Presented master's thesis focuses on the study of hydration of selected biocolloid substances, specifically humic substances (humic acids and fulvic acids), hyaluronic acid with three different molecular weight, chitosan and dextran. Interaction of biocolloids with water was studied by different methods. The effect of solubility, structure, functional groups in molecule on sorption and hydration ability of these biocolloids was investigated. In the case of hyaluronan the influence of molecular weight was also study. Differential scanning calorimetry DSC and perfusion calorimetry give results of heat of hydration, enthalpies and temperature of crystallization and melting. Thermogravimetric analysis TGA was used to determine the original moisture content of the samples.

This diploma thesis deals with an experimental analysis of knee joint replacement lubrication. The experiments were realized at a knee joint simulator which can apply conditions according to certain standard and survey the phenomena by using fluorescence microscopy. The aim of thesis is to clarify the influence of particular components of synovial fluid on the lubrication process. The intensity of fluorescence expresses dimensionless parameter of a lubrication film thickness. There was a fundamental study with mineral oils before the experiments with the synovial fluid. The study allows to have a look at contact transformation during walk. Results are shown in graphs as dependency of intensity on time, including pictures showing phenomena in the contact zone. Experiment results show that protein -globulin creates a layer on the surface. There is albumin on the layer and it makes the lubricating film thicker. The protein interaction is supported by hyaluronic acid and fosfolipids which stabilizes the created structure. According to lubrication is behaviour of film related to a complex structure of synovial fluid. Thesis gives more information about behaviour of synovial fluid and can be used for future development of knee replacements.

Hyaluronová kyselina je v současné době velmi významná biomolekula používaná v medicíně a kosmetice, a její výzkum je důležitý pro další budoucí použití. Zaměření této práce je studování povrchových vlastností hyaluronové kyseliny, jejích hydrofobních derivátů a roztoků SDS pro srovnání, za použití tenziometrie. Tyto sloučeniny byly zkoumány ve vodě a v roztocích ve formě sodné soli. Sledované vzorky byly měřeny dvěmi metodami v různých koncentračních rozmezích při laboratorní teplotě pomocí nového BPA-800P bublinového tenziometeru. Byly navrženy takové experimenty, aby se zjistily využitelné možnosti tohoto přístroje pro další výzkum. Výsledky práce ukazují rozdílnosti jednotlivých povrchově aktivních látek v závislosti na jejich koncentraci a použitém prostředí. Hyaluronová kyselina nevykazuje povrchovou aktivitu, zatímco její deriváty a SDS ano.

The study deals with interactions of polyelectrolytes polystyrene sulfonate and hyaluronic acid with nitrogenic amphiphilic substances, represented by lysine and albumine. To study the interactions pH-metry, conductance, viscositic and turbidity measurement, DLS and reometry were used. All mixtures of different concentrations were measured and the data were compered with data obtained from measurement of samples with amphiphilic sumstances without polyelectrolytes. Observed interactions occured in the aminoacid concentrations between 0 to 20 mmoldm-3, then the PSS interaction groups were fully bonded by lysine and no more interactions were recognized. The same behaviour were observed in albumine solutions with concentration under 2 gdm-3.

The aim of this thesis was to prepare hyaluronic acid micro- and nanoparticles based on electrostatic interactions with oppositely charged molecules. Following parameters were monitored: correlation function behavior, the particle size and zeta potential value. At the beginning, it was necessary to study the behavior of hyaluronan in solution by dynamic light scattering measurement. Micro- and nanoparticles were prepared by mixing different volume ratios of negatively charged hyaluronan and positively charged polyarginine or cetyltrimethylammonium bromide. Micro- and nanoparticles were prepared in aqueous solution as well as in 0,15 M sodium chloride solution (physiological solution). In the case of the hyaluronan solution a polydisperse character of hyaluronan was detected. It was found that the dissolution of hyaluronan in the physiological solution gives us the smaller particle size in opposite to particle size obtained from the same concentrations of hyaluronan dissolved in water. Furthermore, it was found that systems composed of hyaluronan and polyarginine create particle size of about 100 nm. Whereas systems consisting of cetyltrimethylaminoum bromide and hyaluronan form larger particles, in units of hundreds of nanometers, the particle size in physiological solution were smaller than the same systems dissolved in aqueous solution.

