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Preconcentration Techniques for Determination of Uranium Using the Modified Sorbents.
Moos, Martin ; Řehůřková, Irena (referee) ; Kanický, Viktor (referee) ; Kráčmar, Stanislav (referee) ; Sommer, Lumír (advisor)
The work has been focused on the preconcentration techniques for determination of uranium on the modified sorbent, with the final use of ICP-OES and ICP-MS. First ICP-OES at a wavelength of 385.958 nm was used for determination of uranium. Parameters were optimized and the effect of mineral acids (HCl, HNO3), tensides (Septonex, Ajatin) and organic reagents like (4-(2-pyridylazo)resorcinol, ammonium pyrrolidinedithiocarbamate, 8-hydroxyquinoline-5-sulphonic acid and 1,2-dihydroxyanthraquinone-3-sulphonic acid) was observed. For ICP-MS an isotope 238U was chosen. As an internal standard 209Bi (200 ?g•l-1) was used. Modified silica gel was used (Silicagel tethered with C18, C8 alkyls and phenyl) and two types of Amberlite XAD 4 and XAD 16 for the preconcentration of uranium (VI). Commercially produced Amberlite was modified as follows: Drying at 100 °C (24 hours), milled, minced sorbent was selected and fractionated to particles size ranging from 0.32 to 0.63 ?m and finally activated in methanol. The deionised water and Septonex (5•10-3 mol•l-1) at pH 8 were used for Amberlite conditioning. The optimal sorption of uranium (VI) proceeded at pH 8 in the presence of 4-(2-pyridylazo)resorcinol or ammonium pyrrolidinedithiocarbamate with Amberlite XAD-16 moreover with 1,2-dihydroxyanthraquinone-3-sulphonic acid. All organic reagents have mass concentration five time larger than uranium . For the elution of uranium the mixture of 1 mol•l-1 HNO3 with acetone (ratio of 1:1) was used. Acetone was evaporated and the final analysis performed using ICP-OES. The preconcentration of uranium (VI) using Silicagel-C18 conditioning was performed with ethanol, deionised water and Zephyraminu (5•10-4 mol•l-1) at pH 8. For the Silica-C8 and Silicagel-Phenyl the same conditioning procedure without zephyramin was used. Silica-C18 had the highest sorption efficiency with 1,2-dihydroxyanthraquinone-3-sulphonic acid, Silicagel-C8 with ammonium pyrrolidinedithiocarbamate and Silicagel-Phenyl with 8-hydroxyquinoline-5-sulfonic acid, all organic reagents have mass concentration five time larger than uranium concentration. As an optimal elution mixture acetone and ethanol (ratio of 1:1) in the presence of 1 mol•l-1 HCl was evaluated. Acetone and ethanol was evaporated and the final analysis was performed using ICP-MS. Silicagel-C18 was evaluate as the most effective in the presence of zephyramin and 1,2-dihydroxyanthraquinone-3-sulphonic acid. Sorption was also tested in the presence of 20 ?g•l-1 microelements (Be, V, Co, Ni, Y, Pb, Th, Cd) and in the presence of a defined concentrations of K, Na, Ca, Mg, Al and Fe. Direct analysis of uranium was carried out on samples of water from the river Ploucnice and Turonian aquifers containing uranium, 3,5 and 19,3 ?g•l-1. Different type of water required preconcentration of uranium (VI) on Silica-C18 in the presence of 1,2-dihydroxyanthraquinone-3-sulphonic acid and zephyramin. The analysis results gave statistically satisfactory results, which were confirmed using standard addition of 20 ?g•l-1.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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PRODUCTION OF BETA-GLUCANS AND OTHER POLYSACCHARIDES BY YEAST AND MICROALGAE
Byrtusová, Dana ; Kráčmar, Stanislav (referee) ; Kovalčík, Adriána (referee) ; Márová, Ivana (advisor)
Beta-glukany jsou polysacharidy složeny z monomerů D-glukózy. V dnešní době se -glukany těší zvýšené pozornosti zejména kvůli imunomodulační aktivitě a využitelnosti ve farmaceutickém a potravinařském průmyslu. Saccharomyces cerevisiae je dodnes jediným kvasinkovým zdrojem požívaným v biotechnologické produkci. Avšak některé kvasinky z oddělení Basidiomycetes, které jsou schopny produkce lipidů a karotenoidů, mohou být využity rovněž jako alternativní zdroj -glukanů. Dizertační práce se zabývá možností a optimalizací produkce -glukanů a dalších mikrobiálních sacharidů u karotenogenních kvasinek a mikrořas. Testovány byli zástupci rodů Rhodotorula, Sporobolomyces, Cystofilobasidium a Dioshegia. Z nekarotenogenních kvasinek byly do screeningu zařazeny kvasinky rodu Metschnikowia, askomycetní kvasinky a z mikrořas zástupci zelených a červených řas. Experimentální část cílí rovněž na možnosti koprodukce dalších metabolitů, jako