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Soil application of hydrogels containing rhizobacteria
Hlaváčková, Barbora ; Černayová, Diana (referee) ; Kalina, Michal (advisor)
Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that can influence plant growth by their mechanisms. In recent years, there has been an increase in the use of these soil-supporting products in the form of biofertilizers to increase yields and improve crop growth in adverse conditions in an environmentally friendly way. The aim of this bachelor thesis was to assess the effect of the external addition of different application forms of PGPR on soil physico-chemical characteristics and on the growth of model plants of lactuca sativa without irrigation restriction in the first case and in the second case under irrigation restricted conditions. A particular strain of Azotobacter vinelandii CCM 289 was selected for the preparation of four forms of PGPR treatments, namely bacterial biomass suspended in PBS, alginate gel without bacterial culture, gel with bacterial culture and lyophilized gel with bacterial culture. The effect of the different treatments was compared with the negative control without the addition of PGPR, as different forms of treatments may achieve different efficiencies of rhizosphere colonization, which in consequence may have different effect on plant growth. To assess the effect of PGPR addition, soil physico-chemical characteristics (moisture content, pH) and growth characteristics of lactuca sativa such as number of leaves, height and width of aerial parts of these model plants were measured during the cultivation experiments. After the termination of the cultivation experiments, the basic growth parameters of the model plants from each treatment such as total plant length, length of aerial parts and roots of the model plants, as well as total weight of each plant and weight of their aerial parts and roots were determined. After drying, the total dry weight of individual plants and the dry weight of the aerial parts and roots were characterized. After drying, root density was analyzed by performing root scans and compared between treatments. Soil extracts from individual treatments after the cultivation experiments were analyzed to assess the effect of PGPR addition on the microbial activity of the soil used in the experiments by plate cultivation using Petri dishes with agar medium, and the average well colour development (AWCD) value determined using the BIOLOG EcoPlate kit. The obtained results were compared between the individual treatments and also between cultivation experiments conducted with different irrigation conditions. Physico-chemical characterization (pH, conductivity) was performed on soil extracts from the drained soils after the cultivation experiments. The amount of organic and inorganic fractions was analyzed on the dried soils of each treatment by TGA. The results were compared with each other and the effect of different forms of external addition of PGPR on the physico-chemical characteristics of the selected soil under conditions without irrigation limitation and under conditions with irrigation limitation during the experiments was assessed.
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Utilization of vibrational spectroscopy in structural analysis of microbially produced alginates
Kianička, Michal ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
This bachelor thesis focuses on the characterization of alginates using the main vibrational methods FTIR-ATR and Raman spectroscopy, supplemented by the carbazole method and thermogravimetry (TGA). The main objective was to utilize these analytical tools to study commercially available alginates and determine their monomeric composition. For this purpose, a calibration set was created using the ratios of poly(mannuronic acid) and poly(guluronic acid) standards, which was then used to analyze commercial alginates. The thesis focused on two forms of alginates - acidic and alkaline salts, aiming for a better understanding of their composition and subsequent application. The thesis revealed that the vibrational methods used were effective in analyzing the calibration sets, providing calibration curves for determining the composition of alginates. However, in the actual analysis of commercial alginates, these methods were found to be insufficiently accurate and reliable. Possible explanations include the presence of additional components in commercial samples or the influence of molecular weight. The results of the thesis indicate the need for further research tasks aimed at improving the accuracy and effectiveness of vibrational methods in the analysis of commercial alginates.
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Preparation and characterisation of polysaccharide hydrogels
Kulíšková, Kateřina ; Kadlec, Martin (referee) ; Smilek, Jiří (advisor)
This bachelor thesis deals with the study of preparation and characterization of alginate hydrogels. It focuses mainly on the ionic crosslinking of alginate using divalent metal cations Ca2+, Cu2+, Zn2+, Ba2+, Fe2+ together with the trivalent cation Fe3+ in order to compare the crosslinking abilities of different oxidation states. The exogenous method was used for the preparation of alginate hydrogels and the prepared hydrogels were thus studied in the form of millimetre spheres. In the experimental part, optimization was performed to find out the optimum gelation time, which resulted in the crosslinking of the hydrogels throughout their volume. Ca2+ ions were used as a reference due to their wide application and the extensive experience with these ions at the faculty. The knowledge from the optimization was applied to the preparation of the other ions samples. The viscoelastic properties of the hydrogels were investigated by rheological measurements in the form of amplitude and squeeze tests. All ions, except Fe2+, were found to crosslink the hydrogels throughout the volume. Based on the complex modulus, the order of ions was determined according to the degree of crosslinking, The degree of crosslinking also has major impact on the degree of syneresis.
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Determination of vibality of rhizobacterial cultures
Svobodová, Lucie ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on the study of viability of plant growth promoting rhizobacteria (PGPR). Viability was determined in three strains of Azotobacter vinelandii, namely CCM 289, DSM 87 and DSM 720, using flow cytometry with fluorescent probe PI, SYTOXTM Blue and DAPI after 120 and 72 hours of cultivation. Optimization of the appropriate fluorescent probe for the strain was performed, with the PI probe for strain CCM 289 being the most suitable. PI and SYTOXTM Blue probes can be used for strains DSM 87 and DSM 720. For the following experiments, strain DSM 87 was selected and subjected to the influence of different crosslinking reagents. Using a flow cytometer and staining with a fluorescent PI probe, the viability was verified after application of calcium chloride, barium, copper, ferric, aluminium and calcium sulphate solutions of 2, 0.2 and 0.02 wt. % to the culture. Calcium chloride, barium and calcium sulfate solutions had no significant effect on cell viability. On the other hand, when ferric chloride was used, a trend was observed where dead cells decreased with decreasing concentration of the solutions. This effect was also achieved with aluminium and copper chloride, but the use of the most concentrated solution resulted in the inactivation of a greater number of cells than in the previous case, whereas aluminium chloride resulted in the loss of viability of most of the cells present. Viability was also verified for cells released from the prepared gels. For the experiments, solutions of the aforementioned crosslinking agents were chosen at a concentration of 2 wt.%, and the culture was subjected to gelation under the experimentally determined conditions. A portion of the gels was subsequently left in phosphate buffer to allow for the re-release of cells. To facilitate this release, the enzyme alginase was added to break down the alginate. It was found that a concentration of 2 wt % of the selected crosslinking agents did not affect cell viability, i.e., the cells released from the gel appeared to be viable.
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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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