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Size exclusion chromatography (SEC) separates molecules based on their actual size rather than their molecular weight. This indicates that the smaller a molecule is, the longer it will be held in the column since it can penetrate the pores more effectively than a bigger molecule, resulting in a longer retention time, whereas the larger molecule has a shorter retention time. In order to achieve reliable and accurate results the asymmetry factor of the packed columns should be between 0.6-1.4. The aim for this project was to examine whether or not the batch variations of the base matrix used to derive WorkBeads™ 40S and 40Q displayed either hydrophobic or hydrophilic interactions by running different types of proteins through columns packed with the base matrices. The project was performed using an ÄKTA explorer equipped with an ultraviolet (UV) detector and a refractive index detector (RID). The data was gathered and analyzed with the Unicorn™ by Cytiva. The results from the first experiments showed that lysozyme did not elute as expected or not at all, thus leading to a concern that the there might be some hydrophobic interactions in the base matrix, which is a porous media in the form of spherical particles that have been selected for their physical stability and inertness (lack of reactivity and adsorptive properties), and lysozyme. With this suspicion in mind, the different batches of the base matrix underwent hydrophobic interaction chromatography (HIC), where the results may be interpreted in a way that there might electrostatic interactions instead of hydrophobic interactions. However, due to the gel not being suitable for HIC the results were unreliable. By subsequently running lysozyme and other proteins through the columns at different NaCl concentrations the results showed consistent elution at NaCl concentrations > 150 mM, yet inconsistent at a concentration of 150 mM for lysozyme. The elution order by size showed that although lysozyme has a larger hydrodynamic radius (Rh) than cytochrome c it eluted later, which is theoretically incorrect, but it might be owing to some of the base matrix's characteristics or the lysozyme's dual nature of expressing both hydrophobic and hydrophilic interactions on the base matrix. Because of the inconsistent results from lysozyme, another experiment just at 150 mM was conducted where lysozyme did elute consistently with a KD value < 1. Lastly, a titration was performed on the base matrix where some of it was brominated and some of it was not, which was the reference. The results showed that there could be hydrophobic interactions on the brominated sample and hydrophilic interactions on the reference sample. However, what is more likely is that lysozyme is an unreliable protein to use to determine hydrophobicity on this type of gel. Size exclusion chromatography (SEC) separerar molekyler baserat på deras faktiska storlek snarare än deras molekylvikt. Detta indikerar att ju mindre en molekyl är, desto längre kommer den att hållas i kolonnen eftersom den kan tränga sig in i porerna mer effektivt än en större molekyl, vilket resulterar i en längre retentionstid, medan den större molekylen har en kortare retentionstid. För att uppnå tillförlitliga och nogranna resultat bör asymmetrifaktorn hos de packade kolonnerna vara mellan 0.6-1.4. Syftet med detta projekt var att undersöka huruvida batchvariationerna av basmatrisen som användes för att ta fram WorkBeads™ 40S och 40Q uppvisade antingen hydrofoba eller hydrofila interaktioner genom att köra olika typer av proteiner genom kolumner packade med basmatriserna. Projektet utfördes med en ÄKTA explorer utrustad med en ultraviolett (UV) detektor och en refractive index detector (RID). Data samlades in och analyserades med Unicorn™ av Cytiva. Resultaten från de första experimenten visade att lysozym inte eluerade som förväntat eller inte alls, vilket ledde till en oro för att det kan finnas vissa hydrofoba interaktioner i basmatrisen, som är ett poröst medium i form av sfäriska partiklar som har har valts ut för deras fysikaliska stabilitet och inerthet (brist på reaktivitet och adsorptiva egenskaper), och lysozym. Med denna misstanke i åtanke genomgick de olika bactherna av basmatrisen hydrofob interaktionskromatografi (HIC), där resultaten kan tolkas på ett sätt att det kan förekomma elektrostatiska interaktioner istället för hydrofoba interaktioner. Men på grund av att gelen inte var lämplig för HIC var resultaten opålitliga. Genom att efteråt köra lysozym och andra proteiner genom kolonnerna vid olika NaCl-koncentrationer visade resultaten konsekvent eluering vid NaCl-koncentrationer > 150 mM, men ändå inkonsekventa vid en koncentration av 150 mM för lysozym. Elueringsordningen efter storlek visade att även om lysozym har en större hydrodynamisk radie (Rh) än cytokrom c, eluerade det senare, vilket är teoretiskt inkorrekt, men det kan bero på några av basmatrisens egenskaper eller lysozymets dubbla natur att uttrycka både hydrofoba och hydrofila interaktioner på basmatrisen. På grund av de inkonsekventa resultaten från lysozym, utfördes ett annat experiment enbart vid 150 mM där lysozym eluerade konsekvent med ett KD-värde < 1. Slutligen utfördes en titrering på basmatrisen där en del av den var bromerad och en del av den inte, vilket var referensen. Resultaten visade att det kunde finnas hydrofoba interaktioner på det bromerade provet och hydrofila interaktioner på referensprovet. Men vad som är mer troligt är att lysozym är ett opålitligt protein att använda för att bestämma hydrofobicitet på denna typ av gel.

The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...

Durch den Einsatz von Antibiotika haben sich sowohl Virulenz als auch Re-sistenz der den Darm besiedelnden Enterokokken erhöht und sukzessive zu einem gravierenden Anstieg der Infektionsraten geführt. Aktuelle Daten der ECDC zeigen für Vancomycin-resistente E. faecium-Blutkulturisolate einen Anstieg von 9,1% im Jahr 2014 auf 16,5% im Jahr 2017 (ECDC-EARS-Net, 2019). Untersuchungen zur Pathogenese und zu Mechanismen von Infektionen durch E. faecium sind daher von hoher klinischer Relevanz. In der hier vorliegenden Arbeit wurde untersucht, ob es sich bei durch E. faecium induziertem Zelltod von Makrophagen um einen programmierten Zelltod in Form der Apoptose oder um einen toxisch bedingten Zelluntergang in Form von Nekrose handelt. Als Targetzellen für die Infektionsversuche mit E. faecium (ATCC 6057) dienten J774A.1-Makrophagen. Es konnte nachgewiesen werden, dass Zelltodinduktion nicht durch Überstände, sondern durch Pellets von E. faecium, also zelluläre bakterielle Bestandteile verursacht werden. Darüber hinaus konnte gezeigt werden, dass die Zugabe von Lysozym essentiell für die Induktion des Zelltods durch E. faecium ist. Bei der elektronenmikroskopischen Untersuchung von J774A.1-Makrophagen nach Infektion mit E. faecium und Zugabe von Lysozym waren typische morphologische Zeichen nekrotischen Zelltods mit Vakuolisierung des Zyto-plasmas und Zellschwellung zu beobachten. Apoptotische Merkmale wie Kernfragmentierung oder Zellschrumpfung waren dagegen nicht nachweisbar. Fehlende Schrumpfung bei durch E. faecium infizierten Makrophagen und ein extrazellulärer Nachweis von LDH infolge Ausstroms aus den Zellen durch Nekrose bedingte, poröse Zellwände sind weitere Zeichen für nekrotischen Zelltod und wurden in der vorliegenden Arbeit nachgewiesen. Als weitere Methode zur Differenzierung des Zelltods von E. faecium infizierten Makrophagen wurde in der vorliegenden Arbeit ein Immunoblotting für Caspase 3 durchgeführt. In dieser Untersuchung konnte gezeigt werden, dass bis 4 h nach Versuchsbeginn und bei Zugabe von Staurosporin die aktiven Caspase-3-Formen p17 und p19 im Immunoblotting vorhanden waren, woraus auf durch Staurosporin induzierte Apoptose geschlossen werden konnte. Dagegen war die Spaltung von Procaspase 3 in die aktiven Formen p17 und p19 im Immunoblotting von mit E. faecium infizierten J774A.1-Makrophagen 2 und 4 Stunden nach Versuchsbeginn nicht nachweisbar. Damit konnte ein¬deutig auf einen Zelltod durch Nekrose geschlossen werden. In einer In-vivo-Untersuchung wurden die Ergebnisse der In-vitro-Unter-suchungen hinsichtlich eines nekrotischen bzw. apoptotischen Zelltods nach Infektion von peritonealen Makrophagen mit E. faecium überprüft und bestätigt. Messungen des transmembranen Potentials und der Propidiumiodid-Positivität von peritonealen Makrophagen zeigten erst bei zusätzlicher Gabe von Lysozym eine signifikant höhere Zelltodrate durch Nekrose in der Tiergruppe nach Infektion mit E. faecium.

