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Characterization of collagen from different animal tissues
Mikulíková, Zuzana ; Drábková, Michaela (referee) ; Márová, Ivana (advisor)
The aim of the proposed diploma thesis is to optimize the procedure of collagen isolation from animal tissues and methods for molecular characterization of isolates. In experimental part, isolations of collagens from animal tissues (chicken, hen, carp, and pork skin) were performed. Total protein concentration was determined by Biuret method and by TNBSA method. Both methods were used also in order to study physiological and thermal stability of the isolates under model conditions. Isolated collagens were characterized at molecular level using PAGE–SDS and microfluidic electrophoresis. Further, amino acids composition and microelement content were analyzed. Finally, the stability of isolated collagens in several types of simulated physiological and pathological conditions was tested too. During isolation relatively sufficient collagen yields were obtained (about 15 % of purified collagen per original mass of biological material). In all analyzed tissues Type I collagen was found. Thermal stability of individual samples differed according to biological source type and tissue age. Viscosimetry measurements confirmed higher stability in collagen samples of older animals. Collagenase exhibited the lowest degradation effect to bovine collagen, while selected mixed microbial hydrolases differed according to enzyme preparative type. Incubation of collagen isolates with selected human pathogens confirmed higher resistance of bovine collagen to biofilm formation when compared with the chicken one.
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Characterization of collagen from different animal tissues
Mikulíková, Zuzana ; Drábková, Michaela (referee) ; Márová, Ivana (advisor)
The aim of the proposed diploma thesis is to optimize the procedure of collagen isolation from animal tissues and methods for molecular characterization of isolates. In experimental part, isolations of collagens from animal tissues (chicken, hen, carp, and pork skin) were performed. Total protein concentration was determined by Biuret method and by TNBSA method. Both methods were used also in order to study physiological and thermal stability of the isolates under model conditions. Isolated collagens were characterized at molecular level using PAGE–SDS and microfluidic electrophoresis. Further, amino acids composition and microelement content were analyzed. Finally, the stability of isolated collagens in several types of simulated physiological and pathological conditions was tested too. During isolation relatively sufficient collagen yields were obtained (about 15 % of purified collagen per original mass of biological material). In all analyzed tissues Type I collagen was found. Thermal stability of individual samples differed according to biological source type and tissue age. Viscosimetry measurements confirmed higher stability in collagen samples of older animals. Collagenase exhibited the lowest degradation effect to bovine collagen, while selected mixed microbial hydrolases differed according to enzyme preparative type. Incubation of collagen isolates with selected human pathogens confirmed higher resistance of bovine collagen to biofilm formation when compared with the chicken one.
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Degradation of biomaterials in model physiological conditions
Mikulíková, Zuzana ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of presented work was to study degradation of selected modified protein materials and optimization of methods usable for protein concentration changes in samples that undergo proteolytic degradation. In first part overview of biomaterials used in medicine applications, biocompatibility and of collagen materials and their applications was given. In experimental part some methods for proteolysis determination were tested using standards of albumin, collagen and selected amino acids. As a part of this work proteolysis of selected modified collagen materials was observed. First, following methods for total protein analysis were tested: ninhydrin method, biuret method, Hartree-Lowry, UV-VIS spectrophotometry and TNBS method (trinitro-benzen-sulphonic acid). For collagen proteolysis determination two of these methods were optimized: biuret method (peptide group detection) and TNBS (primary amino group detection). These two methods were applied on enzyme degradation of three collagen samples: soluble collagen standard, collagen standard type I (insoluble) and modified collagen material for clinical application. In soluble and modified collagens collagenase effect exhibited decrease of peptide bounds and, simultaneously, increase of primary amino groups according to enzyme concentration and enzyme:substrate ratio. In insoluble collagen analysis was strongly influenced by structure and conformation changes of material during degradation.
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