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Production of carotenoi by yeasts of the genus Cystofilobasidium
Vavrysová, Alena ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
Carotenoids are important industrial pigments present practically in all living organisms. The aim of presented work is the study of regulation of carotenoid production in yeasts of the genus Cystofilobasidium in presence of exogenous stress factors. Growth curve of C. capitatum exhibited typical two-stage course with prolonged stationary phase similar to other carotenogenic yeasts. Maximal production of biomass and beta-carotene occurred in 103rd hour. Applied stress factors (2-5% NACl, 2-5 mM H2O2, 0,01-1 mM Se(IV), 0,1-5 mM Cr(III)) exhibited no significant influence on biomass production, which reached on average 8-9 g/l. Positive effect was observed in presence of 5mM Cr where 10 g/L of biomass was produced. Beta-carotene formation was positively influenced by many applied stress factors, the highest yield (695 g/g) was reached in presence of 0,1 mM Se(IV). No simultaneous regulation of ergosterol and carotenes was observed in Cystofilobasidium cells. Production properties of yeast strain C. capitatum CCY 10-1-1 wee compared with those of other carotenogenic yeasts of the genes Rhodotorula and Sporobolomyces. C. capitatum produced similar biomass yield as Rhodotorula sp. in presence of salt. Production of beta-carotene by C. capitatum was slightly higher than in Rhodotorula glutinis, but lower than in Sporobolomyces strains which exhibited substantially lower biomass production. Karyotype of C. capitatum is relatively different when compared with karyotype of other carotenogenic yeasts. Based on summary of our results in seems that yeasts C. capitatum exhibit similar physiological as well as production properties as some Rhodotorula strains. Thus, yeasts of the genus Cystofilobasidium could be potentially used to industrial production of carotenoid pigments as well as yeast biomass rich in carotenoids and some biogenic elements.
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Optimalization of PCR-RFLP method for taxonomy of yeasts
Olivová, Radana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deal with optimalization method PCR – RFLP for taxonomy enlistment of yeasts. Conventional identification methods for yeasts are time-consuming. Molecular biological method based on PCR are instrumental towards fast and precise identification as compared to conventional phenotypic methods. In this thesis molecular biological method PCR – RFLP was used for identification and enlistment of yeasts. This metod follow repeating spacers of ribozomal DNA of yeast, characteristic for each species and strain. By the help of PCR were amplified specific partitions of DNA. These fragments of DNA were split by restriction endonucleases and identified by horizontal electroforesis. In background of this thesis there are information about yeasts, their taxonomy and molecular biological methods.
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Simultaneous co-cultivation of selected strains of carotenogenic yeasts and autotrophic bacteria
Blažková, Jana ; Němcová, Andrea (referee) ; Szotkowski, Martin (advisor)
The submitted diploma thesis was focused on the study of co-cultivation of selected microorganisms, which were carotenogenic yeasts and cyanobacteria. The production of selected metabolites was compared in these co-cultivations. The main metabolites monitored were carotenoids, sterols, coenzyme Q10, chlorophylls and lipids. Furthermore, this work focused on the study and possibilities of optimizing the production of lipids and lipid substances in selected strains of carotenogenic yeasts and cyanobacterial species. The theoretical part is focused on the description of yeasts, especially carotenogenic yeasts, cyanobacteria and the chemical composition of the produced metabolites. Microorganisms such as yeast and cyanobacteria contain carotenoids, which are natural pigments and are classified as antioxidants. As antioxidants, they have significant biological effects, such as effects on human health. Coenzyme Q has a positive effect on the functioning of organs in the human body. Chlorophyll is widely used in the food industry as a green dye. Lipids produced by microorganisms contain a large amount of unsaturated fatty acids, which is currently used in cosmetics or pharmacy. The theoretical part also described the individual analytical methods by which the production of the monitored metabolites could be determined. The experimental part is focused on the production of carotenoids, sterols, coenzyme Q10 and chlorophyll, which were determined by HPLC, lipids and fatty acid profile were determined by GC. The determined metabolites are monitored in different types of co-culture partners (carotenogenic yeast and cyanobacteria) in media with different additions of macroelements (P, N and Mg). This was followed by a co-cultivation experiment using waste oils (frying and coffee oil) and a study of the effect of waste oils added to co-cultivations. Co-cultivation experiments confirmed the ability of carotenogenic yeasts and cyanobacteria to grow together. The best results were obtained with Rhodosporidium toruloides and Anabena torulosa, Rhodosporidium toruloides and Arthrospira maxima.