jsou lipidy, pigmenty a extracelulární polymery. První část experimentu se zabývá vlivem čtyř C/N poměrů (10:1, 40:1, 70:1 a 100:1) na produkci biomasy, -glukanů, karotenoidů a lipidů. Ze všech testovaných kmenů, S. cerevisiae CCY 21-4-102, C. infirmominiatum CCY 17-18-4, P. rhodozyma CCY 77-1-1 a R. kratochvilovae CCY 20-2-26 vykazovaly nejvyšší produkci -glukanů a byly proto vybrány k podrobnější optimalizaci, zejména osmotického stresu, teploty a zdroje dusíku v kultivačním médiu. Dodatečně, kmen R. kratochvilovae CCY 20-2-26 je schopný produkce extracelulárních glykolipidů a S. pararoseus CCY 19-9-6 extracelulárních polysacharidů. Následně bylo stanoveno množství -glukanů u dalších dvanácti kmenů S. cerevisiae a rovněž možnost produkce polysacharidů u mikrořas.
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Complex characterisation of medical herbs and study of their utilization in food industry as a source of functional components
Burdějová, Lenka ; Kráčmar, Stanislav (referee) ; Šilhár, Stanislav (referee) ; Polovka, Martin (advisor)
Presented thesis deals with the complex characterization of extracts from medical plants using spectroscopic and chromatographic techniques, with emphasis on the influence of solvent, post-harvest treatment of the sample, location and year of production on selected parameters. Part of the work is devoted to application of selected extracts from medical plants in suitable form into beverages. The thesis is divided into two parts. At the first part of the work combination of electron paramagnetic resonance, ultraviolet visible spectroscopy, liquid chromatography and inductively coupled plasma optical emission spectrometry was applied for characterization of aqueous, ethanol and dimethylsulfoxide extracts from 10 selected medical plants, which were collected from two different locations during two years (2015, 2016) and post-harvestly treated by two ways (freezing, drying). In total, 39 parameters were determinated: namely total phenolic compounds content, total flavonoids content, 14 specific phenolic compounds, colour characteristics in the CIE L*a*b* system (L *, a*, b *, C*, h°, BI), radical-quenching activity using the cation radical of the 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid), antioxidant/pro-oxidant activity using the spin trapping technique in the presence of 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO)/potassium peroxodisulphate and content of 15 minerals (Al, As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, Zn). The results confirmed that the studied parameters of medical plants are influenced by the extraction system, post-harvest treatment of samples, year of production, origin of samples (locality) and species composition. It has been shown that the type of extraction solvent significantly affects the composition of the extracts. On the basis of these analyzes, for food application – the isolation of the main functional components (phenolic compounds, flavonoids) –50% ethanol, post harvest treatment by drying and samples of Mentha piperita, Melissa officinalis, Hypericum perforatum and Salvia officinalis were selected. The second part of the thesis was focused on the application of selected extracts of medical plants to beverages (syrups). The above mentioned plants, 25% ethanol instead of 50% ethanol (due to the limitation of the ethanol content in final raw material), were selected for the application. At first, extraction of medical plants was optimized. Suitable ratio of material and solvent (1:10), extraction time (8 h) and herb absorbency (50–62 %) were determined. The prepared extract after filtration was applied to the syrup concentrate in different ratios to produce one-species syrups and further sensorially tested to select the suitable recipe, the best flavour and basis for the production of two-species syrups. The mint syrup was the most tasty and generally the most acceptable one-species syrup, and sage syrup was selected as the basis for two-species syrups. Further, the most appropriate recipes of the two-species syrups were chosen, the most sensorially acceptable one was selected and further tested if it would be appropriate to colour them. Preferentially, it has been found that it is not necessary to colour two-species syrups. On the basis of the sensory analysis, it has been found that peppermint and sage-peppermint syrups were the most tasty and acceptable syrups, which could be, after further testing, produced industrially to enrich the food market.
