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.

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.

This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.

This diploma thesis is focused on controlled production of lipolytic enzymes, bioactive substances and lipids by carotenogenic yeasts. Theoretical part deals with characterization of lipolytic enzymes, carotenoids, lipids and their properties, possibility of production and application. In experimental part the enzymes, carotenoids and lipids were produced by red yeasts Rhodotorula mucilaginosa, Cystofilobasidium macerans and Sporidiobolus salmonicolor by submerged cultivation in mineral medium with different additions: glucose, glycerol, fat, fat with glucose, fat with polysorbate 80, fat with glycerol, fat with polyethylene glycol, fat with higher and lower addition of palmitic acid, enzymatic fat hydrolysate, acidic hydrolysate a basic hydrolysate. The activity of extracellular lipase was monitored in medium after 96-hour cultivation. Concentration of -carotene, total carotenoids, ergosterol and ubiquinone was determined by HPLC, concentration of fatty acids and amount of fat by GC. Production had differed depending on used yeasts and substrate. As the best producer of carotenoids Cystofilobasidium macerans was found, ergosterol was highly produced by Sporidiobolus salmonicolor. The production of ubiquinone was almost equivalent in all yeasts and lipolytic activity was the highest in Sporidiobolus salmonicolor. The patricular medium sample with high lipolytic activity was further separated and analysed by ultrafiltration and PAGE-SDS electrophoresis. This diploma thesis was done within the international project ,,LipoFungi“.

Several yeast strains and microalgae were selected for this diploma thesis. -glucans, lipids, carotenoids, ergosterol and coenzyme Q were determined in selected producers, and the cultivation conditions for yeast strains were optimized to gain enhanced production of -glucans. Microalgae cultivations were carried out according to the instructions of the Collection of Autotrophic organisms (CCALA). Selected microalge strains include Desmodesmus acutus, Dunaliella salina, Arthrospira maxima and Cyanothece sp. Selected yeast species include Rhodotorula glutinis, Cystofilobasidium macerans and Sporidiobolus metaroseus. Edible yeast Saccharomyces cerevisiae was cultivated to compare with other yeast strains because of it's verified production of -glucans. -glucans were then determined by the enzymatic kit K-YBGL Megazyme, carotenoids, ergosterol and coenzyme Q were analyzed by HPLC/PDA and fatty acids were analyzed by GC/FID. The best producer of yeast -glucans was R. glutinis and S. metaroseus, and the best conditions for the production of -glucans and other metabolites was the C/N ratio of 70. Within the microalgae species, only -glucan production was observed, the best producer was D. acutus.

This diploma thesis deals with valorization of waste animal fat using carotenogenic yeast and conversion to biomass enriched with valuable substances. These microorganisms have the ability to utilize various waste substrates and convert them into interesting products such as carotenoids, lipids, ergosterol and ubiquinone. Four strains of yeast (Rhodotorula glutinis, Cystofilobasidium macerans, Rhodotorula mucilaginosa, Sporidiobolus pararoseus) were used. The theoretical part is focused on characterization of yeasts, enzymes and lipases, produced metabolites, yeast and description of analytical methods. In the experimental part, cultures were performed at four C/N ratios on media with different carbon sources, which were glucose, glycerol, fat, fat with added emulsifier, enzymatically hydrolyzed fat and fat with enzyme addition. Biomass was determined gravimetrically for all samples. Carotenoids, ergosterol and ubiquinone were analyzed on an HPLC/PDA equipment. The intracellular lipid content was determined by GC/FID. The production of these substances was different in individual strains depending on the carbon source and the C/N ratio. Rhodotorula glutinis is the most suitable for producing biomass and carotenoids. The highest amounts of ergosterol and ubiquinone were produced by Cystofilobasidium macerans. Sporidiobolus pararoseus had the largest lipid content in biomass. This diploma thesis was elaborated within the international project "LipoFungi".

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.

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.

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.

Yeasts are like other organisms constantly exposed to environmental influences. Their survival depends on the skills to adapt to environmental changes, including the ability to use various alternative sources of nutrients. In presented PhD thesis carotenogenic yeast belonging to the genera Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates, and also subjected to several types of exogenous stress effects and mutations in order to increase the production of microbial biomass enriched with specific metabolites. As alternative nutrient sources derived from waste substrates from agricultural and farm production apple peel, pulp, corn germ and more were tested. Yeasts were also exposed to osmotic, oxidative and combined stress (benefits of various concentrations of NaCl and H2O2 to the culture media), followed by metal ions of selenium and chromium in concentrations of 0.01 mM, 0.1 mM and 1 mM. The effect of mutagen methanesulfonic acid ethyl ester was tested too. In all experiments the adaptivity of cells, morphological changes, color pigments produced by the media while some important fungal metabolites production and changes in chromosomal DNA fragmentation were analyzed. In order to evaluate potential changes in the yeast genome after treatment with mutagen and stress factors methods for isolation of intact chromosomal DNA and DNA analysis by pulsed field gel electrophoresis was optimized. The amount of produced metabolites was mainly analyzed by RP-HPLC with UV/VIS and MS detection. The work has been shown that most strains are able to use waste substrates and produced selected target metabolites. Biomass, for example, in R. aurantiaca on apple fiber was about 7 g/l and in C. capitatum cultivated on modified whey reached to 9 g/l. Amount of produced carotenoids by R. aurantiaca cultivated on wheat germ and maize after enzymatic hydrolysis by F. solani was 1.01 mg/g and S. roseus on pasta 4.3 mg/g. The values of ergosterol synthesis in R. aurantiaca are on the apple shells around 4.8 mg/g, in S. roseus on pasta with the enzymatic hydrolysis of P. chrysosporium 8.9 mg/g. The best substrate for biomass production and induction of carotenoids are waste substartes containing a mixture of simple and complex carbohydrates enriched with the addition of nitrogen compounds. Potential cytotoxic effect of stress factors of low concentrations was demonstrated. Red yeast genome was able to distribute by optimized PFGE, the karyotype of tested yeasts contain 11 or more chromosomes with visible differences between yeast species and genera. During exchange internship the ability of recombinant yeast S. cerevisiae to convert xylose to xylitol, which would be achieved by increasing the production of bioethanol as alternative fuel sources was studied. It turned out that both ligninocellulose materials to bioethanol production, as well as various waste substrates for microbial synthesis of carotenoids would reduce costs for industrial production of yeast metabolites, as well as to reduce the negative burden on the environment.