The theoretical part of this diploma thesis deals with molecularly imprinted polymers. Properties of the monomers and templates used for the polymerization are described. Part is devoted to the preparation of molecularly imprinted polymers and interactions leading to the formation of imprinted polymers. Imprinting technologies and strategies, template removal methods, detection methods, and the advantages and disadvantages of the molecular imprinting are included. Also the applications in various fields, focusing mainly on the area of imprinting proteins and pathogens are discussed in details. The experimental part deals with optimization of the preparation of a molecularly imprinted polymer based on dopamine subsequently used especially for the isolation and detection of Staphylococcus aureus and Enterococcus faecalis. The polymer was coated over a microscope slide, into the 96-well microplate and on a surface of magnetic particles. Fluorescence spectrometry and fluorescence microscopy were used for analyte detection. The developed technique was capable of isolating the target (Staphylococcus aureus and Enterococcus faecalis) at relatively low concentrations (1.103 CFU.ml-1).

This bachelor thesis deals with the problem of ocular prostheses. This problem is in thesis widely researched. Emphasis is placed on protein adsorption onto synthetic surface. Experimental part of the thesis focuses on lysozyme adsorption onto polymethylmethacrylate surface. Protein adsorption was lower than the specified limit of detection. According to experimental data was this amount lower than 5,37 μg/cm2 . In the thesis was conducted survey on secondary education students' awareness of ocular prosthesis. The results show slightly greater awareness of science-oriented students.

This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

eingereicht von DI Gregor Tegl Abweichender Titel laut Übersetzung der Verfasserin/des Verfassers Zusammenfassung in deutscher Sprache Universität für Bodenkultur Wien, Dissertation, 2016 OeBB

This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

eingereicht von Mag. Doris Schiffer Abweichender Titel laut Übersetzung der Verfasserin/des Verfassers Zusammenfassung in deutscher Sprache Universität für Bodenkultur Wien, Univ., Dissertation, 2015 OeBB

Makrophagen bilden als Phagozyten die vorderste Front des angeborenen Immunsystems. So können sie Bakterien aufnehmen und inaktivieren, bevor diese ihre Pathogenität entfalten. Grampositive Bakterien, wie beispielsweise Staphylococcus aureus und Streptococcus pyogenes, sind in der Lage, programmierten Zelltod in Makrophagen auszulösen und hierdurch die Immunantwort des Wirts zu umgehen. In der vorliegenden Arbeit wurde gezeigt, dass darüber hinaus auch zahlreiche andere Staphylokokken- und Streptokokkenspezies zytotoxisch auf J774A.1-Makrophagen wirken, was zur Pathogenität dieser Bakterien beitragen könnte. Enterokokken sind ein wichtiger Bestandteil der menschlichen Darmflora. Sie gewinnen allerdings zunehmend Aufmerksamkeit als Auslöser lebensbedrohlicher Infektionen, vor allem bei immunsupprimierten Patienten. In der vorliegenden Arbeit wurden J774A.1-Makrophagen mit verschiedenen Enterokokkenspezies und einer Reihe von E. faecium-Stämmen infiziert, welche sich hinsichtlich ihrer Antibiotikaresistenz und dem Vorhandensein des Oberflächenproteins Esp unterschieden. Dabei stellte sich heraus, dass Enterokokken unabhängig von der Spezies in der Lage sind, in Makrophagen nekrotischen Zelltod auszulösen. Weiterführende Untersuchungen mit E. faecium zeigten, dass Lysozym für den durch E. faecium ausgelösten Zelltod essenziell ist, wobei der Zelltod weder durch Caspasenhemmung mit zVAD-fmk, Aufnahmehemmung durch Cytochalasin D oder intrazelluläre Abtötung der Enterokokken mit Rifampicin verhindert werden konnte. Das Vorhandensein des bakteriellen Oberflächenproteins Esp oder bestimmter Antibiotikaresistenzen erhöhte die Zelltodraten nicht. Diese Ergebnisse deuten darauf hin, dass es einen gemeinsamen, bislang unbekannten zytotoxischen Faktor von Enterokokken gibt, der im Beisein von Lysozym freigesetzt wird. Weiterführende Arbeiten, die auf den Ergebnissen der vorliegenden Arbeit aufbauen, deuten darauf hin, dass es sich dabei um zytotoxisch wirksame, reaktive Sauerstoffspezies (ROS) handeln könnte, die unter dem Einfluss von Lysozym aus Enterokokken freigesetzt werden. Macrophages are phagocytes and form the front line of defence in the innate immune system. Thus they are able to absorb and inactivate bacteria before these can become pathogenic. Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, are able to induce programmed cell death in macrophages – thus evading the immune reaction of the host. In this study it was shown that, in addition to this, numerous other species of staphylococci and streptococci also have a cytotoxic effect on J774A.1-macrophages, which could contribute to the pathogenicity of these bacteria. Enterococci constitute an essential part of the human intestinal flora. However, they are becoming increasingly apparent as the cause of life-threatening infections, especially in immunocompromised patients. In this study, J774A.1-macrophages were infected with various species of enterococci and a range of E.faecium strains, which differed from each other in respect to their resistance to antibiotics and to whether the enterococcal surface protein Esp was present. It became evident that enterococci, independent of species, are able to induce necrotic cell death in macrophages. Further research with E. faecium showed that lysozyme is essential for cell death induction by E. faecium. When lysozyme was added it was neither possible to prevent cell death by inhibition of caspase through zVAD-fmk, nor by inhibition of bacterial uptake using cytochalasin D, nor by intra-cellular killing of the enterococci by rifampicin. The presence of the bacterial surface protein Esp, or of specific antibiotic resistances, did not increase the rates of cell death. These results indicate that that there is a common, up to now unknown cytotoxic factor in enterococci, which is released in the presence of lysozyme. Further investigations, using the results of this research as their basis, indicate that this factor could be cytotoxically active, reactive oxygen species (ROS), which are released from enterococci when lysozyme is present.

Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first investigate the physical properties of the encapsulating materials (protein fibrils and protein – polysaccharide complexes). Subsequently, microcapsules with tunable release rate and mechanical strength were developed. Firstly, the effect of steady shear and turbulent flow on the formation of protein fibrils from lysozyme was studied. We determined the conversion and size distribution of fibrils obtained by heating lysozyme solutions at pH 2. The formation of fibrils was quantified using flow-induced birefringence. The size distribution was fitted using decay of birefringence measurements and Transmission Electron Microscopy. The morphology of Lys fibrils and kinetics of their formation varied considerably depending on the flow applied. With increasing shear or stirring rate, more rod-like and shorter fibrils were obtained, and the conversion into fibrils was increased. Secondly, we have investigated the surface rheological properties of oil – water interfaces stabilized by fibrils from lysozyme (long and semi-flexible, and short and rigid ones), fibrils from ovalbumin (short and semi-flexible), lysozyme – pectin complexes, or ovalbumin – pectin complexes. We have compared these properties with those of interfaces stabilized by the native proteins. The surface dilatational and surface shear moduli were determined using an automated drop tensiometer, and a stress controlled rheometer with biconical disk geometry. Results show that interfaces stabilized by protein – pectin complexes have higher surface shear and dilatational moduli than interfaces stabilized by the native proteins only. At most of the experimental conditions, interfaces stabilized by protein fibrils have the highest surface rheological moduli. The difference between long semi-flexible lysozyme fibrils or short rigid lysozyme fibrils is not pronounced in interfacial dilation rheology but significant in interfacial shear rheology. The complex surface shear moduli of interfaces stabilized by long semi-flexible fibrils are about ten times higher than those of interfaces stabilized by short rigid fibrils, over a range of bulk concentrations. Interfaces stabilized by short and more flexible ovalbumin fibrils have a significantly higher surface shear modulus than those stabilized by the somewhat longer and more rigid short lysozyme fibrils. Finally, encapsulation systems are developed using layer-by-layer adsorption of food-grade polyelectrolytes on an emulsion droplet template. The first encapsulation system was built with alternating layers of ovalbumin fibrils and high methoxyl pectin. By varying the number of layers, the release of active ingredients can be controlled: increasing the number of layers of the shell from four to eight, decreases the release rate by a factor six. The other encapsulation systems were built with alternating layers of protein – pectin complexes and protein fibrils. Two types of proteins (ovalbumin and lysozyme) and three types of fibrils were used: short and semi-flexible from ovalbumin, short and rod-like, and long and semi-flexible from lysozyme. At low number of layers (less than five), microcapsules from ovalbumin complexes and fibrils were stronger than microcapsules prepared from lysozyme complexes and fibrils. Increasing the number of layers, the mechanical stability of microcapsules from lysozyme complexes and fibrils increased significantly, and capsules were stronger than those prepared from ovalbumin complexes and fibrils with the same number of layers. The contour length of the Lys fibrils did not have a significant effect on mechanical stability of the lysozyme complexes and fibrils capsules. These results show that mechanical properties of this type of capsule can be tuned by varying the flexibility of the protein fibrils.

Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first investigate the physical properties of the encapsulating materials (protein fibrils and protein – polysaccharide complexes). Subsequently, microcapsules with tunable release rate and mechanical strength were developed. Firstly, the effect of steady shear and turbulent flow on the formation of protein fibrils from lysozyme was studied. We determined the conversion and size distribution of fibrils obtained by heating lysozyme solutions at pH 2. The formation of fibrils was quantified using flow-induced birefringence. The size distribution was fitted using decay of birefringence measurements and Transmission Electron Microscopy. The morphology of Lys fibrils and kinetics of their formation varied considerably depending on the flow applied. With increasing shear or stirring rate, more rod-like and shorter fibrils were obtained, and the conversion into fibrils was increased. Secondly, we have investigated the surface rheological properties of oil – water interfaces stabilized by fibrils from lysozyme (long and semi-flexible, and short and rigid ones), fibrils from ovalbumin (short and semi-flexible), lysozyme – pectin complexes, or ovalbumin – pectin complexes. We have compared these properties with those of interfaces stabilized by the native proteins. The surface dilatational and surface shear moduli were determined using an automated drop tensiometer, and a stress controlled rheometer with biconical disk geometry. Results show that interfaces stabilized by protein – pectin complexes have higher surface shear and dilatational moduli than interfaces stabilized by the native proteins only. At most of the experimental conditions, interfaces stabilized by protein fibrils have the highest surface rheological moduli. The difference between long semi-flexible lysozyme fibrils or short rigid lysozyme fibrils is not pronounced in interfacial dilation rheology but significant in interfacial shear rheology. The complex surface shear moduli of interfaces stabilized by long semi-flexible fibrils are about ten times higher than those of interfaces stabilized by short rigid fibrils, over a range of bulk concentrations. Interfaces stabilized by short and more flexible ovalbumin fibrils have a significantly higher surface shear modulus than those stabilized by the somewhat longer and more rigid short lysozyme fibrils. Finally, encapsulation systems are developed using layer-by-layer adsorption of food-grade polyelectrolytes on an emulsion droplet template. The first encapsulation system was built with alternating layers of ovalbumin fibrils and high methoxyl pectin. By varying the number of layers, the release of active ingredients can be controlled: increasing the number of layers of the shell from four to eight, decreases the release rate by a factor six. The other encapsulation systems were built with alternating layers of protein – pectin complexes and protein fibrils. Two types of proteins (ovalbumin and lysozyme) and three types of fibrils were used: short and semi-flexible from ovalbumin, short and rod-like, and long and semi-flexible from lysozyme. At low number of layers (less than five), microcapsules from ovalbumin complexes and fibrils were stronger than microcapsules prepared from lysozyme complexes and fibrils. Increasing the number of layers, the mechanical stability of microcapsules from lysozyme complexes and fibrils increased significantly, and capsules were stronger than those prepared from ovalbumin complexes and fibrils with the same number of layers. The contour length of the Lys fibrils did not have a significant effect on mechanical stability of the lysozyme complexes and fibrils capsules. These results show that mechanical properties of this type of capsule can be tuned by varying the flexibility of the protein fibrils.

