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Production of microbial enzymes and their stabilization by encapsulation
Hazuchová, Eva ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The present thesis deals with the production of microbial enzymes and their subsequent stabilization through encapsulation. The theoretical part focuses on microbial enzymes, especially extracellular hydrolases, their producers and characteristics. Within the theory is also discussed the possibility of the application of enzymes in the field of pharmacy and medicine. Experimental work was focused on the actual production of microbial enzymes and methods for their to stabilization. The production of proteolytic and lipolytic enzymes in dependence on time and the used culture substrate were followed. The highest enzyme production was observed in Aspergillus oryzae when cultured on wheat bran at the third day of cultivation. In the experimental part was further carried out the identification, isolation and purification of enzymes. A substantial part of the experiment was to stabilize produced microbial enzymes by encapsulation. Enzymes were entrapped into alginate particles with encapsulation efficiency in the range of 55-70 %. The highest efficiency exhibited encapsulated enzymes from Aspergillus oryzae. Subsequently, long-term stability of the encapsulated enzyme in two environments (in water and gel) was followed during six weeks incomparison with free enzyme. During storage of free enzyme a significant decrease in enzyme activities occured, especially between the fourth and sixth week of storage. On the contrary, in encapsulated increased enzyme activities were observed. Empty particles exhibited higher stability during storage in the gel than in water. In this thesis potential use of enzymes in the pharmaceutical industry as agents promoting digestion was tested too. According to the results, particles with encapsulated microbial enzymes could be considered as suitable for some pharmaceutical applications.
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Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
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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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Sensory quality of selected types of sweets
Michalíková, Veronika ; Němcová, Andrea (referee) ; Vítová, Eva (advisor)
This diploma thesis deals with the sensory evaluation of selected types of non-chocolate sweets. In the theoretical part the production technology of non-chocolate sweets is described, including raw materials needed. Furthermore, basics of sensory analysis with characteristics of sensory evaluation and selected sensory methods are mentioned. The experimental part deals with the sensory evaluation itself; the selected sensory methods were applied on samples of gummy sweets. Four colours/flavours (strawberry, orange, lemon, apple) from five producers were chosen for evaluation, together 20 samples. The aim was to assess the differences in sensory quality of samples of the same type from different producers. Untrained assessors, from Faculty of Chemistry BUT, were invited for sensory evaluations. Significant differences were found among the samples, glossy appearance and pleasant characteristic taste contributed to the positive evaluation, on the contrary the rigid texture, bland taste and odour and possible off-flavour contributed to the negative evaluation.
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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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Compression and Quality Assessment of ECG Signals
Němcová, Andrea ; Tkacz,, Professor Ewaryst (referee) ; Kudrna,, Petr (referee) ; Vítek, Martin (advisor)
Ztrátová komprese signálů EKG je užitečná a v současnosti stále se rozvíjející oblast. Stále se vyvíjí nové a nové kompresní algoritmy. V této oblasti ale chybí standardy pro hodnocení kvality signálu po kompresi. Existuje tedy sice mnoho různých kompresních algoritmů, které ale buď nelze objektivně porovnat vůbec, nebo jen zhruba. V oblasti komprese navíc nikde není popsáno, zda mají na výkon kompresních algoritmů vliv patologie, popřípadě jaký. Tato dizertační práce poskytuje přehled všech nalezených metod pro hodnocení kvality signálů EKG po kompresi. Navíc bylo vytvořeno 10 nových metod. V rámci práce byla provedena analýza všech těchto metod a na základě jejích výsledků bylo doporučeno 12 metod vhodných pro hodnocení kvality signálu EKG po kompresi. Také je zde představen nový kompresní algoritmus „Single-Cycle Fractal-Based (SCyF)“. Algoritmus SCyF je inspirován metodou založenou na fraktálech a využívá jednoho cyklu signálu EKG jako domény. Algoritmus SCyF byl testován na čtyřech různých databázích, přičemž kvalita signálů po kompresi byla vyhodnocena 12 doporučenými metodami. Výsledky byly porovnány s velmi populárním kompresním algoritmem založeným na vlnkové transformaci, který využívá metodu „Set Partitioning in Hierarchical Trees (SPIHT)“. Postup testování zároveň slouží jako příklad, jak by měl vypadat standard hodnocení výkonu kompresních algoritmů. Dále bylo statisticky prokázáno, že existuje rozdíl mezi kompresí fyziologických a patologických signálů. Patologické signály byly komprimovány s nižší efektivitou a kvalitou než signály fyziologické.
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Authenticity of natural plant component in cosmetics products
Kubalová, Michaela ; Fialová, Lenka (referee) ; Němcová, Andrea (advisor)
The purpose of this thesis was to study the authenticity of selected natural ingredients in cosmetic products. These were specifically cosmetic products that contained citruses, mint or lavender. Commercially available isolation kits were used for DNA isolation. The presence of plant origin DNA was verified by PCR method using primers specific for the ITS2 region of plants. The presence of limonene, a significant allergen contained in said plants, was determined in the samples by PCR method using primers for limonene synthase. At the same time, its presence was verified by HPLC method. In addition, two primers were tested for lavender and monitored for their efficacy, with no significant difference in the usage.
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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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Production of lipid substances by Metschnikowia yeasts grown on some waste substrates
Gonová, Dominika ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
Oleaginous yeasts posses the ability to accumulate increased amount of lipids under appropriate conditions. These microbial lipids vary in the composition of fatty acids which results in their wide application in the biotechnological industry. This master thesis focuses on the lipid production and fatty acids composition from waste substrates by the yeasts Metschnikowia depending on various cultivation conditions. The influence of temperature, the ratio of carbon and nitrogen in medium, and the concentration of different carbon sources was studied. The cheap and easy available waste substrates as glycerol and animal fat were used for the cultivation. The production characteristics of the yeasts were monitored by various technique including gas chromatography, Raman spectroscopy and fluorescence microscopy FLIM. Moreover, the partial optimalization of the pulse field gel electrophoresis was applied in order to characterize the karyotype of the yeasts Metschnikowia. All the studied strains were able to use the waste substrates and at the same time to produce lipids. The amount of lipids and mainly their compositions vary depending on the yeast strain and on the culture conditions. Nevertheless, the ability of the yeasts to produce significant amount of unsaturated fatty acids by manipulation of culture conditions was proved. The maximum lipid yield was achieved by M. pulcherrima 149 on glycerol medium and by M. andauensis 129 on medium containing waste animal fat.
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