of chemically etched surfaces of MET0–MET3 revealed supramolecular structure of commonly occurred structure (radical spherulites) but also supramolecular structure of form (sheaf-like structure). The latter was proved by XRD together with the fact that the content of form decreases with decreasing molecular weight. The mentioned structure differences were not visible on DSC curves because the amount of structure was small and melting temperature, temperature of crystallization and the degree of crystallinity remained the same for all four types of mPP. The structure of the original materials was also characterized after isothermal crystallization performed on differential scanning calorimetry (DSC) and under polarizing optical microscope (POM). The first was performed at 120–126 °C and the latter at 130 °C (Tic). The materials obtained on DSC always contained the structure and its amount increased with increasing Tic whereas higher content of form was always in MET0 with respect to MET3. The structure was proved by XRD and also by DSC heating run followed immediately after the isothermal process. The latter revealed two endotherms belonging to melting of and forms. The presence of form was on the surfaces proved by CLSM. The formation of structure was in-situ observed on POM and the amount of it decreased with decreasing molecular weight. The spherulite growth rate increased with decreasing molecular weight whereas the rate of crystalline portion expressed as half-time of crystallization decreased with decreasing molecular weight.
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The influence of molecular weight of polypropylene on isothermal crystallization of nanocomposites
Krajčik, Ladislav ; Poláček, Petr (referee) ; Bálková, Radka (advisor)
This work deals with the study of the influence of metallocene type polypropylene (mPP) molecular weight on isothermal crystallization of its nanocomposites filled with hydrophobic and hydrophilic nanosilica (1 and 2 vol.%). Real amount of nanosilica was determined by thermogravimetry. Isothermal crystallization was performed on differential scanning calorimeter (DSC) at 119–125 °C and observed in-situ under optical polarizing microscope (POM) at 128 °C using hot stage. The increase of mPP crystallinity degree in time was determined on DSC and spherulite type and growth rate was determined using POM. Supramolecular surface structure of the original and isothermally crystallited nanocomposites was observed directly or after chemical etching (mixture of mineral acids with KMnO4) on confocal laser scanning microscope. The crystal structure of mPP was in all tested materials verified by X-ray diffraction.
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Physical ageing of polypropylene
Weiss, Jiří ; Kučera, Jaroslav (referee) ; Tocháček, Jiří (advisor)
Physical aging of commercially produced polypropylene homopolymers Mosten was studied. The work is focused on monitoring the changes in physical properties of polymer in the time period 90 days from the date of preparation of the test specimen. The aging of polymers was realized at storage temperatures of 23 and 80 °C. The changes in crystallinity, yield strength, modulus, elongation and impact strength using DSC method, tensile tests and impact tests according to Charpy were monitored. Molecular weight distribution was determined by means of GPC at each polymer. Relationships between the changes in physical properties and polymer molecular weight were sought.
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The Study of Biopolymers Stability Using Light Scattering Techniques
Kratochvíl, Zdeněk ; Sedláček, Petr (referee) ; Kalina, Michal (advisor)
The stability of chosen biopolymers (or simple carbohydrates and amino acids) was in-vestigated using light scattering methods. Samples were prepared by dissolving the sub-stance in deionised water or in diluted acetic acid. Firstly, the effect of increasing concen-tration on zeta potential was observed and the optimal concentration for the next measure-ments was determined. Afterwards, the stability and the molecular weight of used sub-stances were studied during long-time storage in different time intervals. It was found out that chitosan, CMC, hyaluronate and sodium alginate were subjected to degradation whereas increase of the molecular weight and the zeta potential was observed in case of BSA. In the end, the samples were exposed to several different external effects, namely the influence of different temperatures, UV radiation and increasing ionic strength. The results showed that rather higher temperature has a significant impact on biopolymers stability. However, used biopolymers were hardly affected by UV radiation except BSA whose molecular weight increased considerably. Decrease of the zeta potential absolute values and the molecular size usually occurred with increasing ionic strength. The change of molecular weight was not detected.
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Synthesis of Mineral Oils by Oligomeration of 1-alkenes
Porubský, Tomáš ; Kučera, František (referee) ; Petrůj, Jaroslav (advisor)
The theoretical part presents a summary of existing information and procedures for the oligomerization of 1 alkenes. It also introduces suggestion of the new method for preparing poly(1-hexene) by controlled or living polymerization with nitroxyl radicals. In the experimental part was synthesized oligomer of 1-hexene with DBP or AIBN radical initiators and coordination Ziegler-Natta catalysts, which consisted of TiCl4 and organometallic compounds TIBA or TNHA. GC/MS analysis of the radical polymerization reaction mixture, showed no oligomer formation even after 6 hours. Effect of Ti/Al ratio, temperature and type of organoaluminium compund on conversion and molecular weight were investigated. Reaction products were analyzed by 1H NMR and GC/MS spectrometry. Results showed that the obtained oligomer was a mixture of oligomers with a degree of polymerization 2-9 and average molecular weight of 220-270 g/mol. Monomer conversion reached values of 72 98 %.
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