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Utilization of advanced techniques of electron microscopy in altrastructural analysis of microbial cells and microbial biotechnology products
Havlíčková, Anna ; Sedláček, Petr (referee) ; Krzyžánek,, Vladislav (advisor)
In the theoretical part, this thesis deals with direct and indirect mechanisms of plant growth-promoting rhizobacteria, which is followed by a section on the selected model organism Azotobacter vinelandii. The second part of the theoretical section deals with electron microscopy starting with the possible interactions of the electron beam with the sample, through types of electron microscopes to various methods of preparing biological samples. The experimental part discusses the cultivation of selected bacterial cultures, the gelation of the cell cultures using chosen cross-linking agents (calcium chloride, malic acid, glucono--lactone with calcium carbonate) and, above all, various methods of preparation and analysis of pure and encapsulated cultures by the means of electron microscopy. In particular, cryogenic scanning electron microscopy and scanning transmission electron microscopy imaging were selected. Using both techniques, poly-3-hydroxybutyrate granules, which were confirmed to be elastic even at cryogenic temperatures, were observed inside the bacteria. Furthermore, it was found that cross-linking of extracellular alginate occurs even in samples without the addition of cross-linking agents. However, the hydrogel network present in these samples was significantly thinner than in samples with the addition of all cross-linking agents used. In this work, the density of the hydrogel network cross-linked with glucono--lactone was also compared. When using this agent with the addition of calcium carbonate, a significantly denser hydrogel network was observed in comparison with using the agent alone. Last but not least, when comparing the obtained results with the literature, it was found that the A. vinelandii bacteria form capsules, during the formation of the capsules the shape of the cells changes from an oval to a spherical bacterium and also the size of the given cell is reduced. From the resulting images, it was found that these capsules are made up of two layers, to which literature refers as electron-transparent intine and electron-dense exine.
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Utilization of novel strategies of alginate crosslinking in the development of next-generation bioinoculants
Gašparová, Dominika ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
Tato diplomová práce se zaměřuje na různé strategie síťování alginátu, s cílem posunout vpřed vývoj bioinokulantů nové generace. Zkoumání různých strategií gelování se aplikuje na pět komerčně dostupných alginátů a izolovaný bakteriální alginát. Experimenty zahrnující přípravu alginátového makrogelu sloužily ke stanovení výskytu a kvality síťování, které byly analyzovány amplitudovými testy. Výběr vhodných síťovacích činidel a stanovení jejich optimálních koncentrací byly klíčovými cíli. Síťovací činidla, která byla vybrána jako vhodná pro makro- i mikroenkapsulační procesy, byly ty obsahující chlorid zinečnatý, chlorid hlinitý, chlorid barnatý, síran vápenatý a chlorid železitý. Optimální podmínky síťování byly stanoveny při teplotě 7~°C a optimální délka doby síťování v rozmezí od 30 do 120 minut. Byly provedeny komparativní analýzy šesti vybraných síťovacích činidel a to prostřednictvím analýzy mechanických vlastností skrz squeeze test, termogravimetrickou analýzou, botnáním gelu a obrazovou analýzou. Síťování barnatým síťovacím činidlem prokázalo slibné výsledky při zachování obsahu vody během rehydratace, zatímco sítování zinečnatým síťovacím činidlem dosáhlo nejvyššího obsahu sušiny. Obrazová analýza odhalila menší gelové perly tvořené síťovacími činidly s obsahem hliníku a barya, což naznačuje jejich účinnost. Mikroenkapsulované gelové perly, zejména ty síťované s baryem, prokázaly potenciál díky jejich menší velikosti. Vrcholem pokusů bylo síťování bakteriální kultury, kde hliníková a barnatá síťovací činidla dosahovala nejmenších gelových perel. Obzvlášť síťování barnatým činidlem dosahovalo nejmenších velikostí s nejmenší odchylkou, indikujíc jeho efektivitu.