This master thesis is focused on the evaluation of the influence of particle surface properties on the results of microrheological measurements with biopolymer solutions. Hyaluronan has been chosen as negatively charged polymer, chitosan as positively charged polymer and glycerol and its solutions of various concentrations were used as homogenous model systems. Dynamic light scattering and single particle tracking microrheology were chosen from passive microrheological techniques. Particles with various surface modifications (neutral, positive surface charge and negative surface charge) were used for the experiments. The results of microrheological techniques were then compared with classic rheology and moreover the glycerol results were compared with tabulated values.

The bacteria of genus Streptococci are among the most significant producers of hyaluronic acid in industrial scale. One of the typical representatives of that group is Streptococcus equi subsp. zooepidemicus. The production of hyaluronic acid in Streptococcus equi subsp. zooepidemicus is heavily influenced by cultivation conditions and by genetic alterations. The present work describes the deletion of genes covR and covS responsible for transcriptional regulation of stress response. According to Galeas a kol. [35] the deletion of these genes in S. pyogenes led to the hyaluronic acid capsule increase. As the S. pyogenes and S. equi subsp. zooepidemicus share approx. 80 % of genome, it was assumed, that the deletion of genes covR and covS in Streptococcus equi subsp. zooepidemicus genome would lead to the higher hyaluronic acid production. The new strain SEZ covRS was obtained by allelic replacement mutagenesis. The cultivations performed in laboratory-scale fermenters in rich Wheat E1 medium showed approx. 9% higher production over parental strain. Therefore, the covRS regulation system plays the same role in Streptococcus equi subsp. zooepidemicus and indirectly regulates the biosynthesis of hyaluronic acid.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysic and Physical Chemistry Candidate: Bc. Martina Dvořáková Supervisor: Doc. Ing. Alice Lázníčková, CSc. Consultant: Mgr. Martina Hermannová, Ph.D. Title of diploma thesis: Optimization of preparative LC-MS method for fractionation of oligosaccharides of hyaluronanu This diploma thesis deals with optimization of LC-MS method for analysis of hyaluronan oligosaccharides in preparative mode. The theoretical part summarizes available information about biological and chemical properties of hyaluronic acid. Hyaluronic acid is easily enzymatically degradable by mammalian hyaluronidases that produce hyaluronan oligosaccharides. The biological function of these degradation products depend on their molecular weight. High-performance liquid chromatography is mainly used for separation and purification of hyaluronan oligosaccharides. A new method for the determination of hyaluronan oligosaccharides is based on a combination of separation techniques and mass spectrometry. The experimental part deals with optimization of ionisation conditions for electrospray ionization mass spectrometry in positive and negative ion mode. In the first step, we focused on setting of capillary voltage, cone voltage, desolvation temperature, flow...