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The Use of Separation Methods for the Study of Selected Drugs and Pesticides in Water
Burešová, Jitka ; Hajšlová, Jana (referee) ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
This work is focused on the study of penetration of pesticides and pharmaceuticals in the aquatic environment. The aim was to develop and verify the optimal method for long-term monitoring of the occurrence of selected pesticides and pharmaceuticals in surface waters. Two frequently used analgesics ibuprofen and diclofenac, and carbamazepine that is used to treat epilepsy, were chosen from the group of drugs. A group of 41 pesticides were selected for monitoring too. The attention was focused on a group of urea pesticides and phenoxyacetic acids, as well as on selected pesticides falling to the group of priority substances or identified as environmental quality standards. Some of pesticides were selected because their consumption in the Czech Republic exceeds 30 tons a year. Liquid chromatography with tandem mass spectrometry (LC / MS / MS) was used for the determination of selected analytes in surface waters. Two optimized analytical method were developed. Ten analytes were determined in negative ion mode and 34 analytes in positive ion mode. Solid phase extraction (SPE) was used for purification and concentration of the samples. Developed methodology was validated and used for monitoring of selected compounds in samples of surface waters from the Morava River basin. Samples were collected on the rivers Morava and Svratka monthly for two years. Samples from the river Blata and stream Nivnička were collected for one year. Surface water samples were also collected from the river Litava. Four samples were taken throughout the length of the flow during one day. Smaller streams were chosen for monitoring because of the probability of occurrence of pesticides. They flow abundantly agriculturally cultivated areas. Drugs ibuprofen and diclofenac were detected in all taken samples, carbamazepine mostly. Among the most commonly occurring pesticides were bentazon, diuron, isoproturon, carbendazim, MCPA, MCPP, propiconazole, tebuconazole and 2,4-D.
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The factors influencing sensory quality of processed cheese analogues
Sůkalová, Kateřina ; Kráčmar, Stanislav (referee) ; Diviš, Pavel (referee) ; Buňka,, František (advisor)
The presented thesis deals with the monitoring sensory quality of processed cheese analogues, focusing mainly on the taste (flavour) and related content of volatile (aroma active) substances. Model samples of analogues were produced by a standard procedure at Tomas Bata University in Zlín. The experimental part was divided into two experiments, which differed in the composition of model samples of analogues. In the first experiment, the traditional fat (butter) was completely replaced by selected vegetable fats (palm, coconut, mixed), in the second experiment only a part (1% w/w - expressed on the total weight of the sample) of butter was replaced by vegetable oils (apricot, flax seed, currant, grape seed). Solid phase microextraction in conjunction with gas chromatography with flame ionization detection was used to determine volatiles. Methods based on valid international standards were used to evaluate the sensory quality of samples, focusing mainly on flavor, namely evaluation using scales (ISO 4121), profile test (EN ISO 13299) and ranking test (ISO 8587). The aim of the work was to assess the effect of the addition of various vegetable fats/oils on the above parameters, at the same time their changes were monitored during 6 months of storage (at 6 ° C). The results showed that the vegetable oil used affects both the sensory quality and the content and composition of volatile substances of analogues. Significant differences between samples were mainly in taste, aroma and overall acceptability. The decreasing overall acceptability of the samples can be expressed in experiment I by the order: product with butter coconut palm mixed fat; in the case of experiment II: product with butter apricot = flax = grape currant oil. Mixed fat, currant and grape seed oil proved to be unsuitable for the production of analogues, on the contrary, analogues with coconut fat and apricot oil were evaluated best. Based on the results of sensory analyses, it was shown that the samples maintain a good sensory quality min. for 3 months while maintaining a low storage temperature ( 6 ° C). An analogue with coconut fat, whose taste, aroma and acceptability were rated as very good, and apricot oil, whose taste, aroma and acceptability were even rated as excellent, could enrich the food offer on the market.
