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Characterization of yeast and algae metabolites by instrumental techniques
Kurowska, Klára ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
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).
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Selected bioengineering characteristics of lactic acid bacteria
Šťásková, Lucie ; Vítová, Eva (referee) ; Babák, Libor (advisor)
The diploma thesis deals with the growth of biomass and production of selected metabolit–lactic acid by thermophilic bacteria Bacillus coagulans. The resulting selected metabolite was determined by HPLC method. Cultivations of this genus were performed on synthetic media, where the influence of carbohydrate used as carbon source was tested. Lactose was more suitable fot growth of biomass and glucose for production of lactic acid. On natural whey media the influence of different conditions were tested. The highest yields of biomass and production of lactic acid were observed on enriched whey medium. The last part deals with comparing the production of biomass and metabolites, depending on the volume of media. There were compared selected bioengineering characteristics of all cultivations.
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Development and optimization of methods for detection of selected fruit species in foodstuffs
Šlosárová, Katarína ; Wikarská, Monika (referee) ; Fialová, Lenka (advisor)
Various types of adulteration of food products are becoming more frequent in the food industry than ever. That is no different for fruit products. This bachelor thesis deals with the detection of mango in fruit purées for infants through the use of qPCR and HPLC in three selected commercial products and their prepared replicas. Within the framework of molecular biology, different primers specific for mango were tested. Several primers, which were the most suitable for the demonstration of mango in fruit purées, were selected based on the course of the qPCR reaction, the size of the amplified product, specificity, and sensitivity. Using these primers, the presence of mango was proven in all six analysed samples. Regarding the instrumental part of the work, a rapid and simple method of extraction of phenolic compounds for their analysis by HPLC was tested, which has been previously successfully used on another plant matrix. This method was proved to be unsuitable for the extraction of phenolic compounds from fruit purées for the purpose of their authenticity verification using HPLC [1].
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Content of rutin in the biological waste material from plantations of elderberry
Boháčová, Hana ; Vránová, Dana (referee) ; Vespalcová, Milena (advisor)
This thesis is focused on determining the concentration of rutin in twigs elderberry (Sambucus nigra L.). The theoretical part provides an overview of the properties, occurrence and use of elderberry in folk medicine, medicine and food. Furthermore, there are the most important substances contained in elderberry, especially flavonoids, including rutin and quercetin as well. There are briefly described their properties, biological effects and possibilities of determination. The content of the experimental part of the thesis was developed cheap extraction methods to obtain the maximum yield of dried twigs rutin elderberry. Rutin content was observed in twigs cultivated elder varieties and wild elderberry. The identification and quantification of rutin in extracts was optimized and validated method of high performance liquid chromatography (HPLC). As a comparative method to develop extraction was used a pressurized hot water extraction (PHWE). There were tested three particle sizes of shredded twigs. The particles were extracted differently hot water varying duration. Most rutin was found in samples milled at 4 mm particles. The optimum time and temperature of extraction was 7 hours at 80°C. The highest amount of rutin in bred elderberry was determined in a sample Mladší 2014, digestion using 1,49 ± 0,004 mg.100g-1 and method PHWE 5,75 ± 0,015 mg.100g-1. There was determined the most rutin in a sample Září 2014 in the wild variant of elderberry. By digestion of ground twigs was obtained 0,90 ± 0,022 mg.100g-1 of rutin, using PHWE 2,98 ± 0,022 mg.100g-1.
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Solubilization of water-insoluble vitamins into vesicular systems based on ionic amphiphilic pairs
Kolomá, Nikola ; Mravcová, Ludmila (referee) ; Mravec, Filip (advisor)
This thesis deals with the characterization and preparation of catanionic vesicular systems and their interaction with water-insoluble vitamins. Catanionic vesicles systems was prepared from two differently charged sufractants CTAB and SDS in formation ion pair amphiphile. Stability of vesicles was secured by adding cholesterol with positively charged surfactant DODAC. Were selected vitamin A and vitamin E for solubilization into these vesicles. The next part of this work was focused on determining the efficiency of incorporation these vitamins into the HTMA-DS vesicular system. The characterization of these systems was improved by measurement on a UV-VIS spectrometer, DLS and by HPLC with UV-VIS detector. During measurement by UV-VIS and HPLC the efficiency of solubilization of vitamin A and vitamin E into vesicles was determinated. In both cases higher efficiency was determinated for vesicles with incorporated vitamin A. Higher efficiency for both vitamins was determinated by HPLC. The reason is probably the higher sensitivity of the metod, work in labs with dark glass and preparing individuals samples into vials, which were inserted into the device for individual dosing by injector. The work serves primary evaluation results in view of the vesicular system for use in pharmaceutical application like carrier non-polar vitamins.
