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Determination of Natural Compounds in Select Plant Materials and Less Obvious Kinds of Fruit
Hohnová, Barbora ; Márová, Ivana (referee) ; Ventura, Karel (referee) ; Omelková, Jiřina (advisor)
Flavonoids are natural compounds widely distributed in plant kingdom. They are inseparable from human diet because they showed a protective effect against cancer, stroke and coronary heart diseases related to their antioxidant properties. Therefore, rapid and efficient extraction procedure prior to chromatographic analysis is required. The liquid extraction at elevated temperature and pressure – Pressurized fluid extraction (PFE) and Pressurized hot water extraction (PHWE), present fast, effective and environmentally friendly extraction methods for the determination of flavonoids in plant materials. PFE and PHWE followed by reversed phase high-performance liquid chromatography with UV-visible detection have been utilized for the determination of a group of flavonoids (rutin, myricetin, quercetin, luteolin, apigenin and kaempferol) in the leaves and berries of less common plants. The matrices were extracted by methanol, ethanol and water at higher temperature 40-120 oC and pressure 15 MPa during 15 minutes. The obtained results were compared with conventional Soxhlet extraction and ultrasound-assisted extraction, the same solvents were used. The most effective extraction of selected flavonoids was achieved by PHWE. PFE showed the extraction yields comparable to those of the Soxhlet extraction, and the lowest extraction power was displayed by ultrasound-assisted extraction.
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Encapsulation of natural antioxidants
Štindlová, Jitka ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
In theoretical part of this diploma thesis the basic properties of antioxidants, especially anthocyanins and phenolic compounds are described, as well as the basic features and principles of free radicals formation. The theoretical part also describes some possible ways of encapsulation of antioxidants into polysaccharide and lipid particles. In the experimental part basic characteristics of extracts from selected lyophilized fruits and vegetables (carrots, apples and mixed berries) are described. As group parameters of plant extracts the total antioxidant activity, content of flavonoids and phenolics, carotenoids, anthocyanins and ascorbic acid were determined. In experimental part also various encapsulation techniques were tested, encapsulation effectiveness of each technique was evaluated and the stability and size of the created particles were determined. As the best encapsulation method in terms of encapsulation efficiency in most of samples/parameters ethanol injection was found. On the other hand the particles prepared by ethanol injection are relatively unstable in terms of zeta potential, which is followed by their tendency to aggregate. As the most stable particles prepared by thin layer evaporation (TLE) and reverse phase thin layer evaporation (RP-TLE) were evaluated. Particles prepared by TLE, RP-TLE and chitosan-alginate particles exhibited a negative charge, while particles prepared by ethanol injection stayed uncharged and chitosan particles have a positive charge.
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Controlled production of polyhydroxyalcanoates by bacterial strain Ralstonia eutropha using different substrates
Šnajdar, Ondřej ; Duroňová, Kateřina (referee) ; Márová, Ivana (advisor)
This bachelor’s thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Ralstonia eutropha. Production of PHA on different substrates has been studied to lower the costs of feedstock. In theoretical part the review has been done about the most important types of PHA, production strains and possibilities of cultivation of Ralstonia eutropha using different substrates. In practical part there has been studied production of PHA on different vegetable oils, including waste oils from different sources (restaurants, homes, food companies). Incorporation of different precursors for copolymer production control was studied too. The highest yields of poly-3-hydroxybutyrate (P3HB) have been achieved using waste oils. For economical and ecological reasons these oils are very suitable substrates. Using concentration 20 g/l of waste rapeseed oil from university canteen there has been produced 13,32 g/l of biomass containing 58,63% of P3HB in 84th hour of cultivation. The highest yield of PHA in precursors study has been achieved applying 1% propanol in 24th hour of cultivation. The PHA increase has been 97%. This PHA was composed from 91% of 3HB and 9% of 3-hydroxyvalerate.
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Use of DGGE to analysis and identification of selected microorganisms
Jankeje, Kristína ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.
