|
|
Influencing of molecular mass of hyaluronic acid by fermentation process
Wikarská, Monika ; Jílková, Jana (referee) ; Velebný, Vladimír (advisor)
The biological function of hyaluronic acid (HA) – the polysaccharide, which consists of repeating units of N-actylglucosamine and glucuronic acid – is extremely broad and often contradictory, depending on its molecular weight (MW). The bacterial production provides HA of high molecular weight and purity. The objective of this thesis is to control the molecular weight of produced HA by the addition of hyaluronidases during the process of fermentation, resulting in reduction of viscosity of the medium followed by increase of yield. Hyaluronic acid was prepared by batch fermentation of the microorganism Streptococcus equi subsp. zooepidemicus. During the process of fermentation, the enzyme hyaluronate 4-glycanohydrolase (BTH) at various concentrations and different times of cultivation was added to bioreactors. The HA yield, the molecular weight and the polydispersity by the SEC-MALLS were monitored, together with the residual glucose concentration and the dynamic viscosity of the production medium. The formation of low and medium molecular weight HA was achieved. The addition of 10 U (BTH) / g (HA) had no significant effect on the molecular weight and therefore the HA function, but the corresponding drop of viscosity was noteworthy for the simplification of the product purification. Potential increase in HA yield was not confirmed because of the very low concentration of residual glucose and the method of precipitating low and medium MW HA.
|
|
|
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].
|
|
|
Preparation and expression of p53 protein isoforms using the GATEWAY expression system
Wikarská, Monika ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor)
The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.
|
|
|
Encapsulation of vitamins and provitamins into nanoparticles
Martinusíková, Katarína ; Wikarská, Monika (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis is focused on the encapsulation of vitamins in liposomes and the subsequent observation of the properties of this form. The theoretical part is focused on the history of vitamins, their classification into groups, but mainly the general characterization of all vitamins. It describes their function in the human body, the consequences of their deficiencies and also the sources of their income. It also describes the technique of encapsulation in liposomes and their characterization and use in the food industry. The experimental part is focused on the preparation of specific liposomes of vitamins and their mixtures. These prepared liposomes are subsequently characterized by properties such as size, stability and encapsulation efficiency. Long-term stability of prepared liposome particles over 2 and 5 weeks were also investigated. In the conclusion of this thesis are summarized all the results of observations and the subsequent suggestion of the possible use of this technique in food products.
|
|
|
Development of antimicrobial wound healing nanostructure biomaterials
Hanák, Jan ; Wikarská, Monika (referee) ; Márová, Ivana (advisor)
The bachelor thesis is focused on optimizing the production of nanoparticles with a bioactive component - especially on enzymes and antibiotics encapsulated into lipozomes. Nanoparticles with active substances could be used in the field of pharmacy or cosmetology. In the theoretical part the composition of the skin, skin injuries, and wound covers are described. The materials used for the preparation of nanoparticles and nanofibers are characterized as well as processes to produce such nanomaterials. Enzymes, antimicrobial drugs, and methods for determining antimicrobial activity are mentioned in this section, too. The practical part deals with the characterization and optimization of the formation of lipozome particles with encapsulated enzymes. The formed particles were divided into two groups. The first type of particles were pure lipozomes, while the second ones enriched by polyhydroxybutyrate to increase the encapsulation efficiency and improve the stability of the particles. The enzymes bromelain and lysozyme were selected for the highest encapsulation efficiency. The encapsulation of these two enzymes took place in two ways - by thin film hydration and by sonication method. Both methods showed high encapsulation efficiency, but the sonication method proved to be unsuitable due to enzyme denaturation. The proteolytic activity of the released enzymes from the particles was measured. The stability of the particles was monitored in a physiological and aquatic environment by measuring the zeta potential for up to 28 days. Moreover, the enzymes, ampicillin, and particles created by sonication were subsequently subjected to antimicrobial tests by using two test bacterial strains. Finally, alginate particles with encapsulated enzymes were also formed, then the encapsulation efficiency and proteolytic activity of the enzyme were measured. In this study also other methods of encapsulation of active substances are outlined, which could be possibly used to produce nanomaterials suitable for wound healing.
|
|
|
Detection of undeclared fruit species in foodstuffs using methods of molecular biology and instrumental methods
Svobodová, Petra ; Wikarská, Monika (referee) ; Fialová, Lenka (advisor)
This thesis is focused on the detection of apple component in fruit-based baby food. The theoretical part deals with the fruit itself – the apple; food adulteration as a reason for the control of food composition and methods, both instrumental and those of molecular biology, by which food adulteration can be detected. In the experimental part, commercial products containing apple puree were purchased and model mixtures were prepared as replicas of the purchased products. DNA was isolated from these samples, its amplifiability was verified, and the presence of the apple component was detected after the selection of suitable species-specific primers. Methanol extracts for HPLC were prepared from the above-mentioned samples using a procedure previously used for the extraction of phenolic substances from apple pulp [1]. Using this analysis, apple in fruit purees was to be identified using phloridzin as a marker for the presence of the apple component. The result was a successful detection of the apple component in both commercial products and model mixtures using qPCR. However, the tested method of phenolic substance extraction proved to be unsuitable for use in the analysis of fruit purees, as phloridzin was not detected either in the apple puree extract or in the model mixtures to which this puree was added.
