The presented diploma thesis deals with the analysis and production of selected microbial metabolites of the yeast Rhodotorula kratochvilovae in comparison to various species of microalgae. The theoretical part summarizes the knowledge about the conditions for optimal growth and production of secondary metabolites. Analytical methods, by which metabolites were quantified and evaluated, were also described. In the experimental part of this thesis, the amount of accumulated biomass, exoglycolipids, intracellular lipids, phycobiliproteins and carotenoids according to various nutrient sources in the culture medium was studied. Specifically, the yields of these metabolites were investigated in the utilization of various sources of nitrogen (yeast extract, urea, ammonium sulfate, ammonium chloride and potassium nitrate), carbon (glucose, mannose, xylose, glycerol and lactose) and at variable C / N ratios (20; 40; 70; 100 and 120: 1) ideally selected sources of observed nutrients in partial experiments. The last part of the experiment was focused on comparison of the analysis of metabolites of autotrophic algae (chlorophylls, phycobiliproteins and lipids).

Presented diploma thesis is focused on the optimisation of Haematococcus pluvialis cultivations in different photobioreactors and on biotechnological production of astaxanthin. Theoretical part summarized the knowledge about optimal growth and production conditions of secondary metabolites. Followed research was focused on actual cultivation systems and on the possibilities of metabolite and nutrient monitoring. In experimental part the growth characteristic of the strain from Březova nad Svitavou (HMP-CCALA 375) was analyzed under optimal cultivation conditions on white and red light. During culture growth the profile and the concentration of carotenoid pigments were determined. The best yield of biomass was achieved in the cultivation on white light (0,939 g/l),carotenoids lutein and -carotene were observed as dominant pigments. In the next experiments optimal growth medium, temperature and light intensity were determined for cultivations of four chosen HMP strains from Germany, America, Africa and Switzerland. The most suitable cultivation medium was found BBM, oppositely the worst results were obtained with BG11. In previous experiments cultivation temperature 22 °C was determined as optimal value for comparative strain HMP – CCALA 375. Selected four strains were cultivated at 22 °C, as well as at 25 °C. Higher temperature was more optimal mainly for Switzerland, German and Africa strains. By analyzing of light intensity influence, it was found that the best increase of biomass was induced by the adaptation of culture on lower illumination (50 µmol photones•m^-2•s^-1) followed by higher light intensity (100 - 150 µmol photones•m^-2•s^-1). HMP from Switzerland showed the best growth results during all cultivation experiments, so this strain could be perhaps useful for industrial production of astaxanthin. In the last part of work, the influence of stress conditions on astaxanthin production by strain from Březova nad Svitavou (HMP – CCALA 375) was studied. Followed stress factors were used: high intensity of light (1 000 µmol photones•m^-2•s^-1), low nitrogen concentration (32,96 mg/l), addition of sodium chloride (0,5%), influence of sodium acetate (2,2 mM) and combination of sodium chloride and sodium acetate (0,5% NaCl, 2mM NaAc). Due to strong illumination (1 000 µmol photones•m^-2•s^-1) the best yield of astaxanthin was obtained (more than 20 mg/g). According to literature [103, 105] significant amount was also observed by addition of sodium acetate (9,2 mg/g). Oppositely minimal astaxanthin production was showed in presence of salt stress (3,8 mg/g). In followed experiments should be studied the influence of stress combinations on HMP – CCALA 375 strain as well as on other suitable strains of H. pluvialis with the aim to achieve the maximal yield of astaxanthin significant for large scale cultivation.

