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Determination of beta-carotene in barleycorn by HPLC
Puč, Vojtěch ; Maleček, Miroslav (referee) ; Prýma, Jaroslav (advisor)
This diploma thesis deals with the natural antioxidants present in cereals, especially in barley (Hordeum vulgare). A close attention is paid to the study of carotenoids determination was conducted. In the experimental part, the method of beta-carotene determination was optimized using high-performance liquid chromatography, diode array detector and mass detector (HPLC/DAD/APCI-MS). The method was used for the beta-carotene and lutein determination in the samples of barleycorn, malt and green barley. This method involves the sample saponification, extraction by diethylether, followed by separation on ODS Hypersil 250x4,6 mm, 5m column, using MTBE/MeOH (20:80) as mobile phase and spectrophotometric detection (450 nm). Quantitative analysis was implemented in the HPLC/DAD system. The MS detector was used for identification of analytes. A number of still unpublished data about the content of beta-carotene and lutein in several varieties of malting barley, malt and green barley are stated in this thesis. The highest content of beta-carotene was found in the green barley sample of variety Malz, harvested in first grow phase (8,49 mg/kg of the dry matter). The content of beta-carotene in barleycorn is relatively low (0,07-0,14 mg/kg of the dry matter). The content of beta-carotene is several times higher in the malt produced from barleycorn (0,24-0,56 mg/kg of the dry matter). The diploma thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.
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Study of metabolic changes in carotenogenic yeasts cultivated under different conditions
Starečková, Terezie ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
The aim of this diploma thesis realized as a comparative study was the study of regulation of carotenoid and ergosterol production in several carotenogenic yeast strains. Yeasts were exposed to exogenous stress factors. Salt stress and oxidative stress (hydrogen peroxide) were reached by addition of NaCl and hydrogen peroxide into production media. Complex changes on metabolome (e.g. pigment and ergosterol production, RP-HPLC), proteome and genome were followed. Proteome changes were analyzed by PAGE-SDS and 2D electrophoresis. To isolation and analysis of chromosome DNA pulsed field gel electrophoresis (PFGE) was used. Six yeast strains were enrolled into the comparative study; three strains of the genus Rhodotorula and three strains of the genus Sporobolomyces. While yeasts Rhodotorula sp. were characterized by enhanced biomass as well as carotenoid production in normal and stress conditions, production of biomass by Sporobolomyces sp. was substantially lower. Carotenoid production in Sporobolomyces sp. was higher than in Rhodotorula sp.; the highest increase of was beta-carotene production was observed in Sporobolomyces salmonicolor cells stressed by salt (4x higher than in control) or peroxide (5x higher). Proteins were isolated from yeast cells by combination of mechanical and chemical disruption by glass beads and NaOh or SDS. Better yields were obtained by NaOH. Two staining methods were tested in PAGE-SDS protein analysis. Coomassie Brilliant Blue staining exhibited lower sensitivity, silver staining led to better visualisation of minor protein fractions too. 1D protein profiles was difficult to evaluate, therefore, 2D electrophoresis of selected strains (R.glutinis, R.rubra) was done. In yeast genome analysis by PFGE at minimum 7 DNA fractions were observed. These results probably are not final, further study will be needed for detailed characterization of red yeast genome.
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Production of carotenoids and enriched yeast biomass on rape-seed waste substrates
Mikheichyk, Nadzeya ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the group of the most widely used natural pigments with significant biological effects. Recently, microbiological production of carotenoids prior synthetic production is examined. The aim to the study was to analyse the properties of carotenogenic yeasts on waste rapeseed substrate. The main purpose of this thesis was conceived as a comparative study of three strains of carotenogenic yeasts: Rhodotorula glutinis, Sporobolomyces roseus a Cystofilobasidium capitatum. Yeasts were cultivated on different nutrient media, in which selected waste material was used as a nutritional source - rape substrate of two types with different lipid content. For all strains the growth characteristics on rapeseed substrate of the first type with a lower content of lipids were performed. All tested strains were able to use waste substrates as a source of nutrients. Most of strains produced increased amounts of carotenoids, in some cases accompanied with a relatively high production of biomass. As a potentially best producer of enriched biomass yeasts Rhodotorula glutinis and Sporobolomyces roseus were showed. They exhibited the highest production of biomass and metabolites in the media with the ratio glucose:rapeseed cake 3:1 and 1:1. The results showed that rapeseed cake can be used as a potential carbon source for growth of carotenogenic yeasts and production of carotenoids and selected lipids to some limit ratio of waste. Further processing of rapeseeed cake could be a suitable target for following study in related works.
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Use of LC/MS technique to analysis of some physiologically active natural compounds
Trčková, Marie ; Čáslavský, Josef (referee) ; Kočí, Radka (advisor)
Presented work is focused on application of combined instrumental method RP-HPLC/ESI-MS in analysis of several groups of natural compounds with positive physiological activities. Especially some antioxidants were studied in commonly and abundantly consumed food. Moreover some other substances than standard compounds were observed in complex dies. In conclusion the HPLC/ESI-MS method is comparatively advantageous in phenolic analysis, while another type of ionisation would be used in case of carotenoid compound.
