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Biotechnological production of PHA employing Cupriavidus malaysiensis
Drábková, Kateřina ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
The subject of this diploma thesis is the study of the production of polyhydroxyalkanoates (PHA) employing Cupriavidus malaysiensis. The aim of this thesis is to obtain and characterize the polymer with the most advantageous properties. The theoretical part deals with polyhydroxyalkanoates and their biotechnological production. Then selected microorganisms producing 3-hydroxybutyrate and 4 hydroxybutyrate, P(3HB co 4HB), are described. After that, the theoretical part deals with the production of PHA employing Cupriavidus malaysiensis. The bacterial strains of Cupriavidus malaysiensis DSM 19416, 19379 and 25816 were used in the experimental part. The strain Cupriavidus necator H16 was used as a control strain. First, the ability to utilize various carbon sources by the given bacterial strains was determined. Then, the aim was to select the optimal carbon source, a precursor for the synthesis of a polymer with high content of 4-hydroxybutyrate, and to determine the ideal culturing conditions for the growth of the microorganisms, which have been studied, and the production of P(3HB co-4HB). A two-stage culture and bioreactor culture was also performed to produce the desired copolymer. Furthermore, the production of terpolymer of 3-hydroxybutyrate, 4 hydroxybutyrate and 3-hydroxyvalerate, P(3HB co 4HB co 3HV), was studied by single stage and two-stage cultivation. In the last part, the produced polymers were characterized by gas chromatography with flame ionization detector (GC-FID), size exclusion chromatography with multi-angle static light scattering (SEC-MALS), differential scanning calorimetry (DSC) and thermogravimetry.
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Monitoring the influence of stress factors on yeasts of the genus Metschnikowia using molecular techniques
Kuljovská, Tereza ; Kovalčík, Adriána (referee) ; Němcová, Andrea (advisor)
Molecular techniques are used widely nowadays, especially in the identification of various species of microorganisms, and provide reliable and accurate results in a relatively short period of time. This work deals with the characterization of Metschnikowia yeasts exposed to various stress factors and the monitoring of their genomic DNA by PCR-DGGE and PFGE methods. The work was focused on the analysis of ribosomal rDNA, specifically the ITS1 and 5,8-ITS2 regions and genes encoding the domains D1/D2, that are part of the large 26S rDNA ribosomal subunit, which are commonly used in the characterization of fungal eukaryotic communities. Three types of stress factors were selected for the experiments: osmotic stress (NaCl environment), oxidative stress (addition of H2O2 to the medium) and nutritional stress (addition of hemp flour / leaves and flowers as a carbon source). The analysis was performed for particular strains Metschnikowia andauensis, Metschnikowia pulcherrima, M. chrysoperlae, M. shanxiensis, M. sinensis and M. zizyphicola. The results showed that addition of the NaCl, H2O2 and hemp components at higher concentrations to the production media does not disrupt the ribosomal DNA when detected by PCR-DGGE. Mutations have not been observed by comparing these strains with yeast that was cultivated under optimal conditions. Despite stress factors, PFGE analysis of karyotypes showed that DNA of some yeast species does not prove any damage and remain intact. Remaining strains proved certain degree of damage, and bands were not detected on the gel for these strains. In the given circumstances, it can be stated that the high adaptability of these species to a stress environment makes them promising biotechnology producers. These yeasts have great potential for usage in agriculture as a tool for biocontrolling of fruit or vines.
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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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Study of the presence of phenolic substances and their antioxidant activity in food products containing rosehip (Rosa canina L.)