L’acide hyaluronique (AH) est un polymère osidique aux propriétés mécaniques et biologiques d’intérêt pour le secteur de la santé et des cosmétiques. L’AH est produit à l’échelle industrielle par mise en oeuvre de souches de streptocoques groupe C et D. A l’issue de la production, une part de l’AH est libre dans le milieu de culture complexe, l’autre est associée à la capsule des cellules. La purification de l’AH est délicate en raison, d’une part, de la complexité des milieux de production et, d’autre part, des exigences de la pharmacopée européenne. L’objectif de cette thèse a été de proposer un enchaînement d’opérations et de conditions opératoires permettant une purification de l’AH à partir d’un milieu de production. Au départ, deux méthodes analytiques originales de quantification et d’évaluation de la taille de l’AH en milieux complexes ont été mises au point afin de pouvoir évaluer les critères de performance des procédés de séparation. Ensuite, quatre opérations permettant, premièrement l’extraction, deuxièmement l’élimination des cellules productrices d’AH, troisièmement la purification, et enfin, le séchage d’AH ont été établies. L’étude de l’extraction de la capsule d’AH assistée par SDS ou au TCA a permis d’établir que l’AH lié aux cellules ne représente que 7 % de l’AH produit. Il en a été déduit que cette étape n’est pas nécessaire. L’étude de la séparation des cellules du milieu de culture a montré que la microfiltration tangentielle n’est pas adaptée en raison, d’une part, d’une rétention trop élevée de l’AH (même avec des diamètres de pores supérieurs à 0,22 µm) et d’autre part, d’un colmatage trop rapide des membranes. Cette étape doit être réalisée par filtration frontale sur adjuvant de filtration (Célite). La purification de l’AH a été envisagée par diafiltration (DF) avec des membranes d’ultrafiltration. L’étude des conditions opératoires (PTM, débit d’alimentation, concentration en AH et seuil de coupure), par plan d’expériences, a permis de sélectionner des conditions qui maximisent le flux de perméat ainsi que la rétention d’AH. Ces conditions ont été appliquées en diafiltration au cours de laquelle les performances de séparation (rendement, pureté, productivité) ont été étudiées. Une méthodologie, basée sur les équations classiques de bilans de matière et de flux de perméat en DF, a alors été proposée. Cette démarche avait tout d’abord pour but de prédire lesdites performances en prenant en compte la composition complexe du milieu, de culture bactérienne mais aussi de faciliter l’agencement des étapes du procédé de purification. La diafiltration permet de satisfaire les exigences de pureté de la pharmacopée, mais pas les niveaux de certaines molécules (protéines et acides nucléiques). L’étude de l’adsorption de ces molécules sur charbon actif, en fonction de la température, de la force ionique, du pH et du type de charbon actif, a permis d’établir les conditions les plus favorables de l’élimination de ces molécules. Les équations classiques de modélisation des isothermes correspondant le mieux aux résultats expérimentaux ont été sélectionnées et utilisées avec les équations de performance en DF, afin d’associer au mieux ces deux étapes. Enfin, concernant le séchage de l’AH, deux méthodes, l’une par lyophilisation et l’autre par précipitation en solvant organique, ont été comparées. Ces dernières permettent d’obtenir moins de 20 % d’humidité et de conserver les propriétés des molécules d’AH / Hyaluronic acid (HA) is a biopolymer with mechanical and biological properties interesting the health and cosmetic areas. The industrial HA production is realized by implementation of streptococcus strains (group C or D). At the end of the production phase, a part of HA is free in a complex culture medium while the other remains associated to the cells in a capsule. The two main difficulties of the purification of HA lie, firstly, on the complexity of the production medium and, secondly, on the high requirements of the European Pharmacopoeia for such an application. The aim of this thesis was to provide an appropriate sequence of separation processes and a set of operating conditions for HA purification from a complex production medium which complies with the Pharmacopea. Initially, two original analytical methods for quantifying and evaluating the size of HA in complex environments have been developed in order to evaluate the performance criteria of the separation processes. Then, the HA extraction from the capsule, the elimination of cells, the purification and drying of HA were using classical separation processes and studied. The HA extraction by SDS or TCA showed that the HA bound to cells corresponds to 7 % of HA production. As a consequence, it was concluded that this step was not necessary. The cell separations from the culture medium by tangential microfiltration was unsuitable because of an excessive retention of HA (even at pore diameters higher than 0.22 µm) and a too fast membrane clogging. Good performances were observed with dead-end filtration using Celite. Then, the purification of HA by diafiltration (DF) with ultrafiltration membranes was considered. The operating conditions (PTM, feed rate, HA concentration and cutoff) allowing to maximize the permeate flow rate and the HA retention were selected from design of experiments methodology. The performances of purification by diafiltration (DF) in these conditions (yield, purity and productivity) were monitored. A method for predicting the performances according to the complex medium composition, based on the conventional mass balance equations and the DF permeate flow rate, was then proposed. This was done in order to facilitate the arrangement of the purification steps. In DF, the requirement of the pharmacopoeia in term of overall purity was obtained (> 95 %) but the proteins and nucleic acids levels were still above the threshold. This is why the adsorption of these molecules on activated carbon was studied as a function of temperature, ionic strength, pH and the type of activated carbon. The isotherm equations that better fitted the experimental data were selected and used combined to the equations of DF performances in order to find an appropriate chaining. Finally, the AH lyophilization and precipitation in organic solvent were compared. Both drying methods present less than 20 % moisture and preserve the properties of the molecule

