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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Production of Selected Microbial Metabolites and Energy Using Different Waste Materials
Petrik, Siniša ; Rychtera, Mojmír (referee) ; Němec, Miroslav (referee) ; Pekař, Miloslav (referee) ; Márová, Ivana (advisor)
Pro zpracování a nakládání s odpadními substráty lze použít řadu postupů a možností. Stále se rozšiřující spektrum metod a technologií umožňuje další využití materiálů a energie ve formě obnovitelných zdrojů. Jedním z řešení pro zpětné získávání některých odpadních materiálů je využití tzv. bílé (průmyslové) biotechnologie, která zahrnuje praktickou aplikaci metabolických aktivit celé řady různých mikroorganizmů včetně jejich specifických biologických drah k produkci látek s vysokou přidanou hodnotou. V předložené práci screeningového typu bylo pro zhodnocení odpadních surovin využito několik druhů mikroorganizmů kultivovaných za různých specifických podmínek včetně kultivace na odpadních materiálech získaných zejména ze zemědělství a potravinářství. Cílem bylo získání vybraných typů průmyslově cenných metabolitů, případně energie. Předložená studie byla zaměřena na srovnání růstu a produkčních vlastností několika kmenů karotenogenních kvasinek rodu Rhodotorula, Sporobolomyces a Cystofilobasidium, kultivovaných v médiích s obsahem glycerolu (technický a odpadní glycerol), dále v médiích obsahujících pšeničnou slámu, hydrolyzovanou slámu zpracovanou v hydrotermálním procesu při vysoké teplotě a zbytky po filtraci hydrolyzátu. Dalším testovaným odpadním substrátem byla syrovátka. Všechny testované kvasinky byly schopny využít glycerol jako jediný zdroj uhlíku. Produkce biomasy při kultivaci na technickém glycerolu se více či méně přibližovala kontrole (cca 7 - 10 gl-1), zatímco při kultivaci na odpadním glycerolu byla produkce vyšší (10.9 - 14.5 gl-1). Produkce karotenoidů a ergosterolu byla vyšší v glukózovém médiu než v médiu s obsahem glycerolu. Všechny testované kvasinky byly rovněž schopny produkovat neutrální lipidy, a to v rozmezí 11 - 15 %, s výjimkou C. capitatum, kde produkce dosahovala více než 22 % obsahu neutrálních lipidů. Pšeničná sláma a produkty z ní připravené se ukázaly být využitelnými substráty s vysokým potenciálem pro produkci biomasy i metabolitů, a to zejména u kmene S. roseus. Syrovátka, jako odpadní produkt mlékarenství, byla účinně využita jako substrát pro kokultivaci karotenogenních kvasinek a bakterií mléčného kvašení. Kokultivační proces může vyvolat nadprodukci pigmentů a ergosterolu, přičemž získaná biomasa díky obohacení o bakterie L. casei dosahovala vyšší kvality. Za účelem energetického využití mikrobiálního metabolismu formou mikrobiálních palivových článků, tzv. „Microbial Fuel Cell“ byla aplikována směsná kultura bakterií získaných z čistírny odpadních vod. Tyto mikroorganizmy hrají významnou roli při výrobě elektrické energie a současně také při čištění odpadních vod. Elektřina je generována přímo z organických látek přítomných v kultivačním médiu a lze ji použít pro provoz čistírny samotné a případně i pro další aplikace.
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Use of Waste Substrates to Production of Enriched Yeast Biomass
Starečková, Terezie ; Demnerová, Kateřina (referee) ; Vávrová, Milada (referee) ; doc.PharmDr.Petr Babula, Ph.D. (referee) ; Márová, Ivana (advisor)
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.
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Random mutagenesis and selection of red yeast mutants capable to utilize particular waste substrates
Čačková, Katarína ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.
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Microbial production of carotenoid pigments using waste substrates
Němcová, Andrea ; Ing.Martina Čarnecká, Ph.D. (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments produced by bacteria, yeasts, filamentous fungi and plants. They exhibit significant biological effects and are widely used in the food industry, pharmacy and cosmetics. The aim of this diploma thesis proposed as a comparative study was regulation of carotenoid and ergosterol production in red yeasts using several waste substrates as whey, corn germs, wheat, apple fiber and pasta. To selected production media extracellular hydrolytic enzymes degrading polysaccharide were added. These enzymes were obtained from the cultivation media of four fungal strains. In this study three carotenogenic yeast strains were used: Rhodotorula glutinis, Sporobolomyces roseus and Cystofilobasidium capitatum. All strains were cultivated simultaneously and changes in biomass and carotenoid production in different production media were monitored and compared. As the best waste substrate apple fiber was utilized, particularly in Rhodotorula glutinis, which exhibited mainly biomass production increase. In Sporobolomyces roseus increased production of biomass and carotenoids have been reported in media with hydrolyzed fiber and pasta as well. Beta-carotene production in this strain reached 4776,38 mg/g of dry weight. The strain Cystofilobasidium capitatum exhibited in waste media a decerase of biomass production accompanied with increased production of carotenoids, especially in wheat mush and pasta medium hydrolyzed by enzyme preparative from Aureobasidium pullulan. It can be concluded that hydrolyzed waste substrates are very hopeful as cheap nutrient sources for yeast strains producing carotenoids and ergosterol. Nevertheless, further study of substrate processing for individual strains is needed.
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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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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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Characterization of waste rice substrates and their potential utilization by microorganisms
Kapar, Jiří ; Duroňová, Kateřina (referee) ; Márová, Ivana (advisor)
The aim of this bachelor thesis was to characterize some cereal substrates. Based on the results of group parameters the most suitable substrate for cultivation of model industrial microorganisms was proposed. Analysis of total phenolics, reducing sugars and total sugars was performed spectrophotometrically. Using high-performance liquid chromatography with refractometry index detection content of individual mono-and disaccharides was measured. Saccharides were extracted from cereal substrates by water and then acid hydrolysis was applied. Two rice substrates were then used as nutrition sources for cultivation of two model industrial microorganisms: yeast Rhodotorula glutinis and bacterium Bacillus subtilis. Before use to production media substrates were processed by chemical and enzyme hydrolysis and analyzed. Production of microbial biomass in media with modified waste substrates was evaluated.
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Use of diffrent nutrition source by selected carotenogenic yeasts
Čačková, Katarína ; Starečková, Terezie (referee) ; Márová, Ivana (advisor)
Carotenoids are the most common naturally occurring terpenoid pigments. Because of their positive biological functions, carotenoids represent a group of important molecules for the pharmaceutical, chemical, food and feed industries. This work was realized as a comparative study focused on use of some waste substrates as nutritional sources for production of carotenoid pigments by red yeast Rhodotorula aurantiaca. Yeast were cultivated in different media with selected waste substrates – apple fiber, whey, wheat etc. To some media hydrolytic enzymes prepared from some fungal strains were added. Most of waste substrates seems to be useful for R. aurantiaca cultivation. As the best nutritional sources for enriched biomass and carotenoid production apple fiber or wheat processed by mixed preparative of extracellular hydrolases from Fusarium solani were found.
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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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