This diploma thesis was focused on the study of lysozyme and lysozyme-like proteins, either of similar function (antibacterial) or molecular weight (14 - 17 kDa), of synanthropic acaroid mites. In general, animals utilize lysozymes for defensive (antimicrobial) or digestive purposes but also as a digestive enzyme. Some chitinases or other enzymes that act similarly to lysozyme can be utilized for similar purposes. Chitinases belong to house dust mite allergens. One of major mite are historically named lysozyme-like proteins which name relates to their size similar to lysozyme. Bacteriolytic activity has also 14.5 kDa (UniprotKB Q8MWR6) protein. The species selected for the study were domestic mites Dermatophagoides farinae, D. pteronyssinus and Lepidoglyphus destructor. Presence of lysozyme was detected by direct detection with polyclonal antibody using immunohistochemistry and dot blots. Immunohistochemistry proved presence of lysozyme epitopes in the feces of D. farinae, D pteronyssinus a L. destructor. Dot blot analysis demonstrated the presence of imunoreactivity of antibody in spent growth medium extracts (SGME) of all three species. This implies that lysozyme is synthesized in the midgut. The presence of lysozyme and lysozyme-like proteins was proved using 2D electrophoresis and MALDI TOF/TOF...

Antimicrobial proteins contained in the albumen represent maternal effects, including the non- genetic component allocated into the egg during its oogenesis. Especially for species, whose broods are exposed to environmental influences until completation, these proteins play a crucial role in the viability of embryos due to their potential to influence the risk of microbial infection, which is considered one of the main causes of reduced hatchability. Also, it is assumed that these proteins, beacause of their specific traits, may influence phenotype of chicks, especially its size and immunity in the early postembryonal stage. In my thesis I focused on three antimicrobial proteins of avian egg white - avidin, lysozyme and ovotransferrin, which vary in their antimicrobial activity. For a better understanding of causal relationships between the concentrations of these proteins in the albumen and their effect on hatching success or offspring phenotype, a series of manipulation experiments and correlative measurements were performed. These experiments were held on the eggs of two precocial species - Japanese Quail (Coturnix japonica) and Mallard (Anas platyrhynchos). Our results indicate a crucial role of antimicrobial proteins in reducing the risk of bacterial infection and their natural concentration...

Proteins are a class of biologically relevant macromolecules that are ubiquitous in all living organisms and fulfil specific functions in these. Essential for most proteins is the coupling between their three-dimensional structure and their biological function. Different external extreme conditions like heat or pressure might lead to the loss of the protein's complex structure, so-called unfolding, and thus of its activity. To protect proteins from these extreme conditions different mechanisms have evolved in organisms to counteract these perturbations. A detailed knowledge of those factors that govern protein stability and how these are affected by external perturbations and modulated by different cosolvents might give a better understanding of how life has evolved under such conditions and what conditions are still tolerable for life to exist. In order to study specific contributions to the protein stability in a controlled way, com binations of different extreme conditions can be employed in an experiment to induce unfolding. In the framework of this thesis, proteins under extreme conditions were studied by small angle X-ray scattering (SAXS). This technique is well suited to determine the size and shape of proteins and thus to investigate their unfolding due to perturbations. Furthermore, using concentrated solutions it can be used to explore the interactions between proteins. This gives the possibility to learn about the factors that govern interactions in dense protein solutions as they are present in the cell's interior. SAXS measurement were performed on Different types of proteins at various extreme conditions to access the influences of the specific properties of the local and global structure on the protein folding. Therefor, SAXS data were obtained for various mutants of staphylococcal nuclease (SNase), a small globular protein, at high pressures and different pH values. This approach allowed to detect the influence of the charge properties of a single amino acid on the global folding state. Moreover, the structure of highly destablized SNase mutant was investigated as a function of solution pH. In addition, specific features of the linear, nonglobular ankyrin repeat domain were determined as a function of pressure and urea concentration. SAXS studies on highly concentrated solutions of the protein lysozyme were conducted at high pressure conditions. These measurements allowed it to determine the pressure-dependent protein-protein interaction potential. For this interaction potential a nonlinear pressure dependence was observed which can be interpreted by the collapse of the second hydration shell of water at 2 kbar. In addition, the influence of small biologically relevant molecules known to affect the protein stability especially under high hydrostatic pressures were studied. The effect of the osmolyte TMAO, which present in deep sea organisms to compensate the effects of high pressure and urea, as well as the influence of urea, glycerol, and mixtures of TMAO/urea and TMAO/glycerol was analyzed. This SAXS measurements on proteins with TMAO added revealed a different pressure dependence of the interaction potential than for pure buffer. In contrast no effect was observed for urea and only a slight one for glycerol. For a 1:2 TMAO:urea mixture as it is present in deep sea organisms, a cancelling of the effects of the two substances was found.

This dissertation describes a systematic study on oxidized starch microgel particles. It begins with the preparation and characterization of oxidized starch gels in terms of some important physical-chemical properties, with the aim to select an optimum gel for further investigation of protein uptake. The gel with the highest degree of oxidation DO100% is chosen for lysozyme uptake because of its high protein uptake capacity and low swelling capacity. In addition, DO30% gels have been used in many experiments, since DO30% starch allows for preparation of well-defined spherical microgel particles and because it is enzymatically degradable. The two main aspects of interest are the protein binding affinity and protein saturation. Neutral pH and low salt concentration are found to be the optimum protein uptake conditions for high protein saturation. For more detailed studies, spherical microgels with a narrow size distribution have been made by optimizing the preparation process. The mobility of lysozyme molecules inside those microgel particles has been investigated. The main conclusion is that high salt and high pH increase the mobility of lysozyme in the gel particles. It implies that high pH and high salt concentration are potential triggers for lysozyme release from the gel. Subsequently, the kinetics of protein release by high pH and high salt concentration is presented. For the aim of application, the antimicrobial activity of lysozyme containing starch gel particles against some bacterial strains is determined. Finally, the deposition of poly-lysine/poly-glutamic acid complex layer around microgel surface is used to stabilize the microgel particle and optimize our system.

This dissertation describes a systematic study on oxidized starch microgel particles. It begins with the preparation and characterization of oxidized starch gels in terms of some important physical-chemical properties, with the aim to select an optimum gel for further investigation of protein uptake. The gel with the highest degree of oxidation DO100% is chosen for lysozyme uptake because of its high protein uptake capacity and low swelling capacity. In addition, DO30% gels have been used in many experiments, since DO30% starch allows for preparation of well-defined spherical microgel particles and because it is enzymatically degradable. The two main aspects of interest are the protein binding affinity and protein saturation. Neutral pH and low salt concentration are found to be the optimum protein uptake conditions for high protein saturation. For more detailed studies, spherical microgels with a narrow size distribution have been made by optimizing the preparation process. The mobility of lysozyme molecules inside those microgel particles has been investigated. The main conclusion is that high salt and high pH increase the mobility of lysozyme in the gel particles. It implies that high pH and high salt concentration are potential triggers for lysozyme release from the gel. Subsequently, the kinetics of protein release by high pH and high salt concentration is presented. For the aim of application, the antimicrobial activity of lysozyme containing starch gel particles against some bacterial strains is determined. Finally, the deposition of poly-lysine/poly-glutamic acid complex layer around microgel surface is used to stabilize the microgel particle and optimize our system.