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Study on relaxation properties of hydrogels using rheological techniques
Lorenc, Pavel ; Klučáková, Martina (referee) ; Smilek, Jiří (advisor)
Submitted bachelor’s thesis deals with the study of relaxation and time stress of viscoelastic substances (hydrogels) using selected rheological techniques, especially with regard to oscillation tests. Hydrogels with different types of crosslinking were prepared and their properties were compared during the measurement. Agarose hydrogels (physically), alginate hydrogels (physicochemically), polyvinyl alcohol hydrogels (chemically) and hyaluronic acid hydrogels (ionically). In this bachelor's thesis, methods for measuring the relaxation properties of these hydrogels using a rheometer were designed and subsequently applied. We examined the relaxation properties of hydrogels using time-dependent tests that examined changes in amplitude over time. We performed a test that had 3 intervals, in which we monitored the response of hydrogels to the changes by changing the amplitudes of deformation with time. The relaxation properties of these hydrogels were studied, which showed significant differences in the behavior of hydrogels in measurements.By these methods, it was found that the structure of hydrogels and concentrations have an influence on relaxation. From the measured results, it was shown that the best relaxing properties have chemically crosslinked polyvinyl alcohol hydrogel, the lowest ability to relax was shown by physico-chemically crosslinked alginate hydrogel.The results from the measured data were finally compared, assessed and commented on the differences between the individual hydrogels.
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Development of a new type of energy drinks containing natural active components
Vysoká, Marie ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The aim of this thesis is the development and preparation of a new energy drink. The theoretical part of the thesis focuses on the comparison of the drinks available in market, the presentation of the main active ingredients and the proposal of enriching the new energy drink. It also describes the possibility of using an encapsulation and the method used to determine specific compound in energy drink or encapsulated particles. In the experimental part of the work, at the beginning, the main content of energy drinks were characterized, for instance caffeine, sugar, polyphenol and vitamin C. Subsequently the energy drink based on selected types of loose teas that were enriched with the encapsulated extract of acai, acerola, maca, goji and ginseng was designed. All extracts contained large amounts of polyphenols and antioxidants. In addition, acerola extracts contained very high amounts of vitamin C. Two types of particles, alginate particles and liposomes were prepared for encapsulation. For all particles, encapsulation efficiency, size, stability, and antioxidant activity were determined. Long-term stability was then monitored within three months. The particles were also exposed to artificial gastric, pancreatic and bile juice. All tested particles showed very good encapsulation efficiency. In terms of long-term storage stability in an aqueous environment. Particularly liposome particles, which also showed high stability by zeta potential measurement, were suitable. On the contrary, in case of alginate particles, when stored in an aqueous medium, the release of active substances occurred. Finally, an energy drink was created and subjected to sensory analysis. The best energy drink was based on black or green tea with alginate particles acai, goji and ginseng.
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Encapsulation of probiotics and prebiotics for use in nutritional products for children
Šnajdarová, Karolína ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The Diploma thesis deals with designing of probiotic dietary supplement for children with strains Lactobacillus acidophilus and Bifidobacterium breve and with prebiotics. Used prebiotics were Inulin, Chia fiber, Bamboo fiber, Chlorella + Spirulina and Yakon syrup. The theoretical part is focused on probiotics, prebiotics and their biological influence. In experimental part the possibilities of encapsulation into alginate particle and lyophilisation of probiotic cells were observed to find their good form to final nutritional product for children. Several types of probiotic with addition of prebiotics were tested in model conditions of gastrointestinal tract. It was found that addition of prebiotic highly increases viability of probiotic cells and their resistance to model conditions of gastrointestinal tract. In this case, the best prebiotic was found in Yakon syrup. The prebiotics were also characterised in terms of nutritional composition, amount of total and reducing sugars, oligosaccharides, proteins, lipids, polyphenols and chlorophyll were obtained. Finally, Chia fiber, Chlorella + Spirulina and Yakon syrup were chosen as prebiotics with best characterisation/properties. In conclusion, a dietary supplement with lyophilized alginate particles containing probiotic cells and with the most appropriate prebiotics were designed. Forms of the product were powder and gummy-bear.
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Isolation and characterization of natural active components and their application in food supplements
Šmídová, Veronika ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The diploma thesis is focused on the characterisation and isolation of naturally occurred active compounds, especially on lipophilic compounds. The next part of the thesis deals with enzymes and their encapsulation into alginate particles to suggest new enzymatic dietary supplement for children with optimal nutrient composition. The theoretical part is focused on the issue of child nutrition, accordingly the necessary nutrients that children need to grow without any health difficulties. It is also focused on the availability of enzymatic food supplements for children. Finally, the theoretical part deals with the lack of chosen natural substances and their effects in connection with cancer. In the experimental part, four types of oils obtained from seeds were characterized. The oils were obtained by two methods: cold pressing with a Yoda kitchen press and extraction in an organic solvent with Soxtherm. Seeds were linseed, sesame, pumpkin, and cumin. With these oils were characterized natural active substances, polyphenols, flavonoids, antioxidants, carotenoids, chlorophylls, and fatty acids. Liposomes were also prepared from selected extracts, in which an effect on intestinal tumour cells were observed. Furthermore, some enzymes were selected, and they were encapsulated into alginate particles with average size 450 µm. In these particles was specified encapsulation efficiency as well as the proteolytic activity after testing in digest juices. The last part of this thesis was focused on the optimization of a complete enzymatic food supplement with addition of alginate particles as enzyme carriers. Samples of these supplements were finally subjected to a sensory analysis.