<p>U&nbsp;radu&nbsp;su&nbsp;ispitane&nbsp;biohemijske&nbsp;promene&nbsp;na&nbsp;plodovima&nbsp;paprike&nbsp;tokom&nbsp;sazrevanja &nbsp;plodova&nbsp;i&nbsp;tokom&nbsp; dozrevanja&nbsp; plodova&nbsp; posle&nbsp; branja.&nbsp; U&nbsp; cilju&nbsp; ispitivanja&nbsp; promena&nbsp; tokom&nbsp; sazrevanja &nbsp;plodovi&nbsp;su&nbsp;uzorkovani&nbsp;u&nbsp;različitim&nbsp;fazama&nbsp;zrelosti&nbsp;tj.&nbsp;kad&nbsp;su&nbsp;plodovi &nbsp;bili&nbsp;zelene,&nbsp;braon,&nbsp;crvene&nbsp;i&nbsp;tamno&nbsp;crvene&nbsp;boje.&nbsp;Svaka&nbsp;od&nbsp;pomenutih&nbsp;grupa&nbsp;zrenja&nbsp;je &nbsp;dozrevana&nbsp;sledećih&nbsp;petnaest&nbsp;dana&nbsp;sa&nbsp;i&nbsp;bez&nbsp;prisustva&nbsp;svetla.&nbsp;Posle&nbsp;perioda&nbsp;dozrevanja &nbsp;plodovi&nbsp;su&nbsp;osu&scaron;eni&nbsp;i&nbsp;samleveni.&nbsp;&nbsp;</p><p>U&nbsp; uzorcima&nbsp; svežih&nbsp; plodova&nbsp; paprika&nbsp; ispitan&nbsp; je&nbsp; sadržaj&nbsp; vode,&nbsp; pepela,&nbsp; ukupnih&nbsp; mono&nbsp; i &nbsp;oligosaharida,&nbsp;mikro&nbsp;i&nbsp;makroelemenata,&nbsp;boja&nbsp;plodova&nbsp;(vizuelnom&nbsp;ocenom&nbsp;i &nbsp;instrumentalnim&nbsp;merenjem),&nbsp;sadržaj&nbsp;pigmenata&nbsp;i&nbsp;sastav&nbsp;etarskih&nbsp;ulja,&nbsp;dok&nbsp;je&nbsp;u&nbsp;suvim&nbsp;uzorcima&nbsp;određena&nbsp;boja, sadržaj&nbsp;mikro&nbsp;i&nbsp;makro&nbsp;elemenata,&nbsp;pigmenata&nbsp;i&nbsp;sastav&nbsp;etarskih ulja. &nbsp;U&nbsp;semenima &nbsp;paprika&nbsp;ispitan&nbsp;je&nbsp;sadržaj&nbsp;vode,&nbsp;pepela,&nbsp;ulja&nbsp;i&nbsp;neophodnih&nbsp;mikro&nbsp;i &nbsp;makro &nbsp;elemenata.&nbsp;</p><p>Dobijeni&nbsp; rezultati&nbsp; pokazuju&nbsp; da&nbsp; se&nbsp; tokom&nbsp; sazrevanja&nbsp; de&scaron;avaju&nbsp; biohemijske &nbsp;promene &nbsp;na&nbsp;plodovima&nbsp; paprika.&nbsp; Povećava&nbsp; se&nbsp; sadržaj&nbsp; suve&nbsp; materije,&nbsp; dolazi&nbsp; do&nbsp; promena&nbsp; u&nbsp; sastavu&nbsp;pigmenata&nbsp;i&nbsp;etarskih&nbsp;ulja.&nbsp;Tokom&nbsp;15&nbsp;dana&nbsp;dozrevanja,&nbsp;ispitani&nbsp;parametri plodova&nbsp;paprika pokazuju&nbsp;da&nbsp;se&nbsp;promene&nbsp;na&nbsp;plodovima&nbsp;nastavljaju&nbsp;i&nbsp;posle&nbsp;ubiranja &nbsp;plodova. &nbsp;U&nbsp;zavisnosti&nbsp;od &nbsp;stepena&nbsp;zrelosti,&nbsp;povećava&nbsp;se&nbsp;sadržaj&nbsp;pigmenta,&nbsp;a&nbsp;menja&nbsp;se&nbsp;i &nbsp;sastav etarskih&nbsp;ulja.&nbsp;</p> / <p>The&nbsp; aim&nbsp; of&nbsp; this&nbsp; study&nbsp; was&nbsp; to&nbsp; investigate&nbsp; biochemical&nbsp; changes&nbsp; of&nbsp; paprika&nbsp; fruits during ripening&nbsp; and&nbsp; postharvest&nbsp; treatments.&nbsp; Fruits&nbsp; were&nbsp; collected&nbsp; in&nbsp; different&nbsp; stages of development i.e. when green, brown, red and deep red color. Each of these&nbsp; color‐type&nbsp; fruits where&nbsp; ripped&nbsp; after&nbsp; harvest&nbsp; (collection)&nbsp; during&nbsp; next&nbsp; fifteen&nbsp; days&nbsp; with&nbsp; or&nbsp; without&nbsp; light.&nbsp; Thereafter, postharvest treatment fruits were dried and milled.</p><p>In samples of fresh paprika fruits the content of water, ash, mono‐ and&nbsp; oligosaharides, micro and macro nutrient,&nbsp; color of fruits (by visual&nbsp; evaluation and&nbsp; instrumental measuring), ASTA value,&nbsp; chlorophyll&nbsp; and&nbsp; carotenoid&nbsp; content,&nbsp; certain&nbsp; constituents&nbsp; of&nbsp; essential&nbsp; oil&nbsp; were determined and activity of peroxidase and &beta;‐glucosidase enzymes, while in dried fruits the color,&nbsp; content&nbsp; of&nbsp; micro&nbsp; and&nbsp; macroelements&nbsp; and&nbsp; composition&nbsp; of&nbsp; essential&nbsp; oils&nbsp; were determined. In seed,&nbsp; the content of water, ash, oil and micro and macroelements were determined.</p><p>Based on the results it could be concluded that, during ripening, the biochemical&nbsp; changes are occurring on paprika fruits. The increase in dry matter content is observ ed along with the changes in pigment and essential oil composition. During the&nbsp; postharvest&nbsp; time, all evaluated parameters indicated that&nbsp; ripening is in progress after the harvest of paprika fruits. Changes in both the&nbsp; pigment and the essential oil content is dependent of a ripening stage.</p>

