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Study of genome of Metschnikowia yeasts by molecular methods
Schneiderwindová, Nicole ; Skoumalová, Petra (referee) ; Němcová, Andrea (advisor)
Yeasts of the genus Metschnikowia belonging to the family Metschnikowiacea are yeasts characterized by vegetative propagation through multilateral budding. These are yeasts widely distributed in nature. More than 35 species occurring have been defined in the wild. They most often occur on flowers, fruits, but also on insects or human skin. They have a wide range of uses due to their antifungal effects in agriculture and the cosmetics industry. This bachelor thesis deals with the study of usage of molecular methods to characterize selected species of yeasts of the genus Metschnikowia. It focuses on a detailed description of the yeast cell structure, karyotype and methods of reproduction in the theoretical part of the work. In the practical part on optimization and description of molecular methods including pulse gel electrophoresis methods used to separate the yeast genome and their subsequent observation of changes in individual parts of genome. First, the yeast was cultured under special conditions that are characteristic of Metschnikowia yeasts, then yeast DNA was isolated using methods suitable for DNA isolation, which was further examined by the PFGE molecular method. The DNA isolation procedure was first optimized for individual yeast strains, as it was necessary to verify the required ratio of low melting agarose to isolated DNA. That was because of it was important for the resulting gel blocks to be suitable for measurement by PFGE analysis. By optimizing the method was possible to create ideal blocks of isolated yeast DNA, which were subsequently subjected to PFGE analysis. Several measurements of PFGE analysis were performed at different time intervals in order to separate small and large yeast chromosomes. The CHEF standard of the yeast Hansenula wingei and the standard of the yeast Schizosaccharomyces pombe were used for the measurements. According to the measurement results, it can be determined that the yeast DNA isolation procedure and subsequent analysis by pulsed gel electrophoresis were successful, as the number of chromosomes of all used yeast species of the genus Metschnikowia was determined.
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Preparation of nanoparticles and nanofibers for application in anti-acne products
Tilšarová, Kamila ; Veselá, Mária (referee) ; Skoumalová, Petra (advisor)
The diploma thesis was focused on the preparation and characterization of nanoparticles and nanofibres with active substances from chosen herbs with the aim to apply this materials to the products against acne. Various types of extracts were tested on the content of polyphenols, flavonoids and antioxidation activity. These extracts were encapsulated to the liposomes and fibres of polyhydroxybutyrate. Prepared liposomes and fibres were tested mainly on antioxidation activity and antimicrobial activity against the strain Propionibacterium acnes. Then, liposomes were applied to cosmetic emulsions. These creams reported high antioxidation activity and excellent stability determined by analytical centrifugation. Prepared nanofibres also reported high antioxidation activity and antimicrobial effect as well. Finally, particles and fibres were tested in contact with human cells. In appropriate concentration, there was no cytotoxic effect and tested materials can be used in applications on problems with acne.
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Gold nano systems for the detection of molecules using surface-enhanced Raman scattering (SERS)
Benešová, Markéta ; Bernatová,, Silvie (referee) ; Skoumalová, Petra (advisor)
This diploma thesis was focused on the production and use of gold nanosystems to enhance Raman scattering. Metal nanoparticles, when interacting with electromagnetic radiation, form so-called localized plasmons, which can enhance Raman scattering. SERS (surface-enhanced Raman scattering) is a non-destructive analytical technique used in this work to measure the concentration and chemical changes in the rhodamine B molecule. Rhodamine B was subjected to photocatalytic degradation using two types of photocatalysts before measurement: TiO2-(H) and TiO2-(H)-Ag. The quantitative dependence of rhodamine B photodegradation on the presence of photocatalysts in a given period time was sought using photodegradation processes. The results of measurements using the SERS method were compared with the results obtained by UV-VIS spectroscopy. From the measured data, it was found that the photocatalysts significantly accelerate the photodegradation processes, because the Raman signal of rhodamine B decreased, while the signal decrease was most pronounced for the catalyst with added TiO2-(H)-Ag, less prominent but still statistically significant signal decrease was observed for the TiO2-(H) catalyst. In the control sample without the addition of photocatalyst, no decrease in signal was observed. In the next phase of the thesis, a sandwich immunoassay was designed that uses SERS to detect E. coli bacteria or other specific microorganisms in the sample. The first component of the sandwich immunoassay is gold nanoparticles, which carry a so-called Raman reporter, which has a clear Raman response in the spectrum, and gold nanoparticles amplify this signal, and antibodies, thanks to which the particles specifically bind to the microorganism. Another component is either gold layered slides or magnetic nanoparticles, which are modified with antibodies and serve to immobilize microorganisms. This system can be a fast and very accurate way to identify a given microorganism in a sample.