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Use of selected fluorescence techniques to characterization of microbial cells
Vaněk, Martin ; Pekař, Miloslav (referee) ; Márová, Ivana (advisor)
Carotenoids are one of the most abundant natural pigment. Furthermore, they have great potential for wide industrial utilization. Nowadays, prefered (and cheaper) synthetic method counters progressively improved biotechnological production. Far more cheaper production should be reached via implementing methods of on-line optimization, such as flow cytometry. In this thesis the dependence of yeasts autotofluorescence intensity on carotenoids concentration in dry mass was studied. The dependence was established by means of linear regression. Moreover, the dependence between light scatter and the amount of dry mass was established by means of model utilizing forward scatter, side scatter and number of cells in unit volume. Yeasts' cells was also studied under the lenses of laser scanning confocal microscope. The goal was to localize carotenoids inside cells. Experiments undertaken suggest that they are localized inside the lipidic granules, but more experiments are required for a proof.
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Production of selected metabolites by carotenogenic yeasts cultivated on food wastes
Turková, Lucie ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
The production of selected metabolites, especially carotenoids, ergosterol and coenzyme Q was observed in four red yeast strains (Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces shibatanus a Sporobolomyces roseus) cultivated on viticulture waste substrates such as skins and stem. In these substrates the content of carbohydrates, fat, tannins and polyphenols was analysed. Cultivations were done also on chemically and enzymatically hydrolysed substrates. For enzyme hydrolysis the extracellular enzyme cocktails of the fungi Phanerochaete chrysosporium and Penicillium lilacinum were used. The highest production of metabolites was observed in S. roseus cultivated on stem hydrolysed by enzymes from P. chrysosporium. The yield of -carotene reached 616,1 g/g dry mass, carotenoids 753,6 g/g dry mass, ergosterol 415,5 g/g dry mass and coenzym Q 77,9 g/g dry mass. S. roseus reached very high yields also in the other series of cultivation. The lowest production of metabolites was observed in S. shibatanus. The yield coefficients of biomass and -carotene were calculated based on consumption of reducing sugars.
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Potential use of waste lipid substrates and glycerol to production of carotenoids by yeasts
Kostovová, Iveta ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of this study was use of lipid waste substrates and waste glycerol as carbon sources for red yeasts. Six yeast strains, namely Rhodotorula glutinis, Rhodotorula rubra, Rhodotorula aurantiaca, Cystofilobasidium capitatum, Sporobolomyces roseus and Sporobolomyces shibatanus were used for cultivation. All studied yeast strains were able to utilize glycerol and rapeseed oil as a carbon source. Cultivation in oil medium was in most of strains accompanied by decreased production of carotenoids. Increased production of ergosterol, lycopene and biomass was observed in some strains only. Most of yeast strains cultivated in glycerol medium produced increased amount of biomass as well as carotenoids. The best producer of bi-omass and pigments was yeast strain Sporobolomyces roseus CCY 19-6-4 grown in medium containing glucose and glycerol in a 1:1 ratio.
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Production of carotenoids by red yeasts grown on some waste substrates
Kubáčková, Martina ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the most widespread and abundant classes of natural pigments with utilised in food industry, pharmacy and cosmetics. Presented work was realized as a comparative study of some substrates for cultivation of red yeast Rhodotorula glutinis, which produces carotenoid pigments. Yeasts were cultivated in different media with several waste substrates (for example potato fiber, apple waste, cereals, grains, etc.) as nutrition sources. To some substrates hydrolytic enzymes isolated from the fungi genus Fusarium were added. Majority of waste substrates were acceptable for cultivation. However, the best conditions for production of carotenoid enriched biomass (6,4 g/l of biomass enriched 3,2 mg /g of beta-carotene) exhibited media with hydrolytic fungal enzymes.
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Kvalitativní hodnocení ekologicky a konvenčně pěstovaných tykví (Cucurbita maxima Duch.)
Kneblová, Lenka
This graduation thesis was developed at the Department of Vegetable Growing and Floriculture in year 2013/2014. The work solves the issue of the pumpkin production (Cucurbita maxima Duch.) in the system of ecological and conventional production including quality parameter evaluation. The differences in the quality between products coming from organic and conventional agriculture are explained in the literary part. The study also characterizes the ecological production legislative. The theoretical part deals with the description and cultivation of the pumpkins. The experimental part is focused on the evaluation of content substances, such as vitamin C, dry matter, carotenoids. Another of the monitored parameters are color change and weight loss during storage. In the measured parameters were found significant differences in the content of dry matter and discoloration.
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Monitoring flavonoidních látek a karotenoidů ve vybraných doplňcích stravy
Hynštová, Veronika
Dietary food supplements are among the most rapidly growing sectors in the food product industry. The majority of consumers trust in the safety and efficacy of these products. For these reasons is a quality control required and analytical methodologies for this must be used. For identification and quantitative analysis four flavonoids diosmin, hesperidin, rutin and troxerutin in food supplements was used HPLC/MS method. For identification and quantitative analysis three carotenoids betacarotene, lutein and zeaxanthin in food supplements was used HPLC/UV/ViS/DAD method. Separation of flavonoids was achieved on the column ZORBAX POROSHELL 120 EC-C18 (50 x 4,6 mm, 2,7 um) and separation of carotenoids on the column ZORBAX SB CN (75 x 4,6 mm, 3,5 um). The amount of flavonoids and carotenoids in tablets and capsules was determined altogether in 12 different commercial preparations.
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