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Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions
Murmiliuk, Anastasiia ; Štěpánek, Miroslav (advisor) ; Šachl, Radek (referee) ; Hoffmann, Ingo (referee)
Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions Abstract: This PhD thesis is dedicated to the study of polyelectrolytes and their self-assembly in aqueous solutions. The morphology and ionization state of individual polymer chains were investigated as well as their co-assembly with oppositely charged species. First, the charge regulation of short and long weak polyelectrolytes in aqueous solution was investigated to deeper understand their pH-responsive properties. The changes of ionization degree and charge of oligopeptides composed of 5 amino acids with acid side-chains and 5 with basic side chains were followed upon varying the pH in order to reveal the effect of interactions between the like-charged and oppositely charged groups. It was shown that intra-molecular electrostatic interactions and conformational flexibility caused the suppression of the total charge and enhancement of ionization of the peptides. To get an insight into the distribution of local H+ concentration in the proximity of a polyelectrolyte chain, a modified polyelectrolyte structure was used with a fluorescent pH- indicator, covalently attached at the end of the chain. Ionization of the pH-responsive fluorophore revealed its effective pK which was compared with the local...
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Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.
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Preparation of food supplement containing probiotic bacteria and components with anti-inflammatory effect
Horňáková, Nikola ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The presented thesis discusses the issues of chronic inflammatory diseases of the digestive system and suggests the possibility of the alternative natural remedies improving the health conditions or prolonging the remission phase of IBD. The main goal is to design a probiotic supplement enriched by natural anti-inflammatory agents. Phytochemicals, concretely phenolic compounds, flavonoids, and carotenoids suppress harmful inflammatory processes by direct targeting the function of the immune cells or by inhibiting damaging oxidative stress in general. Therefore, there were several plants potentially rich for these biologically active substances selected. Concretely, blueberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), turmeric (Curcuma longa), peppermint (Mentha piperita), chamomile (Matricaria recutita), cinnamon (Cinnamomum zeylanicum), ginger (Zingiber officinale) and flax (Linum usitatissimum). The suitable parts of these plants were extracted by using a total of three different solvents – water, ethanol, and hexane for obtaining lipidic extracts. The concentration of total polyphenols, total flavonoids, total carotenoids, and the ascertainment of specific polyphenols was determined in the prepared extracts. These characteristics were enhanced by the measurement of antioxidant activity of the aqueous and ethanolic extracts. The interaction of individual samples with probiotic cultures (Bifidobacterium breve, Lactobacillus acidophilus) and the ability of extracts to influence the viability of probiotics in the model digestion process has been examined. The most interesting samples (aqueous extracts of blueberries, mint, chamomile, and cinnamon) were selected for further experiments. The possible cytotoxicity towards human intestinal epithelial cells was tested in vitro by the MTT tests utilizing the CaCo-2 cell line. Extracts showing the highest levels of beneficial phytochemicals and antioxidant activity, supporting the growth of probiotic cultures, and showing minimal cytotoxic effects on human intestinal cells were then co-encapsulated with probiotics into alginate particles of a diameter of 1 mm. Water extracts of mint, chamomile, and cinnamon have been chosen. The encapsulation efficiency of successfully entrapped probiotics and phenolic compounds was determined in prepared particles. Furthermore, the particles were studied during the process of model digestion, when the release of the desired substances in the various parts of the digestive system was observed and assessed whether the components would reach the crucial point of action – the colon. For the use of the proposed probiotic mixture as a dietary supplement, a recommended dose of 1 g has been determined. Lastly, possible adjustments such as lyophilization or sheathing by an extra protective polymerous layer, e.g. chitosan, were suggested to prolong the shelf life of the particles and volatile substances stability.
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Plant-based cosmetic products
Horváthová, Iveta ; Pavelková, Renata (referee) ; Skoumalová, Petra (advisor)
The aim of this bachelor thesis was studying an antimicrobial activity of selected herbal extracts and their encapsulated forms as liposomes. Furthemore, the thesis focuses on determonation of biologically active compounds, natural pigments and antioxidant activity. 6 kinds of herbs were studied - Humulus lupulus, Matricaria chamomilla, Taraxum officinale, Bellis Perennis, Cymbopogon citratus and Rubus Fruticosus. In teoretical part, the general characteristics of herbs and their use are described, it also includes the description of active compounds, microorganisms and used methods. Practical part is focused on characterisation of herbs from the perspective of antioxidant activity, determination of totaly phenolic content and flavonoids, chlorophyll and carotenoids. For analysis of antioxidant activity, concentration of phenolic compounds and flavonoids and antimicrobial activity aqueous and 20% ethanol extracts were prepared. Based on the obtained results, selected extracts were encapsulated into various types of liposomes consisting egg yolk, soy or sunflower lecithin. Generally, ethanol extracts showed higher content of active compounds. For chlorophyll and total carotenoid content determination the highest content of natural pigments was found in Taraxum officinale. Furthemore, antimicrobial activity of extracts and liposomes against 6 strains were determined - Micrococcus luteus, Serratia marcescens, Escherichia coli, Propionibacterium acnes, Staphylococcus epidermidis and Candida glabrata. The highest inhibitory effect was found in Matricaria chamomilla and Cymbopogon citratus samples. Encapsulation effectivity, size, stability and long-term stability of liposomes after a week and two months were determined. From the perspective of zeta potential all the particles were stable and reached a good encapsulation effectivity. It turned out that after two monts of storage the particles decomposed and active compounds into surroundings were released. Selected herbs generally had high content of antioxidants and active compouds. The most suitable lecithin for encapsulation was shown to be sunflower lecithin.