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Addition of probiotics to baby food products
Dudrová, Markéta ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
This Diploma thesis deals with preparation of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium breve enriched with prebiotics meant for application in baby food products. Natural extracts from matcha, moringa, young beat, young barley, chlorella and spirulina were selected as prebiotics. The theoretical part is focused on probiotic bacteria, their biological effects and their effects on the child´s body. The experimental part deals with the cultivation of probiotic bacteria with plant extracts, monitoring their viability and stabilization in an encapsulated form. Mixtures of probiotic cells with prebiotics were encapsulated into alginate particles to increase stability. Some of the alginate particles were processed by freeze drying. Mixtures of probiotic cultures with plant extracts were subjected to model human digestion by the action of model digestive juices in unencapsulated, encapsulated and lyophilized form. Selected extracts of plant materials were characterized in terms of amount of total and reducing sugars, total phenolic substances, individual phenolic substances and antioxidant activity. Further, two baby commercial dietary supplements containing probiotics were selected, which were characterized in terms of cell number and viability. Probiotic products were also subjected to model digestion.
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Isolation of active compounds from algae and their application in cosmetic products
Nováková, Laura ; Byrtusová, Dana (referee) ; Skoumalová, Petra (advisor)
The bachelor thesis is dedicated to the study of active substances and antioxidant effects of cultivated microalgae. We compared the results with a commercial alga that is freely available. In the theoretical part we focused on description of algal metabolites in cosmetics and a brief description of the methods used in their testing. In the experimental part there were analyzed three types of extracts (aqueous, alcohol and oil) from 4 species of algae. It is mainly the content of polyphenols, flavonoids and antioxidant activity that is determined. According to the results, the highest content of active substances was found in oil extracts. All extracts were encapsulated into liposomes. The average size of the nanoparticles was in range of 200 nm to 300 nm. The particles were stable enough also for long-term stability. The largest decrease was observed in the oil extract of the cultivated algae Chlorella vulgaris. The cytotoxicity of oil extracts was realized by MTT test on human cell cultures HaCaT. Finally, we concluded that the prepared liposomal particles could be used for anti-aging cosmetics.
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Production of extracellular hydrolases by specific mould species
Pokrývková, Zuzana ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
This bachelor's thesis is focused on studying the possibilities of producing extracellular hydrolytic enzymes by the fungus Penicillium lilacinum on two substrates. The theoretical part is focused on the characterization of selected hydrolytic enzymes, their characteristics, the possibilities of production and their applications. In the experimental part the production of hydrolytic enzymes by the fungus Penicillium lilacinum was performed in mineral medium and in a medium wherein as substrate yeast strains Sporobolomyces roseus and Rhodotorula glutinis were used. During cultivation process, production of cellulases, amylases, xylanases, lipases, proteases, and mannases was monitored. Samples were taken on 3rd, 5th, 8th, 10th and 12th day of cultivation. Production varies depending on time and substrate type. Amylase, cellulose and xylanase activity was measured in two steps - at the beginning of culture (3 to 5 day) and at the end of cultivation. The rest of enzymes were detected mainly in the 8th to 10th day of culture. The best three samples with the highest value of enzyme activities were further used for lyofilization and purification by ultrafiltration. Further, enzymes were tested for disruption of yeast cell wall. In the conclusion, the yeast protoplasts of yeast strains Rhodotorula glutinis a Sporobolomyces roseus were prepared successfully.