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Isolation, Identification and Characterisation of Microbial Communities of Wine and Selected Foods
Šuranská, Hana ; Španová, Alena (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
Proposed dissertation thesis deals with wine and artisanal cheeses microbiology. The first part is focused on identification of yeasts isolated from grapes and musts during production of white and red wines. The grape varieties were grown under the integrated and organic farming on Moravian vineyard. Yeasts were identified by ITS-PCR-RFLP method (amplifying internal transcribed spacer ITS: ITS1, ITS2 and 5.8S rDNA) and unknown species were subjected to partial sequencing of ITS rDNA region. In total, 524 isolates were divided into 14 different species belonging to six genus were identified from. The first stages of fermentation process were characterised by predominance of non-saccharomyces species especially H. uvarum. Due to increased ethanol concentration strains of S. cerevisiae prevailed in the later phases of the process. Further, partial aim of this study was to isolate and to apply selected autochthonous S. cerevisiae strains as starter culture during controlled industrial wine fermentation process. Genus Saccharomyces was distinguished from other non-saccharomyces species by ITS-PCR-RFLP. Further, in order to distinguish Saccharomyces genus at the species and the strain level, several molecular methods were applied including PCR-fingerprinting (rep- and RAPD-PCR), species-specific primers (multiplex and touchdown PCR), LSU-DGGE and interdelta PCR. Species-specific primers enabled us to distinguish some species of the Saccharomyces sensu stricto complex. Furthermore, interdelta PCR seems to be useful tool for S. cerevisiae strains identification. Among 120 isolated autochthonous strains belonging to Saccharomyces genus, 45 different strains were identified. Based on its sufficient technological properties (osmo- and ethanol tolerance, low H2S production etc.), S. cerevisiae 1-09 strain isolated from grape berries coming from moravian vineyard was chosen. Strain S. cerevisiae 1-09 was tested in small amount of must and after that also during industrial fermentation of red and white wine production. Based on the results of chemical and sensorial analysis, the strain seems to be suitable for application as the starter culture for winemaking process. The final part of this thesis is focused on quantification and identification of the yeasts isolated from artisanal cheeses and their by-products coming from Western Balkan Countries. Isolated species were identified by ITS-PCR-RFLP, partial sequencing and by physiological tests. Among the 20 yeast species found, D. hansenii, C. zeylanoides and Y. lipolytica were found to be predominant. Moreover, we developed culture-independent, semi-quantitative technique based on construction of ITS-clone library from metagenomic DNA to investigate complex fungal communities associated with artisanal cheeses and their by-products. Novel technique is based on direct extraction of total DNA from the sample. This was compared with culture-dependent ITS-PCR-RFLP and culture-independent LSU-DGGE methods. The results highlighted the discrepancies among these methods. Finally, the divergences among applied methods were confirmed by correlation analysis and by indices of general biodiversity and dominance of species. ITS-clone library approach combines the advantages of cultivation-based analysis and LSU-DGGE with semi-quantification of fungal species without the requirement of their cultivation. This study might open new perspectives in direct and complex analysis of yeasts and moulds in food matrices.
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Identification of yeasts from interspecific varieties of grapes
Sadel, Peter ; Omelková, Jiřina (referee) ; Vránová, Dana (advisor)
The main goal of my diploma thesis was to identify and characterize yeasts from must Hibernal and also collection yeasts by using methods called RFLP-PCR (Restriction Fragment Length Polymorphism - Polymerase Chain Reaction). Theory was the first part of my diploma thesis which dealt with wine, yeasts and molecular methods. Theory section was followed by experimental section divided into two parts. The main goal of the first part was to characterize and identify yeasts from must Hibernal by using PCR and RFLP-PCR methods. In the samples there were found yeasts Saccharomyces and Pichia. The second experimental part of my diploma thesis had a goal to extend the database of new yeasts using the same methods mentioned in the first part of experimental section.
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Use of stress factors for the production of lipid substances by yeasts of the genus Metschnikowia
Tručková, Marie ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
This Master´s thesis consists of two parts. The theoretical part is focused on the influence of various stress factors in the cultivation process, such as osmotic stress, oxidative stress, dehydration, cultivation time, pH or temperature on yeasts of the Metschnikowia genus. The practical part deals with osmotic and oxidative stress. Oxidative stress was performed under two different conditions (various cultivation time and temperature). The properties of yeast production were monitored mainly by gas chromatography and marginally also by flow cytometry. The analysis was performed for Metschnikowia pulcherrima, Metschnikowia andauensis, Metschnikowia chrysoperlae, Metschnikowia sinensis, Metschnikowia zizyphicola and Metschnikowia shanxiensis strains. The results showed that both oxidative stress and osmotic stress induce better lipids production. The amount of lipids, especially the fatty acid composition, also varied depending on the strains studied and the culture conditions used. The production of unsaturated fatty acids was further demonstrated in this work. The most suitable medium for the production of lipids and unsaturated fatty acids was the salt medium. It is clear that yeasts of the genus Metschnikowia are highly adaptable yeasts. Therefore, they might be potentially auspicious biotechnology producers.