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The Introduction of Contact Ecotoxicity Tests for the Assessment of Terrestrial Ecosystems
Modlitbová, Pavlína ; Kráčmar, Stanislav (referee) ; Beklová, Miroslava (referee) ; Demnerová, Kateřina (referee) ; Vávrová, Milada (advisor)
This dissertation thesis is focus on using toxicity tests with testing organism - terrestrial isopod Porcellio scaber. This organism is use for toxicity assessment of selected anorganic compunds which could contaminate terrestrial ecosystem. This organism was selected for several reasons - sophisticated test methodology; well known biology of these animals and their ecological relevance. Besides classical endpoints such as mortality, change in mass of test specimens and the effect of the presence and concentration of contaminants onto food consumption; we monitored changes in the morphometric characteristics at the level of tissues and cells, the cytotoxicity and possibly bioaccumulation in various parts of the body of the organism. Selected test substance in this thesis were nanoparticles and inorganic salts. Wherein for chosen gold nanoparticles was carried out a complete study presents several levels of organization of living matter (organism, tissue, cell). For selected salts compound, this study was carried out on the behavior of organisms and classical endpoints.
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Mycotoxins in Brewing Materials and Beer
Běláková, Sylvie ; Vávrová, Milada (referee) ; Márová, Ivana (referee) ; Kráčmar, Stanislav (referee) ; Čáslavský, Josef (advisor)
The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.
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Use of some molecular techniques to metabolic characterization of industrially significant yeasts
Kostovová, Iveta ; Brázda, Václav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Karotenoidy, ergosterol a mastné kyseliny jsou velmi žádané látky využívané v krmivářském, potravinářském a kosmetickém průmyslu. Konvenční zdroje mastných kyselin a karotenoidů jsou závislé na sezónních podmínkách, geografické poloze a na dostupnosti zemědělské půdy, což znesnadňuje pokrýt jejich neustále se zvyšující spotřebu. Velmi slibným řešením je mikrobiální produkce výše uvedených látek pomocí karotenogenních kvasinek, které jsou schopny simultánně produkovat karotenoidy, mastné kyseliny i ergosterol. Předložená disertační práce je zaměřená na molekulární a metabolickou charakterizaci karotenogenních kvasinek a na jejich potenciál pro průmyslové aplikace. Proto první experimentální části práce jsou zaměřeny na kvasinky druhu R. mucilaginosa a R. toruloides, jejich produkční vlastnosti, vliv nutričního stresu a různých zdrojů uhlíku, jakými byly xylóza a glycerol. Kromě podrobné charakterizace jejich produkčních vlastností, byly tyto kmeny také charakterizovány molekulárními metodami, zahrnující sekvenční analýzu ITS1, ITS2 a D1/2 ribozomálního operonu a analýzu mini a mikrosatelitních sekvencí M13 a GTG5. Druh R. toruloides je známý jako vynikající producent mastných kyselin, a proto se v poslední době stal cílovou karotenogenní kvasinkou pro vývoj nástrojů pro jeho genetickou manipulaci. V této práci byly úspěšně připraveny geneticky modifikované klony kmene R. toruloides, nesoucí