Umfassende molekulare Analyse aller Invertebraten-Lyoszyme von dem Modellorganismus C. elegans; Charkaterisierung des ILYS-5 auf Proteinebene mit biochemischen und funktionalen Ergebnissen

[no abstract]

AAn der Vielzweck-Beamline BL9 der Dortmunder Elektronenspeicherringanlage DELTA wurde ein neuer Aufbau zur Messung der Röntgenkleinwinkelstreuung (Small Angle X-Ray Scattering, SAXS) an flüssigen Proben entwickelt und aufgebaut. Dieser ermöglicht Messungen sowohl unter Normalbedingungen als auch unter für biologische Systeme extremen Bedingungen, wie unter erhöhter oder erniedrigter Temperatur (-15°C bis +100°C), sowie unter Hochdruck von bis zu 7000 bar. Mit Hilfe des neuen Aufbaus wurden Messungen an wässrigen Lösungen der Proteine Lysozym, Insulin und Staphylokokken Nuclease (SNase) durchgeführt und um zusätzliche Messungen an externen Strahlungsquellen ergänzt. Die an Lysozym- und Insulinlösungen durchgeführten Messungen ermöglichten eine ausführliche Charakterisierung der Lösungsstruktur der jeweiligen Proteine. Darüber hinaus erfolgte eine Analyse der intermolekularen Wechselwirkungen zwischen den gelösten Proteinmolekülen, sowohl in puren Lösungen als auch in Anwesenheit von Cosolventien (Natriumchlorid, Trifluoroethanol, Glyzerin und Ethanol). Die Messungen an Lösungen des Proteins SNase wurden mit dem Ziel durchgeführt, die Einflüsse von Cosolventien (Urea, Sorbitol, Glyzerin, Kaliumsulfat, Trifluoroethanol und Trimethylamin-N-oxid) auf die druck- und temperaturinduzierte Denaturierung des Proteins zu bestimmen.

Keywords: Emulsion, flocculation, bridging, saliva, salivary protein, salivary peptides, lysozyme, -lactoglobulin, complex formation, LC-MS, SELDI-TOF-MS, proteomics. Upon consumption food emulsions undergo various structural and compositional changes in the mouth. One of these changes is that mixing of an emulsion with saliva induces droplet flocculation In the study described in this thesis we investigated the influence of saliva on emulsions properties, the mechanism of flocculation and the role in sensory perception. Firstly, we started with evaluating the effect of parameters related to emulsions on flocculation (i.e. differently charged surfactants and proteins such as -lactoglobulin and lysozyme used as emulsifiers and oil-volume fraction). Among the obtained results, we observed that the sign and the density of the charge on the surface of the droplets determine the (ir-)reversibility of flocculation upon dilution with water and shearing. Secondly, the effect of saliva-related parameters was analyzed. Among other aspects, it appeared that an increase in salivary protein concentration increased emulsion flocculation, and that extensive flocculation is typically found for unstimulated saliva. This approach shows that both emulsion and saliva properties affect the flocculation behavior of emulsions/saliva mixtures. To investigate the nature of the flocculation, we characterized the salivary protein composition in both the continuous phase of the emulsion/saliva mixture and on the emulsion droplets. Different physical-chemical and biochemical techniques were used. For this approach, we focused on -lactoglobulin and lysozyme stabilized emulsions, which flocculated reversibly and irreversibly, respectively, upon mixing with saliva. A large number of salivary proteins and peptides in the molecular mass (Mr) range between 0.8 kDa and 100 kDa and the salivary mucins MUC5B and MUC7 (Mr > 200 kDa) associated with emulsion droplets of the emulsions. The results also indicate that the emulsifying protein at the oil-water interface determines which salivary components associate with the droplets in the flocs. A hypothesis is formulated that emulsion flocculation is mainly driven by a complex formation involving specific interactions and electrostatic attraction between salivary peptides/proteins and the emulsifying proteins at the droplets surface. The importance of the saliva-induced droplet flocculation was demonstrated with a sensory paneling study. Emulsions stabilized by whey protein isolate, (predominantly composed of -lactoglobulin) showed reversible flocculation and were perceived as creamy. In contrast, emulsions stabilised by lysozyme showed irreversible flocculation and were perceived as dry, rough and astringent. To conclude, this thesis shows that saliva-induced emulsion flocculation is driven mainly by association of salivary peptides and proteins to the droplets surface. Because of this, flocculation is determined by the composition of the droplet interface as well as the composition of the saliva, and can be controlled by variation of emulsion parameters (charge, pH, ionic strength). This interaction between emulsions and saliva may help to improve our understanding an control the sensory perception of emulsions.

Was hat Lysozym aus Hühnereiweiß mit Wein zu tun? Nur wenige Verbraucher werden wissen, dass Lysozym bei der Weinherstellung zur Steuerung des biologischen Säureabbaus eingesetzt werden kann. Eier und deren Bestandteile werden jedoch auch als potentielle Allergene betrachtet. Daher müssen Weine in Zukunft gekennzeichnet werden, sofern Inhaltstoffe aus Eiern im Endprodukt nach-gewiesen wurden. Von der Internationalen Orga-nisation für Rebe und Wein (OIV) wurde 2007 eine Methode für die Bestimmung von Lysozym in Wein in die internationale Sammlung der Analyse-methoden für Wein und Most aufgenommen.

Keywords: Emulsion, flocculation, bridging, saliva, salivary protein, salivary peptides, lysozyme, -lactoglobulin, complex formation, LC-MS, SELDI-TOF-MS, proteomics. Upon consumption food emulsions undergo various structural and compositional changes in the mouth. One of these changes is that mixing of an emulsion with saliva induces droplet flocculation In the study described in this thesis we investigated the influence of saliva on emulsions properties, the mechanism of flocculation and the role in sensory perception. Firstly, we started with evaluating the effect of parameters related to emulsions on flocculation (i.e. differently charged surfactants and proteins such as -lactoglobulin and lysozyme used as emulsifiers and oil-volume fraction). Among the obtained results, we observed that the sign and the density of the charge on the surface of the droplets determine the (ir-)reversibility of flocculation upon dilution with water and shearing. Secondly, the effect of saliva-related parameters was analyzed. Among other aspects, it appeared that an increase in salivary protein concentration increased emulsion flocculation, and that extensive flocculation is typically found for unstimulated saliva. This approach shows that both emulsion and saliva properties affect the flocculation behavior of emulsions/saliva mixtures. To investigate the nature of the flocculation, we characterized the salivary protein composition in both the continuous phase of the emulsion/saliva mixture and on the emulsion droplets. Different physical-chemical and biochemical techniques were used. For this approach, we focused on -lactoglobulin and lysozyme stabilized emulsions, which flocculated reversibly and irreversibly, respectively, upon mixing with saliva. A large number of salivary proteins and peptides in the molecular mass (Mr) range between 0.8 kDa and 100 kDa and the salivary mucins MUC5B and MUC7 (Mr > 200 kDa) associated with emulsion droplets of the emulsions. The results also indicate that the emulsifying protein at the oil-water interface determines which salivary components associate with the droplets in the flocs. A hypothesis is formulated that emulsion flocculation is mainly driven by a complex formation involving specific interactions and electrostatic attraction between salivary peptides/proteins and the emulsifying proteins at the droplets surface. The importance of the saliva-induced droplet flocculation was demonstrated with a sensory paneling study. Emulsions stabilized by whey protein isolate, (predominantly composed of -lactoglobulin) showed reversible flocculation and were perceived as creamy. In contrast, emulsions stabilised by lysozyme showed irreversible flocculation and were perceived as dry, rough and astringent. To conclude, this thesis shows that saliva-induced emulsion flocculation is driven mainly by association of salivary peptides and proteins to the droplets surface. Because of this, flocculation is determined by the composition of the droplet interface as well as the composition of the saliva, and can be controlled by variation of emulsion parameters (charge, pH, ionic strength). This interaction between emulsions and saliva may help to improve our understanding an control the sensory perception of emulsions.