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Monitoring of the influence of using indigenous yeasts for wine production in the conditions of winery
Beníčková, Romana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with identification of yeasts by applying the RFLP-PCR method. Objective of the thesis was to identify the yeasts present in wine from Grüner Veltliner during fermentation. Identification was made by amplification of 5,8S-ITS sequences of DNA by the polymerase chain reaction with primers ITS1 and ITS4. Amplified DNA was submitted to the restriction analysis by restriction endonuclease HaeIII, HinfI and HhaI. By restriction analysis with a specific enzyme, the amplified DNA is chopped into the specific fragments which are characteristic for given kind of yeasts. In the analysed wine, the dominance of autochthonal yeast Saccharomyces cerevisiae was confirmed throughout fermentation. The other identified yeasts in the wine were of kind Pichia. The second part of the thesis was to expand the database by characterization of 28 type-yeasts, using RFLP-PCR analysis. To compare the genetic similarity, program BioNumerics was used, which processed the results of UPGMA cluster analysis using Jaccard´s coefficients.
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Application of chemical and physical stresses in the late phase of growth to selected strains of microorganisms
Langer, Marek ; Holub, Jiří (referee) ; Szotkowski, Martin (advisor)
A wide spectrum of microorganisms is used in the biotechnological industry for production of meaningful metabolites; including carotenoids and chlorofyles with antioxidant effects, fatty acids, ubiquinone and ergosterol. Ways to increase yield of metabolites to maximum capacity are being researched to increase efficiency and economical sustainability in industry. One of the ways is the application of chemical stress factors on microorganisms as is described in this thesis. In the theoritical part certain microorganisms, their important intracellular metabolites and their metabolism are established. The last subchapter covers the usage of stress factors in industry. In the experimental part each microorganism was subjected to various concentrations of sodium chloride, cobalt(II) sulfate and iron(III) chloride. Significant increase of lipid production was registered after an application of sodium chloride on yeast strain Rhodosporium toruloides. An increase in production of torularhodin in Sporidiobolus pararoseus occured after application of iron salt. After an application of sodium chloride the growth of torularhodin in strains Cystofilobasidium macerans a Rhodosporidium toruloides was significant. Sodium chloride was also a suitable stress factor for lycopene formation in Rhodotorula mucilaginosa. The most meaningful stress factor of algae was cobalt(II) sulfate which increased metabolite production in Scenedesmus obliquus. Cyanobacteria Anabaena torulosa also attained a significant production in the presence of sodium chloride.
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Analysis of glucans in plant and microbial samples
Vít, Radek ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The aim of the diploma thesis is study of glucans production in selected species of yeasts, algae and plants. Cultivation conditions for yeast strains were performed to gain increased production of glucans under different temperature conditions and in media of different composition. Into the set of tested yeasts species strains Saccharomyces cerevisiae (CCY 6646), Sporidiobolus pararoseus (CCY 19-9-6), Phaffia rhodozyma (CCY 77-1), Rhodotorula glutinis (CCY 20-78-26) and Cystofilobasidium infirmominiatum (CCY 17-18-4) were enrolled. Saccharomyces cerevisiae was cultivated as a control strain because of its verified production of -glucans. -glucans were determinated by the enzyme kit K-YBGL Megazyme. For comparison, algal strain Euglena gracilis (CCALA 349), some species of mushrooms (shiitake, oyster mushroom, garden champignon and Jew’s ear) and cereals (wheat, rye, oats, rice and barley) were analysed too. Further, fatty acid content in the yeast cells was determined by the GC/FID. The best producer of yeast -glucans was R. glutinis CCY 20-7-26, which showed the highest biomass production (12-14 g/l) and also a relatively high amount of -glucans (25-30 %), in cultivation at 15 °C in a medium containing yeast extract in combination with ammonium sulphate. The presence of -glucans has been demonstrated in the microalgae, Euglena gracilis CCALA 349, as well as in samples of higher fungi and cereals.
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Factors influencing the quality of red wine
Zechmeisterová, Lucie ; Vránová, Dana (referee) ; Omelková, Jiřina (advisor)
In my thesis, I focused on monitoring of microorganisms in the sample of red grape juice and on the interactions between yeasts, bacteria and filamentous fungi. Three different media were applied for the cultivation of microorganisms; firstly for monitoring of total volume of microorganisms, secondly for yeasts and third time for lactic acid bacteria. The indirect method was used for the determination of the amount of viable cells. This method consists in enumerating of visible macroscopic colonies grown up on agar plates. When the cells grew up, the forms of colonies were analyzed visually and the morphology of microorganisms was detected microscopically. The operating time of enzymes in grape juice in the production of red wine was monitored after application of commercial enzymatic preparation. The enzym action in grape juice was observed on the basis of the process of degradation of high – molecular substrate by enzymes through the use of Ubbelohd´s viscometer. The research findings provided a lot of knowledge about the occurance of microflora in the process of production of red wine. The commercial preparations added to grape juice played a significant role.
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