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Study of antimutagenic properties of selected kinds of honey
Lichnová, Andrea ; Melounová, Jitka (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of antimutagenic properties in selected kinds of honey and propolis. In honey extracts compounds with antimutagenic and antioxidant effect were analysed by spectrophotometry, RP/HPLC/UV-VIS, HPLC/PDA and on-line LC/MS. Further, effect of long-term storage on active compound levels was studied. Antimutagenic activity was tested by simple eukaryotic system - yeast strain Saccharomyces cerevisiae D7. The highest antimutagenic effect and simultaneously the highest stability of antimutagenity values was found in several kinds of multi-floral honey, rape seed honey, honey from eucalypt and orange flowers and in honeydew honey. Samples obtained from trade network exhibited at general lower antimutagenity values when compared with samples from bee-keeper. The highest content of total phenolics was detected in honey with royal yelly, the lowest content was measured in acacia honey. The highest values of total flavonoids exhibited buckwheat and eucalypt flower honey. Total phenolic content was substantially changed during long-term storage (decrease about 50 -70 %), while total flavonoid content was stable and no significant changes during storage were observed. Honey with high antimutagenity values exhibited also higher phenolic and predominantly flavonoid content and high antioxidant activity. Because of relative stability and low changes in most of honey samples during storage it can be concluded that honey belong to foodstuffs which are able to conserve their positive nutritive properties for a long time.
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Characterization of active substances in several kinds of beer
Benešová, Pavla ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of this bachelor thesis is to analyse biologically active compounds, especially of phenolic character, in 10 kinds of alcohol-free beer in comparison with alcoholic beer. In theoretical part basic steps of brewery technology are described including alcohol-free beer production. Characteristic compounds occurring especially in Czech beer e.g. proteins, phenolics, bitter substances and also methods for their analysis are discussed as well. Microfluidic electrophoresis (Experion, BioRad) was used to beer protein analysis. Phenolic compounds, flavonoids, technological characteristics and antioxidative activity were measured by spectophotometry. Individual phenolics of selected beers were identified and quantified by LC/ESI-MS analysis too. In alcohol-free beers similar concentrations of biologically active compounds were found in comparison with alcoholic beers, especially in the case of beer phenolics.
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Use of genotoxicity tests to characterization of some natural substances and particles
Tilšarová, Kamila ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
In production of drugs, cosmetics, in food industry and other biotechnologies, testing of genotoxicity (that are changes in genetic information which are not inhereted) is one of the common steps in the process before distribution to the market. This bachelor´s thesis is focused on the characterization of extracts from chosen natural substances with respect to the content of polyfenols, flavonoides and antioxidant activity. These extracts were encapsulated into liposomes and chitosan particles. The aim was testing possible genotoxicity of whole extracts and particles on possible genotoxicity on the basis of SOS Chromotest performed on bacteria E. coli. This genotoxic assay did not show genotoxic effect of neither pure extracts nor tested liposomes.
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Production and characteritzation of extracellular hydrolases from selected moulds
Skoumalová, Petra ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.
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Study some active substances in Czech beer
Pařilová, Kateřina ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
The aim of this work was to study phenolics and other active compounds in 22 kinds of lager beer and especially to find some differences between Czech beer and beers made in foreign countries. In theoretical part the most useful methods for phenolic and protein analysis are discussed. Further, basic steps of brewery technology are introduced and main beer components with regard to Czech beer specificity are presented. Analysis of total phenolics, total flavonoids, antioxidant activity and most of basic technological characteristics was performed spectrophotometrically. To identification and quantitative analysis of individual phenolics on-line liquid chromatography with photo-diode array (PDA) and mass spectrometry (MS) detection was used. Protein analysis was performed by vertical electrophoresis SDS-PAGE and by Experion microfluidic electrophoresis (BioRad). From the total of 22 samples, 15 Czech beers and 7 beers of foreign marks were enrolled into this study. In these samples levels of brewery characteristics, group antioxidants and individual phenolics were compared. Flask and can beers as well as alcoholic and non-alcoholic beers were compared too. As a model comparative sample the Czech hop of Sládek variety was used. Finally, usefulness of tested parameters to differentiation composition and technology of individual beer samples was evaluated and parameters suitable for Czech beer authenticity analysis were proposed.
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