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Preparation of antimicrobial gels with plant extract and oils
Benešová, Markéta ; Hoová, Julie (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis was focused on studying antimicrobial effects of natural plant extracts and their subsequent application. The research was mainly focused on the antioxidant and antimicrobial activity of plant extracts and liposomal particles with their contents. Theoretical part of thesis was focused on the topic of natural antimicrobial and antioxidant substances, problems with bacterial resistance as well as describing features and attributes of used herbs, and characterization of gels and liposomes. Within the experimental part of thesis, three types of extracts (aquatic, ethanol and oil), prepared from 10 different types of plants, were described. The content of phenolic, flavonoid substances, also antioxidant and antimicrobial activity were then determined. After that, the contents of all extracts were encapsulated into liposomes, and size, stability, encapsulation activity, also antioxidant and antimicrobial activity of those liposomes were determined. The size distribution was wide, but in terms of the measured zeta potential, most of the particles were stable. All prepared extracts and liposomes containing them were tested for their antimicrobial activity. The tests were performed on a gram-positive genus of Micrococcus luteus and a gram-negative type of Serratia marcescens. The most effective oil extracts against both species were eucalyptus and fir, as well as oil extract of cloves, along with aqueous extracts of mustard seeds, cloves and juniper, or ethanol extract of anise which also has high antimicrobial effects. Finally, these extracts and liposomes were applied into the gels, which could be found useful in the pharmaceutical industry, as an alternative to externally used antibiotics, or in the cosmetic industry.
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Encapsulation of active and complementary components of energy drinks
Kšenžighová, Petra ; Vysoká, Marie (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis was focused on the development of an energy drink containing plant powders and active substances in both free and encapsulated form. Green barley, matcha tea, moringa and young wheat were chosen as plant powder samples. The theoretical part describes the active and complementary constituents of energy drinks, their nutritional properties and impact on health. The encapsulation itself is also described in this section. In the practical part, extracts from plant powders were prepared, in which bioactive substances were determined, namely total phenolic content, flavonoids and antioxidants. The total content of carotenoids and chlorophylls was also determined in the samples. By juicing the fruit, fruit juices were obtained in which vitamin C and antioxidant content were subsequently determined. Liposome particles differing in soy and sunflower lecithin were prepared containing caffeine, taurine and vitamin C. Encapsulation activity, colloidal stability, size and long-term stability were determined in the particles. All prepared particles, with the exception of encapsulated taurine in sunflower particles, were stable. Finally, a sensory analysis was performed, where respondents evaluated the prepared energy drinks in terms of taste, consistency, smell and appearance. Based on the results of measurements, a new energy drink was designed.
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Stability of PHA-based particles and fibres in different environments
Tarageľ, Matej ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The aim of this diploma thesis is the preparation of liposome nanoparticles enriched with PHA and PHA nanofibers. The nanostructures served to encapsulate extracts of lipophilic and hydrophilic nature. The characterization of the properties of nanostructures such as polydispersity, size, colloidal stability, long-term stability after exposure to various environments such as seawater, water from the Brno dam and tap water, and finally the cytotoxicity of fibers with extracts was addressed. The theoretical part is focused on different types of water, human skin, coffee and subsequently carotenoids. It continues by describing of possibilities of extraction and preparation of lipophilic and hydrophilic extracts and possibilities of their determination is discussed. Finally, it describes the possibilities of preparation and characterization of PHA based nanomaterials. The practical part deals with the preparation of liposome particles and fibers enriched with PHA with encapsulated extracts, their characterization, and their subsequent exposure to various environments. Monitoring of their long-term stability was carried out, but the release of the encapsulated extracts into the environment to which the nanoparticles and nanofibers were exposed was also measured. Finally, the interaction of nanofibers with live HaCaT cells was monitored, and cytotoxicity assays determined the viability of the cells after interaction with the nanofibers.