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Use of microcalorimetry in the study of hydration of biopolymers
Bola, Tomáš ; Smilek, Jiří (referee) ; Krouská, Jitka (advisor)
This master thesis deals with the using of microcalorimetry in the study of hydration of biopolymers. Lactose has been selected together with the other biopolymers although it is not among biopolymers but disaccharides. Selected biopolymers are alginate, dextrane, chitosan and hyaluronan of two molecular weights. Lactose has been selected for these purposes mainly because it is a model example to determine whether or not the reaction to moisture between the other samples and the saturated salt solution occurs. The biopolymer hydration study, as opposed to the commonly used perfusion calorimetry method using the possibility of measuring with adjustable moisture has been used an isothermal microcalorimetry method where at two constant temperatures the reaction of the sample to the different moisture released by the saturated salt solution was monitored.
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Preparation and characterization of hybrid hydrogels matrix
Magera, Lukáš ; Kalina, Michal (referee) ; Smilek, Jiří (advisor)
Submitted bachelor’s thesis aims for preparation and subsequent optimisation of hybrid hydrogels, which represent material with high application potential because of their unique properties. Hybrid hydrogels have extraordinary mechanical resistance and other beneficial properties (e.g. transportation) due to their unique hybrid network structure, strong interpenetrating network entanglement and efficient energy dissipation system. This work engages in chemically-physically crosslinked hybrid gel, namely the combination of alginate-polyacrylamide. The hybrid hydrogel was formed and then underwent the tests of drying and swelling. The main purpose of the experimental part was to determine viscoelastic properties of hybrid hydrogels using standard oscillation tests. Swelling tests showed that the hybrid network containing limitedly swelling alginate gel reaches lower degree of swelling than the single unlimitedly swelling polyacrylamide gel. Viscoelastic tests unveiled an influence on the mechanical properties by value augmentation of the hybrid gel´s storage modulus in comparison with the reference alginate hydrogel. Hybrid hydrogel reported an increase in the linear viscoelastic area compared to the reference polyacrylamide gel, whose hybrid structure prevents from creating microcracks, that caused earlier rupture of the reference polyacrylamide gel.
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Study of barrier and transport properties of polyelectrolytes using diffusion techniques in hydrogels
Valentová, Kristýna ; Kalina, Michal (referee) ; Smilek, Jiří (advisor)
This diploma thesis was focused on study of barrier and transport properties of selected polyelectrolytes in hydrogel matrices by using diffusion techniques. The study of these properties was performed in horizontal diffusion cells where is observed the change in diffusion probe concentration over time. Diffusion experiments were performed on an agarose hydrogel with the addition of alginate, hyaluronic acid, polystyrene sulfonate, humic acids and as a model probe rhodamine 6G was used. Important parts of this thesis are also the methods which characterize the substances and hydrogel matrices such as rheology and potentiometric titration. The main aim of this diploma thesis was to investigate the effect of interactions between passing model dye (rhodamine 6G) and the appropriate gel (agarose + polyelectrolyte) on the fundamental diffusion parameters (effective diffusion coefficient, lag time, etc.).
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Preparation and characterization of enzyme-containing wound dressings
Jurová, Bronislava ; Szotkowski, Martin (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study and characterization of nanofiber wound dressings from natural biopolymers. Proteolytic enzymes, specifically collagenase and bromelain, were incorporated into these nanofiber covers. The theoretical part deals with a brief description of skin wounds, their healing and skin covers. There are also methods for preparing these covers and the methods used to characterize them. The practical part deals with the optimization of the preparation of nanofiber coatings based on polyhydroxybutyrate, gelatin, alginate and chitosan. These nanofibers were then enriched with active substances and their gradual release into the model environment was monitored. Finally, their proteolytic activity was determined for these substances.
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