Proteine, umgangssprachlich auch Eiweiße genannt, bestehen aus einer linearen Polypeptidkette aus bis zu 20 verschiedenen Aminosäuren. Sie sind nahezu an allen biologischen Prozessen beteiligt und bilden die Grundbausteinen aller Zellen. Ihre letztendlich spezielle funktionelle Eigenschaft resultiert aus ihrer dreidimensionalen Form. Steht eine Proteinlösung in Kontakt mit einer festen Oberfläche oder einer Oberfläche mit fluidem Charakter (Polyelektrolyt-Bürsten), adsorbieren Proteine spontan an der Flüssigkeit/Festkörper-Grenzfläche. Eine große Anzahl von Triebkräften wird für diesen Vorgang diskutiert. Hierzu zählen u. a. Konformationsänderungen des Proteins, elektrostatische Anziehungskräfte, van der Waals-Kräfte und das Modell der counterion release force. Zur Untersuchung der Adsorption der Proteine Hühnereiweiß-Lysozym, Staphylokokken-Nuclease (SNase), sowie Rinder-Serum-Albumin (BSA) auf Siliziumdioxid, Polyelektrolyt-Multischichten (PEM), planaren Polyelektrolyt-Bürsten (PEB) und fluiden sphärischen Polyelektrolyt-Bürsten (SPB) wurden die folgenden Messmethoden in dieser Arbeit angewendet: die Neutronenreflektometrie, die optische Reflektometrie und die CD-Spektroskopie. Adsorption von gelöstem Lysozym und SNase an einer Silizium/Wasser-Grenzfläche Sowohl Lysozym als auch SNase weisen positive Enthalpieänderungen bei ihrer Adsorption aus der Lösung auf oxidiertes Silizium auf. Entropische Kräfte müssen daher bei diesem Vorgang über enthalpische dominieren. Die Wechselwirkung zwischen dem Substrat und den Proteinmolekülen ist attraktiv auf Grund der ungleichnamigen Ladungen. Beide untersuchten Proteine besitzen bei einem pH-Wert von 7 eine positive Nettoladung; das Siliziumdioxid ist demgegenüber negativ geladen. Ein größerer H-Wert bei einer stärkeren Protein-Oberflächenbelegung wird durch eine größere Zahl an repulsiven Protein-Protein-Wechselwirkungen verursacht. Positive Entropieänderungen können zum einen in einer partiellen Dehydratisierung der hydrophoben Substrat- und Proteinoberflächen begründet sein. Zum anderen werden wahrscheinlich durch Protein-Substrat-Wechselwirkungen Lysozym- und SNase-Moleküle partiell entfaltet, so dass deren Konformationsentropie zunehmen sollte. Eine Temperaturerhöhung hat zur Folge, dass die native Proteinstruktur destabilisiert wird und adsorptions-induzierte Konformationsänderungen bevorzugt werden. Proteinadsorption an einer gleichnamig geladenen Polyelektrolyt-Bürste Die variable Proteinbindungskapazität einer planaren PAA-Bürste wurde mit Hilfe der Neutronenreflektometrie und theoretischer Überlegungen charakterisiert. Nachgewiesen werden konnte, dass bei niedriger Ionenstärke große Mengen negativ geladener BSA-Moleküle an einer gleichnamigen PAA-Bürste binden. Bei einer erhöhten Ionenstärke von 0,5 M konnte jedoch mit Hilfe der Neutronenreflektometrie eine Proteinresistenz der Oberfläche beobachtet werden. Diese experimentellen Nachweise entsprechen zuvor durchgeführten Studien an sphärischen PAA-Bürsten-Partikeln. Die Analyse der Neutronenreflektometriekurven zeigt, dass die BSA-Moleküle tief in die PAA-Bürste eindringen. Alle Experimente weisen darauf hin, dass direkte elektrostatische, van der Waals- und hydrophobe Wechselwirkungen nicht für die Proteinaffinität einer PAA-Bürste bei niedriger Ionenstärke verantwortlich sind. Ein einfaches mean field-Modell ist im Rahmen dieser Arbeit entwickelt worden, das den Gewinn von freier Energie durch Freisetzung von Gegenionen, wenn ein Protein-Molekül aus der Lösung in eine gleichnamiggeladene Polyelektrolyt-Bürste eindringt, voraussagt. Diese Gegenionen-„Verdampfung“ ist entropischer Natur und kann über abstoßend wirkende Coulomb-Energien dominieren, wie in dem BSA/PAA-System. Die experimentellen und theoretischen Ergebnisse dieser Studie legen somit eine neue fundamentale Triebkraft für die Proteinadsorption an Grenzflächen nahe. Die Änderung der Ionenstärke einer Proteinlösung im Bereich einiger 100 mM läßt die Struktur und Dynamik eines gelösten Proteinmoleküls unberührt. Das „Schalten“ der Proteinaffinität einer PAA-Bürste durch Änderung der Ionenstärke der Lösung ist wahrscheinlich unabhängig von dem untersuchten Protein, was den Einsatz einer PAA-Bürste in biotechnologischen Anwendung als sehr erfolgsversprechend erscheinen läßt. Zum einen scheint eine einfache und günstige Möglichkeit gegeben, Oberflächen in einem salzhaltigen Medium proteinresistent zu machen. Zum anderen ist die Verwendung als Wirkstoffträger oder Nanoreaktoren in Form von sphärischen Polyelektrolytbürsten denkbar, bei denen die Wirkstoffe (Enzyme) an der Polyelektrolytbürste zunächst immobilisiert und später durch Erhöhung des Salzgehaltes in ihrer Umgebung wieder freigesetzt werden. Besonders wichtig ist hierbei der Aspekt, dass Proteine nach der salzinduzierten Desorption aus der Polyelektrolyt-Bürste ihre Konformation und biologische Aktivität nahezu beibehalten. Adsorption von SNase an einer Polyelektrolyt-Multischicht Polyelektrolyt-Multischichten bieten ein neues Forschungsfeld für die Beschichtung von planaren und kolloiden Substraten. Ihre Herstellung ist einfach. Alternierender Kontakt eines geladenen Substrates mit positiven bzw. negativen Polyelektrolyten führt zu einer selbstanordnenden (self-assembled) Multischicht, die einzigartige Eigenschaften aufweist. Der schrittweise Aufbau eines dreidimensionalen Schichtensystems erlaubt den Einbau einer Vielzahl von Verbindungen und Partikeln, wie Proteinen, Nanopartikeln, Biopolymeren (DNA) und vielen anderen Stoffen. Zu den möglichen Anwendungsbereichen dieser planaren Schichtensysteme zählt der Gebrauch als Matrixmaterial für funktionalisierte oder biologische molekulare Funktionseinheiten, wie Biosensoren, Trennungsmembranen oder ähnliches. In dieser Arbeit wurde die räumliche Verteilung des Proteins SNase, das mit einer planaren Polyelektrolyt-Multischicht interagiert, mit Hilfe neutronenreflektometriescher Messungen bestimmt. Durchgeführt wurden die Messungen bei den Temperaturen 22 °C und 42 °C und bei zwei unterschiedlichen Nettoladungen des Proteins (isoelektrischer Punkt der SNase: 9,5), die durch Wahl des pD-Wertes zu 4,9 bzw. 7,5 erreicht wurde. Die Polyelektrolyt-Multischicht wurde auf einem Silizium-Wafer durch das Auftragen von Polyethylenimin (PEI), Polystyrolsulfonat (PSS) und Polyallylaminhydrochlorid (PAH) in der Reihenfolge Si-PEI-PSS-(PAH-PSS)5 hergestellt. Unter Kontrastvariation wurden für jede Bedingung von Temperatur und pD-Wert zwei unterschiedliche Reflektivitätskurven gemessen. Aus der Analyse der Reflektivitäts-Kurven konnte das Protein-Dichteprofil senkrecht zur Grenzfläche berechnet werden. Die Ergebnisse zeigen, dass die SNase teilweise in die Polyelektrolyt-Multischicht nach der Adsorption bei allen untersuchten Versuchsbedingungen eindringt. Die gemessenen Neutronenreflektivitäten stimmen mit einer Eindringtiefe von 50 Å bei einem pH-Wert von 4,9 und 25 Å bei 7,5 überein. Da SNase einen isoelektrischen Punkt von 9,5 hat, trägt das Protein bei beiden untersuchten pD-Werten eine positive Netto-Ladung und interagiert mit der äußeren letzten PSS-Schicht unter elektrostatisch anziehenden Kräften. Mit ansteigender Temperatur steigt die Menge an adsorbiertem Protein bei beiden pD-Werten an, was die Dominanz einer entropisch treibende Kraft anzeigt. Bei einem pD-Wert von 4,9, bei dem eine relativ hohe Proteinladung vorliegt, ist der temperaturinduzierte Massenanstieg der immobilisierten Proteinmoleküle stärker innerhalb der Polyelektrolytschicht ausgeprägt. Hingegen hat bei einem pD-Wert von 7,5, der näher am isoelektrischen Punkt der SNase liegt, die Erhöhung der Temperatur den Effekt, dass sich die Proteinmoleküle außerhalb der Polyelektrolyt-Multischicht an der Wassergrenzfläche anreichern. Daraus kann geschlossen werden, dass das Eindringen der SNase in eine Polyelektrolyt-Multischicht hauptsächlich auf einem Komplexierungs-mechanismus beruht aufgrund einer Entlassung von Gegenionen. Sphärische Polyelektrolyt-Bürsten als Trägerpartikel für Proteinen Sphärische Polyelektrolyt-Bürsten (SPB) sind eine neue und sehr interessante Art von Kolloid-Partikeln. Sie bestehen aus einem Polystyrolkern, auf dem lange Ketten aus Polyacrylsäure (PAA) oder Polystyrolsäure (PSS) verankert sind. Bei geringer Ionenstärke adsorbieren Proteine spontan auf diesen Partikeln. Eine ihrer besonderen Eigenschaften ist ihre Proteinresistenz bei moderaten Salzkonzentrationen. Daher ist es möglich, durch Erhöhung der Ionenstärke bereits adsorbiertes Protein wieder von den Polyelektrolytketten zu lösen. Dadurch, dass Enzyme auf den SPB geladen und wieder entlassen werden können, bietet sich in Zukunft eine mögliche Anwendung als Wirkstoffträger an. Desweiteren scheint eine mögliche Anwendung als Nanoreaktor, der das immobilisierte Enzym nach einer gewünschten Reaktion wieder aus den SPB entlassen kann, sehr erfolgsversprechend. Mit Hilfe der CD-Spektroskopie wurde in dieser Arbeit die Konformation eines Proteins, welches mit einer sphärischen Polyelektrolytbürste wechselwirkt, untersucht. Rinder-Serum-Albumin (BSA) wurde als Modellprotein gewählt. Es trägt bei neutralen pH-Werten aufgrund seines isoelektrischen Punktes von pH=5 eine negative Nettoladung und besteht in seiner Sekundärstruktur größtenteils aus α-Helices. Die Ergebnisse der CD-Messungen zeigen, dass BSA durch Wechselwirkung mit SPB, d.h. nach einem Adsorptions-Desorptions-Prozess, nur geringfügig seine Sekundärstruktur verändert. Dabei stehen die beobachteten Konformationsänderungen nicht in Zusammenhang mit denen, die in Lösung bei niedrigen pH-Werten gefunden werden. SPB erweisen sich somit als vielversprechende Trägerpartikel für Proteine, da sie eine schonende Proteinumgebung bereit stellen.