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Isolation, characterization and application of active components of prickly pear and aloe vera
Kocúrová, Petra ; Vysoká, Marie (referee) ; Skoumalová, Petra (advisor)
The main goal of this bachelor thesis was the study of antioxidant and antibacterial effect of natural plant extracts, specifically from pulp and peel of leaf of Aloe vera and Opuntia ficus–indica. Liposomes with encapsulated active compounds were prepared and their use in food and cosmetics industry was analyzed. The theoretical part was focused on botanical with and phytochemical description of natural plant and their use in nutrition and medical practice. Also encapsulation and methods used to analyze encapsulated substances were described. In the experimental part basic characteristics of extracts from two kinds of plantusing ethanol as a solvent in its different concentration (0 %, 20 %, 40 %, 80 %) were analyzed. Mainly totalcontent of phenolics, flavonoids and total antioxidant activity were determined. Selected extracts were also encapsulated into liposomes and the encapsulation efficienty, size, stability and long–term stability of liposomes were determined. Antibacterial activity was tested against Grampositive Micrococcus luteus, Staphylococcus epidermidis and Propionibacterium acnes and Gramnegative Serratia marcescems a Escherichia coli. Moreover, antibacterial acvivity against yeast Candida glabrata was tested. Finally, 2 ethanol extracts of plant samples were tested in terms of cytotoxicity by using cell line Caco–2 and profile of selected phenolic compounds ere determined in the selected extract samples by using high performance liqiud chromatography. 20% ethanol solution was chosen as the most efficient extract solvent which contained the highest concentration of total phenolic content and concurrently all tested extracts showed antioxidant activity. However, there was minimal or none antibacterial effect. Prepared liposomes from extracts were stable. Also product for cosmetics and food industry was designed, specifically healing gel with moisturizing effect and paste made of liposomes as food supplement for athletes.
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Development of new type of energy drinks based on coffee with active substances
Dudrová, Markéta ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
This bachelor thesis deals with development and preparation of new energy drinks with active plant component in free and encapsulated form. The theoretical part is focused on characterization of the energy drinks, their components and properties. The experimental part deals with the preparation of plant extracts, their characterization and subsequent encapsulation into liposome particles. All particles were determined for size, colloidal stability, long-term stability, antioxidant activity and encapsulation efficiency. Energy drinks based on various types of coffee were prepared and enriched with free or encapsulated guarana, goji, matcha or cocoa extract. In prepared energy drinks the content of caffeine, sugar, phenolics and antioxidant aktivity was monitored. Finally, selected energy drinks were subjected to sensory analysis, where appearance, color, smell and taste were evaluated.
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PREPARATION AND APPLICATION OF SOME BIOPOLYMERS, NANOPARTICLES AND NANOFIBRES FOR COSMETICS AND FOOD
Bokrová, Jitka ; Pekař, Miloslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation of nanoparticles and nanofibers with natural active ingredient and testing their biological effects. Modern types of application forms were prepared from biomaterials based on one or more natural polymers. Chitosan particles were prepared from cross-linked polymer using ultrasonication. A mixture of soy lecithin and cholesterol was used for preparation of liposomes. Poly-3-hydroxybutyrate was used for preparation of combined liposomes, too. All liposome particles were prepared by ultrasonication. Nanofibers were obtained from polyhydroxybutyrate using electrospinning. Mixtures of low-molecular antioxidants obtained by extraction from natural sources were used as active ingredients. Different types of teas, barks, herbs, spices, fruits and vegetables were selected as sources of natural antioxidants. Total phenolic and flavonoid content and total antioxidant activity of extracts were determined using spectrophotometrical methods. Obtained natural extracts were subsequently used for encapsulation. Prepared application forms were characterized in terms of their physicochemical properties. Particle size was monitored by dynamic light scattering. Colloidal stability of particles in suspension was determined using zeta potential. Spectrophotometry was used to evaluate the efficiency of encapsulation of active compounds into particles. The morphology of the new type of combined PHB liposomes was monitored by electron microscopy. Chromatography was used for quantification of individual components of particles. Morphology of nanofibers and incorporation of active agent into their structure were monitored using FTIR-ATR spectroscopy and electron microscopy. Afterwards, antimicrobial, cytotoxic and genotoxic effects of preparations were evaluated. It was found that the most suitable types of extracts for liposome preparation are aqueous and lipid extracts of natural antioxidants. Prepared particles showed excellent stability and good encapsulation efficiency. The study confirmed that incorporation of polydroxybutyrate into liposome structure does not reduce neither the colloidal stability of the particle, nor the efficiency of encapsulation process. Antimicrobial and antimycotic effect of preparations against model microorganisms Micrococcus lutues, Serratia marcescens and Candida glabrata was detected. It was found that process of encapsulation increases the inhibitory effect of natural extracts of antioxidants. The safety of preparations was assessed using two human cell cultures: epidermal keratinocytes and HaCaT cell line. Assays of cell viability and plasma membrane integrity were used to determine cytotoxicity of preparations. Low toxicity of liposome particles was confirmed by a series of cytotoxic tests. Obtained data showed that association of phospholipid with PHB polymer does not cause a significant increase in cytotoxicity in human skin cells. Genotoxicity testing on model procaryotic organism confirmed zero genotoxic potential of preparations. The new type of combined particles and polymeric fibers cant thus be used as a carrier for active ingredients, complex natural extracts, antimicrobial agents and many others.
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