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Preparation of nanoparticles and nanofibers with antimicrobial components
Kubišová, Veronika ; Slaninová, Eva (referee) ; Skoumalová, Petra (advisor)
This thesis addresses the problem of inadequate current wound therapy and presents a solution in the form of nanomaterial-based wound dressings (coverings). Specifically, it focuses on the development and characterization of various nanofibrous materials with integrated liposome particles that could serve as a source of therapeutic agents and be useful specifically in the field of a wound therapy. The review in the introductory part of the thesis first focused on the mentioned problem, which describes the shortcomings of existing conventional dressing materials. A description of human skin from an anatomical and functional point of view was not omitted, nor was the wound healing process itself. Different types of covering materials were also presented. However, a large part was focused on nanomaterials and their use in the field of the skin wound therapy. The nanomaterials mentioned were mainly liposome particles and nanofibres, as the experimental part of the work was focused on these structures. The description dealt with the characterization of these structures as well as the biopolymers used for their preparation. However, the aim was to prepare nanofibers with liposome content, so the method of forming such systems was described. Various therapeutic agents are also an integral part of the covering materials, especially those that suppress the development of infection and reduce wound pain; therefore, the search focused on the antibiotic ampicillin and the analgesic ibuprofen. The experimental part was devoted to the actual production of nanofibers with liposomes and also to the qualitative demonstration of the presence of liposomes in electrostatically prepared nanofibers. The selected polymeric components of these systems were polyhydroxybutyrate (PHB) and gelatine. However, the nanofibers and liposome particles (as well as combined liposomes with PHB) were first prepared in their own form and characterized mainly in terms of the gradual release of the drug substances. The results obtained were then compared with those of therapeutic drug delivery via combined nanofibrous structures with liposomes. For these combined structures, the aim was to achieve synergy in drug delivery between these systems. The aim of this work was to create a biomaterial covering with the controlled drug release. The drugs contained in these materials were the aforementioned ampicillin and ibuprofen. An important part of the work was then to determine the safety of the prepared materials which were tested for cytotoxicity, where the tests performed were MTT test and LDH test. And the actual wound healing ability of the nanofibers was then monitored in a scratch test or a "wound healing" test. At the end of the paper, recommendations for future work on this topic are given.
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Encapsulation of active components and their applications in food industry
Malčíková, Hana ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The Diploma thesis is focused on encapsulation of bioactive compounds which are contained in selected superfoods for the purpose of application to childrens nutrition supplements. In view of the nature of selected samples, which are seaweed, walnuts, hemp seed and flax seed, the emphasis is put on the content of omega-3 and omega-6 acids. The theoretical part introduces topic of children food, further it describes the nature of selected superfoods and last but not least it contains screening of available childrens food supplements containing omega-3 fatty acids on the market. In the experimental part, an optimization of the appropriate type of extract was made. A 24 hour aqueous macerate and 20% ethanol macerate were selected for next analyzes. Samples were characterized by content of polyphenols, flavonoids and the antioxidant activity and they were used to forming of extracts were encapsulated into liposomes for which their encapsulation efficiency, stability and long-term stability have been tested. Hexan extracts were also prepared for the same reasons. It was found that liposomes of selected superfoods should not be stored for 12 weeks. The samples were used analyzed for the carbohydrate content by the duboise method, a protein content by the Kjeldahl method and the determination of the fatty acid profile by gas chromatography. Furthermore, the content of carotenoids and chlorophylls in algae samples was determined by spectrophotometry and this analysis was verified by high-performance liquid chromatography. The technique of high-performance liquid chromatography was also used to verified a content of polyphenols in the algae samples. Finally, a sensory analysis of prepared alginate supplements containing algae and cold-pressed oils was performer in combinations to achieve a higher omega-3 and omega-6 fatty acid content. The F01-labeled sample containing wakame algae was evaluated as the best one.
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Cosmetics preparations against acne
Měšťánková, Zuzana ; Skoumalová, Petra (referee) ; Němcová, Andrea (advisor)
Hemp is a plant containing a number of active substances that can be used in cosmetics and pharmacy. The subject of this bachelor thesis is the multiplication of active substances by mixing cannabis with herbs that have anti-inflammatory effects. The theoretical part contains a description of the determined antioxidants and cannabinoids. The experimental part focuses on measuring the content of these substances in extracts created from the dry parts of herbs and hemp variety Ferimon. The work is also focused on the preparation of a cream containing cannabis and herbal extracts, which are aimed at inhibiting the formation of acne. The antimicrobial effect of creams and extracts was investigated on bacteria Propionibacterium acnes and Micrococcus luteus. The analysis of the extracts shows that the largest amount of active substances contained a mixture of mountain arnica and hemp, which also showed a good antimicrobial effect against both types of bacteria. The creams showed a low inhibitory effect due to the low content of active substances.
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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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