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Determination of selected red fruit species in plant-based food using multiplex PCR and instrumental methods
Vybíralová, Natálie ; Langová, Denisa (referee) ; Fialová, Lenka (advisor)
In many cases food is becoming the subject of adulteration, including fruit products that contain raspberries, strawberries and blueberries. This bachelor thesis is focused on the detection of strawberries, rapsberries and blueberries in model and commercial baby food products using multiplex PCR and HLPC. The theoretical part of this thesis is focused on composition of red fruits, their importance in human nutrition and especially about isolation of DNA from plant material. The aim of the experimental part of work was the analysis of selected commercial and model mixtures fruit purees containg raspeberries, strawberries and blueberries using instrumental and molecular biological methods. The results of these metods are compared. Commercial purees were bought in retail grocery shop. Model mixtures of these purees were prepared in our laboratory. DNA was isolated from fruit purees after and it’s amplifiability was comfirmed, it was successfully used in multiplex PCR to confirm the presence of raspberries, strawberries and blueberries in fruit purees. In the instrumental part, certain phenolic substences which are specific to red fruits were detected by HPLC in model and commercial mixtures.
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Detection of selected fruit species in plant-based foodstuffs through instrumental analysis and methods of molecular biology
Ondruch, Petr ; Langová, Denisa (referee) ; Fialová, Lenka (advisor)
Food is often subject to adulteration, including fruit products containing strawberries. This work deals with the detection of strawberries in model and real fruit products, so that possible falsification can be detected. The aim of the work was the preparation of model and real fruit products, their analysis using selected instrumental and molecular-biological methods and mutual comparison of the results of these methods. Model mixtures of fruit products were prepared and commercial ones were purchased. The content of polyphenols and flavonoids was determined by UV-VIS spectrophotometry, which were characterized in more detail by HPLC. DNA was isolated from the fruit purees, suitable primers confirmed the amplifiability and formation of a specific product for strawberry, and this product was verified by electrophoretic control. In the instrumental part, certain substances were detected by extraction from model and commercial mixtures and the HPLC method, and the amount of selected substances was determined. The lowest amount of strawberry DNA that could be detected by selected primers in the PCR method was 0.7 ng /µl, in commercial mixtures this DNA could not be detected. In the HPLC method, the strawberry was detected in all samples by focusing on a particular fingerprint and the amount of pelargonidin-3-glucoside in the samples. The instrumental method can be considered more suitable in this case.
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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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The possibility of using of elderberry fruit pomace
Ondrušová, Pavlína ; Diviš, Pavel (referee) ; Vespalcová, Milena (advisor)
This diploma thesis concerns the determination of anthocyanin pigments in the elderberry pomace (Sambucus nigra L). The theoretical part describes in general the plant specification, its chemical composition, botanical characteristics and the possibility of utilization of elderberry in food processing industry and in pharmacy. However, the main concern of this thesis are the anthocyanins. This part is closely focused on chemical structure and factors that have significant influence over their stability. There are briefly summarized the positive effects of these colourants and their use in the food industry. In the experimental part, the elderberry juice and the extracts from the rhizomes were prepared. Two different solvents, a 1: 1 water / ethanol mixture and a mixture of water and methanol in the same ratio were used as extractors. To identify and quantify the dyes in the prepared samples, a chromatographic separation system (HPLC) with a Kinetex EVO C18 column and isocratic elution of the combined mobile phases of water of acetonitrile and formic acid was developed. The analysis only took 12 minutes. Two major dyes cyanidin-3-glucoside and cyanidin-3-sambubioside were identified and quantified by HPLC. The largest amount of anthocyanins was obtained by extracting mixture methanol / water (594.6 ± 0.42 mg.100 g-1). The content of anthocyanin dyes in dry extracts and juice was also determined by the often-used pH-differential method. Both results were compared. The HPLC method provided very accurate results against the less demanding pH-differential assay method, which proved to be suitable for the indicative measurement of anthocyanin content in real samples.
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Chromatographic determination of simple sugars in selected beverages
Jurečková, Zuzana ; Diviš, Pavel (referee) ; Vespalcová, Milena (advisor)
An important part of the drinks are simple carbohydrates, because this thesis focuses on this issue. The theoretical part describes the characteristics , production , metabolism and the use of simple carbohydrates such as glucose, fructose and sucrose. There is also the definition of drinks and their main divisions . The literature review is based on the experimental part of analyzing selected beverages by high performance liquid chromatography with refractometric detection coupled to columns or alkyl ion-exchange phases. In conclusion, the results were compared to determine and comment . Nowadays the emphasis is on the correct functioning of the digestive tract. An important factor that supports the intestinal microflora is a prebiotic carbohydrate - tagatosa This could become tagatosa whose properties , production, metabolism in the body and any use is also included in the search . In the experimental part is listed possible optimization analysis of samples containing tagatosy on both types of columns in the HPLC system. The result is again commented in conclusion.
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