In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen egg white lysozyme (HEWL). The kinetics of protein aggregation is studied mostly experimentally and, when possible, theoretically. The process of fibrilar (or linear) protein aggregation is the process of formation of elongated structures from otherwise compact (globular) proteins. Studying the kinetics of this process for different proteins can lead to a better understanding of the mechanism of the process and to a possible generalisation of this mechanism. The investigation of the morphology of the formed aggregates at different stages of the process of aggregation could also lead to a more complete picture of the detailed mechanism of the process. Last, but not least, is the influence of the protein stability on the type of the formed aggregates and the kinetics of fibrilar aggregation.The specific aims of this thesis are the following: 1) To investigate the kinetics of heat-induced fibrilar aggregation of two model proteins, bovineb-lg and HEWL, in as much detail as possible; 2) To study the morphology of the fibrils formed from both proteins; 3) To study the influence of the environment such as temperature, pH, and ionic strength on the kinetics of fibrilar aggregation and the morphology of the formed fibrils.The heat-induced fibrilar aggregation ofb-lg is investigated at pH 2.0, 80°C, and at various ionic strengths. Fibril formation is followed in situ using static (SLS) and dynamic light scattering (DLS), small angle neutron scattering (SANS), and proton NMR techniques. The fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. Even after prolonged heating, an appreciable fraction of the protein molecules is incorporated into fibrils, only when theb-lg concentration is above some critical concentration that is ionic strength dependent.The linear aggregation ofb-lg upon prolonged heating at pH 2.0 at80°Cappears to be a multistep process. Competing reactions lead to two products: long linear aggregates and low molecular weight "dead end" species. The "dead end" species comprises monomeric non-native protein molecules and cannot form fibrils. Fibril formation involves at least two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon subsequent slow cooling.Based on the obtained experimental data we have derived a kinetic model for the heat-induced aggregation ofb-lg at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils as well as a side reaction leading to the complete denaturation and inactivation of a part of the protein molecules. An analytical solution of the model for the early stages of the aggregation is obtained. The model describes very well the experimental data obtained by in situ SLS. It allows us to obtain molecular parameters for the kinetics of fibrilar aggregation ofb-lg as a function of the ionic strength. It gives us an expression for the apparent critical concentration for fibril formation due to the competition between the complete denaturation of the protein molecules and the formation of long fibrils. We also obtain the size of the critical nucleus for the fibril formation as a function of the ionic strength. In the case of a 13 mM ionic strength the critical nucleus consists of ca. 4 monomers; for all the other ionic strengths studied it is a dimer. This shows the important role that the non-specific electrostatic interaction has for the fibrilar aggregation ofb-lg at pH 2.0. It affects the rate of aggregation: the higher the ionic strength, the faster the aggregation. It also affects the detailed mechanism by which the aggregation takes place: the size of the critical nucleus increases when decreasing the ionic strength from 50 mM to 13 mM.We have also shown that time-resolved SANS can be used with success in studying protein aggregation and that with enough additional information for the aggregation process one can in practice obtain complete information about the aggregation kinetics of the process.Tapping mode atomic force microscopy results indicate that the fibrils formed at pH 2.0 upon heating at 80°Chave a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. The main difference between the fibrils observed at different ionic strengths is their length and curvature. Fibrils obtained at higher ionic strength are shorter and more curved as opposed to longer and straighter fibrils obtained at lower ionic strengths. In case of higher ionic strength the fibril formation is faster, more fibrils are formed and as a result the mean length of the fibrils is shorter. Fibrils obtained at all ionic strengths exhibit similar type of periodic morphology, which suggests that the detailed mechanism of fibril formation might be independent of the ionic strength, but specific forb-lg.In the case of HEWL we study the effect of pH and temperature on the fibril formation. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism (CD)). The stability of HEWL toward heat treatment is greatly influenced by the pH. The lower the pH, the lower the stability of the protein is. The conditions at pH 2.0 are unique in promoting the fibrilar aggregation of HEWL since heating of solutions at pH 3.0 and 4.0 to temperatures just above the midpoint of the unfolding transition of the molecule does not lead to the appearance of fibrilar aggregates.HEWL fibrils are formed after a lag time that is practically concentration independent. This means that the governing process for the fibril formation is the change in the structure of single protein molecules caused by a prolonged exposure to a temperature close to the midpoint of the unfolding transition. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating.The fibril morphology is complex. The fibrils formed at pH 2.0 are long and straight with a length of the order of 5mm and predominant thickness of about 4 nm and consist of stiff rod-like subunits with length either 124 or 157 nm. On smaller scale the fibrils consist of a coiled structure with a period of ca. 30 nm that gives the appearance of the rod-like subunits probably because of defects occurring every 4 or 5 turns.The fibrils consist mostly of full-length HEWL, although, some fragments due to hydrolysis at pH 2.0 and 57°C are probably incorporated into the fibrils. At any rate the hydrolysis of the protein is not the cause of the aggregation since at pH 3.0 no hydrolysis is detected but fibrils do form.In conclusion we can say that for a full and general description of the processes of fibrilar aggregation of globular proteins the type of specific interaction responsible for the aggregation must be identified. The interacting parts of the protein must also be identified. The last and most difficult task is to characterise the conformation of the protein in solution at conditions suitable for aggregation.