|
|
|
Development of cosmetic products with antimicrobial effect
Dribňáková, Barbora ; Wikarská, Monika (referee) ; Márová, Ivana (advisor)
The proposed bachelor's thesis focuses on the preparation and study of nanomaterials with antimicrobial properties. The nanofibers were synthesized from biopolymers using the forcespinning method, and liposomes were prepared using the sonification method. Active ingredients used in this work are tymol, carvacrol, and eugenol. The theoretical section of the thesis discusses the methods for preparing nanofibers and nanoparticles and the materials required for their synthesis. It also covers the encapsulation of active ingredients in nanomaterials. Additionally, this part explores the skin microbiome, antimicrobial effects, and the use of both natural and synthetic antimicrobial agents. The practical part of the thesis focuses on determining the polyphenol content and antioxidant activity of each active ingredient. The gradual release of active ingredients into saline solution and distilled water was determined for nanofibers synthesized from polyhydroxybutyrate with encapsulated active ingredients. Furthermore, it deals with the optimization of nanofiber synthesis from chitosan and polyamide using the forcespinning method. The functionalized liposomes were characterized in terms of their size, stability, encapsulation efficiency, and gradual release of active ingredients. The liposomes showed high stability even after several weeks, and their size ranged from 170.90 ± 1.63 nm to 243.07 ± 7.83 nm. Finally, the antimicrobial activity of the prepared products and active ingredients was tested against Cutibacterium acnes, Staphylococcus epidermidis, and Escherichia coli. The tested active ingredients in pure and encapsulated form, demonstrated antimicrobial effect against both gram-positive and gram-negative bacteria. We assume the possibility of using the prepared nanomaterials in cosmetic products intended for the treatment of acne and other skin diseases, and also its use in the form of preservatives.
|
|
|
Micronization of biopolymer material
Gistingerová, Radka ; Wikarská, Monika (referee) ; Pospíšilová, Aneta (advisor)
This bachelor thesis focuses on the preparation of poly(3-hydroxybutyrate) microparticles for cosmetic products. The particles were prepared by emulsion technology, where a chloroform solution of poly(3-hydroxybutyrate) was dispersed in an aqueous solution of polyvinyl alcohol. The original poly(3-hydroxybutyrate) was also in powder form, but it was thought that its properties could be improved by this treatment. Evaporation of all the chloroform produced poly(3-hydroxybutyrate) particles. By changing the input concentrations of the two components, the particle size distribution was altered. All prepared powders were measured using a scanning electron microscope and measured by laser diffraction. Experiments and subsequent measurements showed that increasing the concentration of the emulsifier solution leads to an overall decrease in particle size, whereas a higher concentration of poly(3-hydroxybutyrate) causes an increase in particle size. To evaluate the potential of the prepared particles in cosmetics, all powders were evaluated sensorially. The evaluators observed almost no differences between the powders, only the original poly(3-hydroxybutyrate) powder was rated slightly worse compared to our prepared powders. The powder that is currently used in cosmetic products was rated very similarly to the powders that were prepared by emulsion technology.
|
|
|
Development of antimicrobial wound healing nanostructure biomaterials
Hanák, Jan ; Wikarská, Monika (referee) ; Márová, Ivana (advisor)
The bachelor thesis is focused on optimizing the production of nanoparticles with a bioactive component - especially on enzymes and antibiotics encapsulated into lipozomes. Nanoparticles with active substances could be used in the field of pharmacy or cosmetology. In the theoretical part the composition of the skin, skin injuries, and wound covers are described. The materials used for the preparation of nanoparticles and nanofibers are characterized as well as processes to produce such nanomaterials. Enzymes, antimicrobial drugs, and methods for determining antimicrobial activity are mentioned in this section, too. The practical part deals with the characterization and optimization of the formation of lipozome particles with encapsulated enzymes. The formed particles were divided into two groups. The first type of particles were pure lipozomes, while the second ones enriched by polyhydroxybutyrate to increase the encapsulation efficiency and improve the stability of the particles. The enzymes bromelain and lysozyme were selected for the highest encapsulation efficiency. The encapsulation of these two enzymes took place in two ways - by thin film hydration and by sonication method. Both methods showed high encapsulation efficiency, but the sonication method proved to be unsuitable due to enzyme denaturation. The proteolytic activity of the released enzymes from the particles was measured. The stability of the particles was monitored in a physiological and aquatic environment by measuring the zeta potential for up to 28 days. Moreover, the enzymes, ampicillin, and particles created by sonication were subsequently subjected to antimicrobial tests by using two test bacterial strains. Finally, alginate particles with encapsulated enzymes were also formed, then the encapsulation efficiency and proteolytic activity of the enzyme were measured. In this study also other methods of encapsulation of active substances are outlined, which could be possibly used to produce nanomaterials suitable for wound healing.
|
|
|
Encapsulation of vitamins and provitamins into nanoparticles
Martinusíková, Katarína ; Wikarská, Monika (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis is focused on the encapsulation of vitamins in liposomes and the subsequent observation of the properties of this form. The theoretical part is focused on the history of vitamins, their classification into groups, but mainly the general characterization of all vitamins. It describes their function in the human body, the consequences of their deficiencies and also the sources of their income. It also describes the technique of encapsulation in liposomes and their characterization and use in the food industry. The experimental part is focused on the preparation of specific liposomes of vitamins and their mixtures. These prepared liposomes are subsequently characterized by properties such as size, stability and encapsulation efficiency. Long-term stability of prepared liposome particles over 2 and 5 weeks were also investigated. In the conclusion of this thesis are summarized all the results of observations and the subsequent suggestion of the possible use of this technique in food products.
|
guest ::
RSS feed.