Hronová, Vendula ; Mikulíková, Renata (referee) ; Kovalčík, Adriána (advisor)
This bachelor's thesis monitored the content of bioactive substances and antioxidant activity in the jams made of the fruit of rose hips (Rosa canina L.). The jams were sweetened with various sweeteners such as cane sugar, beet sugar, erythritol and stevia. Moreover, the rheological properties of jams were determined. The properties of home-made jams prepared from rosehip fruit were compared with the commercial jam. Another aim was to determine whether the by-products received after the production of jams could serve as beneficial sources of bioactive substances after the preparation of water extracts. The content of carbohydrates, polyphenols, flavonoids, and antioxidant activity were determined in jams and extracts spectrophotometrically. Next, the spectrophotometric detection of pectin, the chemical characterization of jams by infrared spectrometry, and the spectrophotometric determination of the jam colour were performed. The concentration of bioactive substances in jams was influenced by the geographical location and the time and weather conditions of harvest. The rheological properties and pectin content of the jams depended on the type of the used sweetener. The assessment of the water extracts prepared from the by-products after jam production and from the dried fruits of rosehip showed the high content of bioactive substances and high antioxidant activity in all tested extracts. The presented work points out that it is possible to prepare jams with superfood properties from the fruit of rose hips and extracts with high antioxidant activity.
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Preparation and characterization of chitosan-based materials
Puchírová, Simona ; Kalina, Michal (referee) ; Kovalčík, Adriána (advisor)
The presented diploma thesis focuses on the characterization of the rheological properties of hydrogels based on agarose, alginate, polyvinyl alcohol and the study of the antimicrobial effect of chitosan added to selected hydrogels. The theoretical part is focused on the creating a literature research dealing with the description of the basic characteristics of hydrogels in general, describes the nature, properties, structure and possibilities of its use. In the experimental part, hydrogels without added active substance and hydrogels modified with chitosan were prepared. Based on rheological measurements, suitable combinations of hydrogels with chitosan were selected. A part of the hydrogel was taken from the samples and monitored in a drying and re-swelling process in distilled water. The hydrogels were tested by Ftir analysis, which verified the presence of chitosan in the samples. Monitoring of the antimicrobial effect of chitosan was performed by the hole diffusion method. Efficacy was tested on bacterial strains of Escherichia coli, Candida glabrata, Micrococcus luteus, Serratia marcescens. Most excised hydrogel disks have formed zones of inhibition of various sizes. Inhibition zones were also formed around hydrogels without chitosan, where the sensitivity of individual microbial cultures to acetic acid is assumed. The antibacterial active of chitosan was verified by studying the separate sensitivity of chitosan solutions and acetic acid.
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Study of volatile compounds of chocolate depending on geographical origin of cocoa beans
Kubínová, Martina ; Kovalčík, Adriána (referee) ; Vítová, Eva (advisor)
Diploma thesis is focused on cocoa beans volatile compounds from various region (Tanzania, Haiti, Ecuador, Madagascar and Peru) and chocolate bars made of them. The main aim was to judge diferences between samples according to geographycal origin. At the same time, samples of chocolates with different content of cocoa solids (50%, 70% „organic“, 70%, 78%, 90%) and a sample of cocoa powder from the common market were analyzed. Volatiles were identified and semiquantified by HS-SPME-GC-MS. A total of 86 volatile substances were identified, they were divided into 7 groups according to the nature of the odor: fruit tones (17), nut and chocolate (16), astringent and sour (12), unspecified (14), sweet nad honey (10), earthy (9) and floral (8). The data were transformed into a 2D graph by analyzing the main component according to selected distinguishing varibles. The difference in the volatile substances of cocoa beans depending on the geographical origin were confirmed using principal component analysis.
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The effect of the quantity and type of nuts used in the production of pâté on the product sensory quality
Mejzlíková, Blanka ; Kovalčík, Adriána (referee) ; Mikulíková, Renata (advisor)
This bachelor thesis deals with the sensory evaluation of pâté with nuts. The 9 samples of pâtés which contained different quantity of nuts and different types of nuts were produced for sensory evaluation. In the theoretical part pâtés are characterised, their history, composition and manufacturing are described. The part of literature search is description and chemical composition of nuts which were used for production of pâtés. Another topic is a description of conditionals and methods of sensory analysis and sensory evaluation of meat products. The practical part comprises production of test samples, samples for sensory analysis, evaluating the heat treatment and sensory analysis. Percentage amount of lipids and composition of fatty acids were determined by gas chromatography. Processing of results of sensory and chemical analysis of samples of pâtés was determined that the best samples were pâtés with cashew nuts and pistachios.