In industry, antioxidants are added to prevent changes that affect quality due to oxidation, such as the development of off flavors, unacceptable odors and discoloration. New resources that are familiar to consumers and may work as antioxidants should be studied. The changes in antioxidant activity were determined for four freeze-dried native wild berry powder species in Utah, including skunkbush (Rhus trilobata), chokecherry (Prunus virginiana), river hawthorn (Crataegus douglasii) and silver buffaloberry (Shepherdia argentea), during cold storage (-20 °C) for 6 months. The total antioxidant activity was stable in most of the berry powders during the six months of frozen storage. Overall the river hawthorn and buffaloberry contained the highest antioxidant activity values, which indicated that they would be more efficient for use as food antioxidants. The effectiveness of two different levels of native Utah river hawthorn and buffaloberry freeze-dried powders in maintaining the quality of ground poultry and ground beef was studied. Meat patties mixed with berry powders were held at 4 °C and analyzed every three days for two weeks. The river hawthorn berry and silver buffaloberry delayed the undesirable changes in flavor or color caused by the oxygen present in the air in both chicken and beef ground patties. However, the plant pigments in the silver buffaloberry increased the redness of chicken patties. Neither berry significantly changed the color of ground beef patties or changed the microbial growth in ground meats. Overall, both river hawthorn and buffaloberry are suitable for using in ground beef product as antioxidants. Milk mineral and sodium tripolyphosphate are antioxidants that could bind free metal ions into insoluble or inactive forms. They can delay the changes of the meat pigment and maintain color in meat products. The purpose of the third study was to examine the effect antioxidants on meat pigment.pH describes the amounts of acid in food. Slowed color changes in the poultry meat pigment with antioxidants was seen both at the pH of living poultry muscle (7.2) and the pH of raw poultry meat (pH 6.0). This suggested that addition of metal-binding antioxidants like milk mineral or sodium tripolyphosphate could help preserve poultry meat pigment and maintain the color of poultry products.

This dissertation is focused on exploring the potential of bacteria for the biosynthesis of natural products with the purposes of generating novel natural product derivatives and of improving the titer of pharmaceutically important natural products. A wide variety of compounds from various sources have been historically used in the treatment and prevention of diseases. Natural products as a major source of new drugs are extensively explored due to their huge structural diversity and promising biological activities such as antimicrobial, anticancer, antifungal, antiviral and antioxidant properties. For instance, penicillin as an early-discovered antimicrobial agent has saved millions of lives, indicating the historical importance of natural products. However, the alarming rise in the prevalence of drug resistance is a serious threat to public health and it has coincided with the decreasing supply of new antibiotics. Bacteria with a tremendous undiscovered potential have still been one of the richest sources of bioactive compounds to tackle the growing threat of antibiotic-resistant pathogens. Nevertheless, the production level of those important compounds is often quite low, and often undetectable using current analytical techniques. To expand the chemical repertoire of nature and to increase the titer of the natural products, researchers have developed various strategies, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. Thus, in my dissertation research, I have exploited a few of these strategies. First, I heterologously expressed some of the biosynthetic genes from the sch biosynthetic gene cluster, resulted in the production of a novel glycosylated angucycline. I was also able to generate another new glycosylated derivative of angucycline through gene disruption of tailoring enzymes. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through the overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. This work thus provides an initial understanding of functional roles of regulatory elements in the biosynthesis of Sch47554 and Sch47555 and several engineered strains with enhanced production of Sch47554. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of the yew tree and optimized the fermentation conditions for an improved yield of zeaxanthin diglucoside up to 206 ± 6 mg/L. With the introduction of an additional copy of the Pscrt gene cluster through an expression plasmid, the engineered strain Pseudomonas sp. 102515/pOKF192 produced zeaxanthin diglucoside at 380 ± 12 mg/L, which is 85% higher than the parent strain. This strain holds a great potential for the production of pharmaceutically important antioxidant agent, zeaxanthin diglucoside.

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The growth of Rhodotorula glutinis in sweet sorghum juice in three levels of three factors of temperature, carbon to nitrogen ratio and pH was evaluated. Accompanying of this growth was the generation of lipids converted to fatty acid methyl ester (FAMEs) and carotenoids. The optimized condition for maximum biomass and carotenoid accumulation was determined to be at 25C, pH of 5.5 and carbon to nitrogen ratio of 10. This condition yielded 22.7 g/L biomass with specific growth rate of 0.213 hr-1. At this condition the carotenoids generation was also maximum with 2.6 mg/gram biomass, comprising of torularhodin, beta-carotene and torulene. The accumulation of lipids following generation of biodiesel was highest at same temperature and pH but carbon to nitrogen ratio of 70, generating 96.3 mg of FAMEs/gram of biomass containing methyl ester of oleic acid, linoleic acid, palmitic acid, stearic acid and linolenic acid.

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No description available.

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