nadměrně exprimované geny pro diacylglycerol acyltransferázu (DGA1) a glycerol-3-fosfát dehydrogenázu (GPD1). Produkce mastných kyselin u modifikovaných klonů nebyla ve srovnání s původním kmenem vyšší. Proto byla další část práce zaměřená na přípravu nadprodukčních mutantních kmenů připravených náhodnou mutagenezí. Kombinace limitace dusíkem a inhibice produkce karotenoidů vedla k úspěšné selekci robustních mutantních kmenů s nadprodukcí karotenoidů vykazující rezistenci vůči difenylaminu. Poslední část práce se zabývá produkčními vlastnostmi méně známých druhů karotenogenních kvasinek náležící do řádů Sporidiobolales a Cystofilobasidales, ve srovnání s relativně dobře prostudovanými karotenogenními druhy R. toruloides a P.rhodozyma. V této studii byly nejlešími producenty mastných kyselin kmeny S.metaroseus CCY 19-6-20 a C. macerans CCY 10-01-02. Nejlepší producent karotenoidů, kmen R. mucilaginosa CCY 19-04-06, navíc produkoval lykopen, který představoval více než 80 % celkového množství karotenoidů produkovaných tímto kmenem.
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PREPARATION AND CHARACTERIZATION OF NATURAL EXTRACTS WITH ANTIMICROBIAL EFFECT
Vysoká, Marie ; Obruča, Stanislav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Celosvětová spotřeba antibiotik v posledních několika desetiletích významně vzrostla, což vedlo ke zvýšenému výskytu genů antibiotické rezistence (ARG), které představují rostoucí vážnou hrozbu pro lidské zdraví na celém světě. Používání přírodních nebo alternativních léčivých přípravků tudíž v posledních několika letech výrazně vzrostlo. Velká pozornost je nyní věnována přírodním extraktům, které by mohly být v jisté formě použity jako jedna z možností léčby bakteriálního onemocnění, a to s vysokou účinností proti patogenům a se zanedbatelnými vedlejšími účinky. Biomasa kvasinek, která obsahuje cenné mastné kyseliny, sacharidy, nukleové kyseliny, vitamíny a minerály, byla testována jako možná alternativa, a to samotná i v kombinaci s rostlinnými extrakty ve formě doplňku stravy. Tato disertační práce je zaměřena na studium antimikrobiální aktivity a nutriční přínosy vybraných rostlinných extraktů, jako je acai, hřebíček, kurkuma, zázvor, ženšen a oregano. Následně byly testovány biologické účinky kvasinkové biomasy rodu Metschnikowia pulcherrima CCY 029-002-145, Cystofilobasidium infirmominiatum CCY 17-18-4, Phaffia rhodozyma CCY 77-1, Rhodotorula kratochvilovae CCY 20-2-26. Tyto kvasinky byly vybrány zejména pro vysoký obsah aktivních látek v produkované biomase. Na závěr byl posouzen potenciální synergický efekt rostlinných extraktů ve spojení s kvasinkovou biomasou ke zvýšení celkového antimikrobiálního účinku, antioxidační aktivity a vlivu na růst buněčných linií Caco-2, B16F1 a HaCaT. Vzhledem k dosaženým výsledkům lze konstatovat, že zařazení imortalizované kvasinkové biomasy v kombinaci s přírodními extrakty do doplňků stravy ve vhodné formě může být prospěšné lidskému zdraví. Existuje přímý vztah mezi životaschopností buněk a apoptózou. Všechny mikrobiální extrakty vykazovaly určité pozitivní výsledky na apoptózu u rakovinných buněčných linií a současně nevykazovaly žádné cytotoxické účinky na nezhoubné buněčné linie při stejných koncentracích.
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