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Biotechnological production of selected biopolymers employing Haloferax mediterranei
Strečanská, Paulína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is to study of production of selected biopolymers, polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by Haloferax mediterranei. The first part contains theoretical background including properties of biopolymers, advantages and purposes of commercial production, characteristics and positive aspects of cultivation of extreme halophile archaea Haloferax mediterranei. The experimental part is focused on study of effects of concentration of waste substrates, such as proteolytic whey hydrolysate and feather hydrolysate on PHA and EPS production. The effect of medium volume and its correlation with acceess of oxygen on biopolymer production were studied as well. PHA, in particul copolymer P(3HB-co/3HV) was analysed by gas chromatography with FID detection. The content of 3HV in PHA to 15,09 % was achieved using medium which contains 25 % proteolytic whey hydrolysate without glucose supplementation. Medium with 10 % of feather hydrolysate and 10 % of feather hydrolysate with 30 g/l glucose proved to be suitable in volume 50 ml in 250 ml Erlenmeyer flask. Haloferax mediterranei was able to produce biomass and accumulate PHA using protein substrate without need of saccharide supplementation.
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Encapsulation of active substances into nanofibers and possibilities of their application
Procházková, Lucie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
The master thesis was based on the optimization of the production of nanofiber covers and to gaine the product for subsequent functional use. The production of nanofiber covers was made by electrospinning and forcespinning from selected materials. Polyhydroxybutyrate, gelatin, chitosan and alginate were used as starting materials. After successful optimization, these materials were enriched with active ingredients ampicillin and ibuprofen for the functionalized use of covers for more effective wound healing. The theoretical part was focused on the issue of skin, healing processes, types of wounds and nanofibers, the characterization of selected starting materials for the formation of nanofibers was also mentioned. The practical part was based on the lengthy optimization of the preparation of fiber covers and later enriched with active ingredients. Furthermore, combined covers made of different materials with contents of both active ingredients were designed. This was followed by the characterization of all prepared covers from the point of view of stability in the short and long term. The gradual release of active ingredients was determined spectrophotometrically and by hifh performance liquid chromatography. It was also important to determine the antimicrobial activity of selected active substances. At the end of all testing, combined coatings containing both active ingredients were used for safety testing with human keratinocyte cells (HaCaT). Safety testing was based on determining the viability of human cells using the MTT test, to verify the LDH test. A scratch test was also performed, a wound healing test after the application of devised combined covers.
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Preparation of DNA with local DNA structures
Lofítková, Ellen ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
In this thesis I focused on quadruplexes and cruciforms formed by DNA. Plasmids pBluescript and others derived from it by inserting oligonucleotid sequences were studied. Sequences forming quadruplexes and cruciforms were found by in silico analysis by QGRS Mapper and Palindrome analyser. Plasmids were transformed into E. coli, isolated and then cleaved with enzymes S1 nuclease and restriction endonuclease ScaI. Cleaving with S1 nuclease predicated presence of local structure. Combined S1/ScaI cleavage did not bring satisfying results due to lost of DNA during purification.
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Biotechnological production of PHA copolymers containing 4-hydroxybutyrate
Kovářová, Radka ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.
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Study on biotechnological potential of thermophilic gram-positive bacterium Brevibacillus sp. Bz
Filimonova, Anastasiia ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
Předmětem předložené diplomové práce je studium biotechnologického potenciálu termofilní grampozitivní bakterie Brevibacillus borstelensis BZ. Teoretická část obsahuje obecnou charakterizaci termofilních organismů a jejich termozymů. Popisuje také adaptivní molekulární mechanismy, které zajišťují termostabilitu těchto proteinů. Závěr teoretické části je věnován biodegradaci odpadních substrátů a polymerů na bázi přírodních a fosilních zdrojů. První část experimentální práce se zabývá produkcí hydrolytických enzymů na různých původních zdrojích a odpadních substrátech. Díky intenzivní enzymatické produkci na původních zdrojích je Brevibacillus borstelensis BZ považován za velmi slibného producenta termostabilních enzymů, konkrétně xylanáz a celuláz. Testovaný bakteriální kmen BZ byl schopen produkce termostabilních enzymů i na odpadních substrátech. Na vybraných substrátech kmen BZ přednostně produkoval xylanázy. Díky tomu byla xylanázám věnována zvláštní pozornost, kdy bylo testováno teplotní a pH optimum. Závěrem experimentální práce byla testována schopnost bakterie Brevibacillus borstelensis BZ degradovat vybrané polymery na bázi přírodních a fosilních zdrojů. Kmen BZ poskytl nový pohled na biologický rozklad polyethylentereftalátu (PET), amorfní frakce kyseliny polymléčné (PLA), semikrystalické PLA a polyhydroxyalkanoátů (PHA). Pozorováním povrchu PET fólie skenovacím elektronovým mikroskopem (SEM) se potvrdilo zdrsnění materiálu, přítomnost rýh a naprosté pronikání bakterie skrz fólii. Pokud jde o polymery na bázi přírodních zdrojů, PHA granule byly zcela degradovány. Studiem morfologie povrchu obou zmíněných PLA bylo prokázáno jasné zhoršení jejich struktury přítomností jam a trhlin na povrchu polymerů.