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Utilization of waste banana peels for packaging materials
Vachta, Jonáš ; Mikulíková, Renata (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with isolation of cellulose from waste banana peels and it’s use as a filler in composite films. The aim of this work was to prepare a bioplastic capable of being an eco-friendly alternative to conventional polymers with a potential for application in the packaging industry. The cellulose was isolated from banana peels using a method combination of alkalic and acidic hydrolysis and bleaching by sodium chlorite. The samples after isolation were characterised by Fourier transform infrared spectroscopy (FT-IR) and their average particle size was determined by microscope observation. Subsequently, composite films were prepared using agar and starch matrix, glycerol as plasticizer and isolated cellulose as filler in various concentrations to assess the effect of added cellulose on the properties of the resulting bioplastics. Characterization of the prepared bioplastics included FT-IR measurement, surface examination by a scanning electron microscope (SEM) and the determination of mechanical properties by measuring tensile strength, elongation at break and Young’s modulus. It was found that the addition of cellulose from banana peels has a positive effect on the mechanical properties of all prepared agar- or strach-based composites, with the exception of elongation at break in agar films, where there was a slight decrease compared to the reference films.
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Study of selected vegetable oils from nuts
Lovásová, Kristína ; Kovalčík, Adriána (referee) ; Zemanová, Jana (advisor)
The bachelor´s thesis deals with the characterization of the selected nut oils. The theoretical part concentrates on the topic of vegetable oils, their composition and properties. It also focuses on the nuts, the specification of the selected nut oils and their use in different industries. It includes an overview of analytical methods suitable for assessing the quality of oils. The experimental part compares the results of the oil characterization using the selected methods. Based on the results, the differences between the samples were evaluated. The properties of the oils were determined on the basis of fat characteristics. These were subsequently specified by the determination of fatty acid composition. Fatty acids were determined using gas chromatography with a flame-ionization detector after conversion to methyl esters by acid esterification with boron trifluoride. The determination of volatile compounds was performed through solid-phase microextraction with gas chromatography separation and mass spectrometry detection. According to the fat characteristics, in particular the peroxide value, it was found that both oils were partially oxidatively degraded, even though they were stored in the refrigerator (4 °C). A total of 13 saturated, 7 monounsaturated and 4 polyunsaturated fatty acids were detected. The total content of fatty acids varied, the higher content was detected in the walnut oil. Significant differences occurred in the composition of unsaturated fatty acids; the hazelnut oil had a higher content of monounsaturated oleic acid, the walnut oil had a high content of polyunsaturated linoleic and -linolenic acids. A total of 29 volatile compounds were identified, the number and content of volatile compounds in samples differed. 20 volatile compounds were identified in the walnut oil, and 19 compounds in the hazelnut oil. The higher content was in the hazelnut oil, the quantitatively most important group were aldehydes in both oils.
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Preparation and characterization of biodegradable composites based on polyhydroxyalkanoates and coffee grounds
Cisáriková, Barbora ; Němcová, Andrea (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis was focused on the preparation and characterization of biodegradable composites based on poly-3-hydroxybutyrate (PHB) and spent coffee grounds (SCG). The aim was to prepare biocomposites based on PHB and coffee grounds. Biocomposites were prepared by solution casting in chloroform. Films with a thickness of about 150-200 µm were prepared with the addition of 20% unmodified and modified coffee grounds. The PHB/SCG films were modified by an addition of a plasticizer and a crosslinking agent or by blending with polybutylene adipate terephthalate (PBAT). The thermal properties of the films were determined by differential scanning calorimetry. The compatibility filler/polymer matrix compatibility was assessed morphologically with the naked eye. It has been found that the direct addition of spent coffee grounds to the PHB is not suitable due to the deterioration of the morphology of the film surfaces and the formation of aggregates. The compatibility between the spent coffee grounds and the PHB was improved after the addition of the plasticizer and the crosslinking agent. However, the highest compatibility between SCG and PHB was achieved in the case of esterified coffee grounds. Another way to improve the resulting morphological properties of the films thus prepared was to use coffee grounds fractions.
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