Ce travail se proposait de développer, à l’échelle du laboratoire, un procédé de production microbienne d’acide hyaluronique (AH), biopolymère d’intérêt pour la cosmétique et la santé, chez une bactérie lactique naturellement productrice, Streptococcus zooepidemicus. Une étude bibliographique a permis d’identifier les points potentiellement critiques pour ce travail qui sont la composition du milieu de culture (identification de nutriments essentiels à la synthèse d’acide hyaluronique), les conditions opératoires et plus particulièrement d’oxygénation (transfert d’oxygène mais aussi maintien du potentiel d’oxydo-réduction), en relation avec les performances de production et le métabolisme des souches considérées. Une étape préliminaire a consisté en un développement et une amélioration de techniques analytiques pour disposer d’outils pertinents de suivi du métabolisme de Streptococcus zooepidemicus. La quantification de la biomasse hors acide hyaluronique ainsi que la détection de l’activité hyaluronidasique ont ainsi été développées tandis que d’autres méthodes chromatographiques et enzymatiques ont été simplement appliquées et validées aux substrats et métabolites considérés. La mise en œuvre de souches « environnementales » prélevées sur des sites infectieux chez le cheval, ainsi que de souches de collection, a permis dans un premier temps de formuler un milieu de culture et de définir des conditions de mise en œuvre exploitables à plus grande échelle pour la production d’AH. Des résultats très encourageants ont pu être obtenus avec des productions d’AH supérieures aux essais de la littérature, tout en mettant en avant des facteurs influents cruciaux tels que la concentration initiale en glucose ou encore l’oxygénation des cultures. L’influence de ces facteurs a été étudiée par la suite par le biais de cultures en bioréacteur de laboratoire en modes discontinu et discontinu-alimenté avec pour résultat l’obtention d’un mode de mise en œuvre et de conditions physico-chimiques de production améliorées. En parallèle, une étape importante de ce travail a consisté en une approche d’amélioration de souches « environnementales » par mutagénèse aléatoire, les performances de ces souches s’avérant initialement décevantes. Des mutants surproducteurs ont ainsi été générés, caractérisés au niveau de leurs performances métaboliques et conservés. En dernier lieu, un des mutants les plus performant a été mis en œuvre dans les conditions et le mode de culture sélectionnés précédemment. Tant le niveau de production d’AH, la productivité associée, que les tailles obtenues ont permis de valider ce travail de développement d’un procédé microbien de production d’AH, tout en identifiant de nouvelles voies d’amélioration / This work intended to develop a laboratory scaleproduction process ofhyaluronic acid (HA), a biopolymer of health and cosmetic interest, using a naturally AH producing lactic acid bacterium, Streptococcus zooepidemicus. A literature review allowed to identify the following critical points: firstly, the composition of the culture medium (identification of essential nutrients for microbial growth and synthesis of HA), secondly, the oxygenation level (oxygen transfer and associated redox modifications), and finally, in relation with, the production and metabolism abilities of the considered strains. A preliminary step was dedicated to the developmentorthe improvementof analytical techniques in order todispose of appropriate tools for the monitoring of Streptococcus zooepidemicus metabolism. The quantification of the biomass without considering capsular HA fraction as well as detection of hyaluronidase activity have been developed while other chromatographic and enzymatic methods have been more basically applied to and validated with the substrates and metabolites considered. The laboratory scale cultures of collection (ATCC) as well as “environmental” strains were initially used to formulate a workable cultivation broth and to define suitable culture conditionsfor a use at a larger scale to produce HA. Very positive results were obtained with higher production of HA in comparison with literature assays, while critical influencing factors such as the initial glucose concentration or the oxygenation levelin cultures were highlighted. The influence of these factors was thoroughly studied with bioreactor cultures in both batch and fed-batch modes leading to improved cultivation conditions and culture mode. In parallel, another important step consisted in the highly performance improvement of initially low HA producing "environmental" strains via random mutagenesis. Very promising overproducing mutants have therefore been generated, characterized in their kinetic and metabolic capabilities and long-term stored. At last, one of the best and most reliable mutant has been cultivated with the best previously selected medium composition and operating conditions. Both the HA production level, productivity and size observed validated the findings of this process development work, while helping to identify new improvement domains and strategies