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Modification of porosity of bacterial cellulose in situ
Ondruchová, Barbora ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the production and modification of porosity of bacterial cellulose in situ using the bacterium Komagataeibacter xylinus. The theoretical part of the work was focused on the review of various methods of culturing Komagataeibacter xylinus and the production of porous samples of bacterial cellulose. The sizes of pores in bacterial cellulose depend mainly on the applied cultivation method. Bacterial cellulose produced statically or dynamically contains pores with the dimensions of approximately 0.02 µm to 10 µm. The difference in porosity in bacterial cellulose prepared by static and dynamic cultivation was confirmed experimentally. The production yields of bacterial cellulose were compared and discussed. Next, the porosity of the bacterial cellulose was modified in situ by the addition of wax particles. Scanning electron microscopy confirmed, that the accumulation of wax particles in the production medium could significantly support the porosity of bacterial cellulose and, at the same time, increase its production.
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Preparation and characterization of modern wound covers
Balášová, Patricie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.
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Biotechnological production of PHB using non-traditional carbon sources
Fialová, Tereza ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
The bachelor thesis was focused on studying the possibilities of using waste rosehip byproducts obtained after the production of liqueurs as secondary carbon sources for the production of poly (3-hydroxybutyrate) (PHB). Cupriavidus necator was used for PHB biosynthesis. All cultures were run as submerged batch cultures in mineral media containing microelements in Erlenmeyer flasks for 72 hours. Rosehip oil obtained by hexane extraction from rosehip seeds and an enzymatic hydrolysate prepared from rosehip waste pulp were used as secondary carbon sources. After 72 hours of fermentation, yields showed that rosehip oil is a comparable carbon source to fructose for culturing of C. necator, in contrast to the enzymatic hydrolysate. Fermentation using 20 g / l rosehip oil produced 10.82 g / l biomass and 6.20 g / l PHB. This is about 21.6% more biomass and 13.1% more PHB than was received by fermentation using 20 g / l fructose. Fermentation of C. necator using an enzymatic hydrolysate with a reducing sugar concentration of 20 g / l gave only 2.92 g / l biomass and 0.12 g / l PHB. Even the combination of enzymatic hydrolysate with fructose did not prove satisfying yields for the production of PHB. Next, in liqueur, rosehip oil and enzymatic hydrolysate was also determined the content of total reducing sugars, phenolics, and total antioxidant activity. Moreover, the fatty acid profile and the content of dyes was determined in rosehip oil. The results showed that all selected rosehip products showed a significant range of total polyphenols (2.04 - 12.4 g GAE /l sample) and antioxidant activity (1.3 - 2.0 mmol TE /l sample).
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Biotechnological production of polyhydroxyalkanoates on wheat bran
Guziurová, Pavlína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotechnological production of polyhydroxyalkanoates (PHA) from lignocellulosic waste – wheat bran. The aim was to determine whether the hydrolysate from wheat bran subjected to different types of pretreatment could be used as a substrate for the production of PHA using selected bacterial strains. The selected strains were Halomonas organivorans, Halomonas halophila and two strains of Schlegelella thermodepolymerans. Halophilic strains were proved as the best producers and were subsequently used for cultivations. The hydrolysates after neutral pretreatment were utilized by the bacteria most efficiently, due to the lowest content of microbial inhibitors (phenolic substances) where the bacteria produced the most PHA. The highest value of produced PHA was determined on the hydrolysate after neutral pretreatment by using the strain Halomonas organivorans, namely 2,82 g/l. The hydrolysate was also used for the production of lactic acid bacterial strains of Lactobacillus. The highest achieved concentration of produced lactic acid was 16,73 g/l by the Lactobacillus casei strain.
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Determination of biological activity of adiponectin, a recombinant protein using cell culture
Pernicová, Iva ; Rittich, Bohuslav (referee) ; Ševčík, Mojmír (advisor)
This thesis deals with the determination of biological activity of adiponectin, a recombinant protein using cell culture. First it was important to acquire the working skills for the cell culture of cell line 3T3-L1. An optimal concentration of inactivated fetal bovine serum in cell culture media was determined. A stimulation of the cell proliferation by HB-EGF, PDGF-BB and bFGF growth factors was observed at various concentration levels. Afterwards the biological activity of adiponectin was determined as an inhibition of growth stimulation with 5 ng/ml PDGF-BB. This biological activity assay for adiponectin was also conducted with lyophilized adiponectin and a growth factor bFGF (0.1 ng/ml). The lyophilization did not affect the biological activity of adiponectin.
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