In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen egg white lysozyme (HEWL). The kinetics of protein aggregation is studied mostly experimentally and, when possible, theoretically. The process of fibrilar (or linear) protein aggregation is the process of formation of elongated structures from otherwise compact (globular) proteins. Studying the kinetics of this process for different proteins can lead to a better understanding of the mechanism of the process and to a possible generalisation of this mechanism. The investigation of the morphology of the formed aggregates at different stages of the process of aggregation could also lead to a more complete picture of the detailed mechanism of the process. Last, but not least, is the influence of the protein stability on the type of the formed aggregates and the kinetics of fibrilar aggregation.The specific aims of this thesis are the following: 1) To investigate the kinetics of heat-induced fibrilar aggregation of two model proteins, bovineb-lg and HEWL, in as much detail as possible; 2) To study the morphology of the fibrils formed from both proteins; 3) To study the influence of the environment such as temperature, pH, and ionic strength on the kinetics of fibrilar aggregation and the morphology of the formed fibrils.The heat-induced fibrilar aggregation ofb-lg is investigated at pH 2.0, 80°C, and at various ionic strengths. Fibril formation is followed in situ using static (SLS) and dynamic light scattering (DLS), small angle neutron scattering (SANS), and proton NMR techniques. The fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. Even after prolonged heating, an appreciable fraction of the protein molecules is incorporated into fibrils, only when theb-lg concentration is above some critical concentration that is ionic strength dependent.The linear aggregation ofb-lg upon prolonged heating at pH 2.0 at80°Cappears to be a multistep process. Competing reactions lead to two products: long linear aggregates and low molecular weight "dead end" species. The "dead end" species comprises monomeric non-native protein molecules and cannot form fibrils. Fibril formation involves at least two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon subsequent slow cooling.Based on the obtained experimental data we have derived a kinetic model for the heat-induced aggregation ofb-lg at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils as well as a side reaction leading to the complete denaturation and inactivation of a part of the protein molecules. An analytical solution of the model for the early stages of the aggregation is obtained. The model describes very well the experimental data obtained by in situ SLS. It allows us to obtain molecular parameters for the kinetics of fibrilar aggregation ofb-lg as a function of the ionic strength. It gives us an expression for the apparent critical concentration for fibril formation due to the competition between the complete denaturation of the protein molecules and the formation of long fibrils. We also obtain the size of the critical nucleus for the fibril formation as a function of the ionic strength. In the case of a 13 mM ionic strength the critical nucleus consists of ca. 4 monomers; for all the other ionic strengths studied it is a dimer. This shows the important role that the non-specific electrostatic interaction has for the fibrilar aggregation ofb-lg at pH 2.0. It affects the rate of aggregation: the higher the ionic strength, the faster the aggregation. It also affects the detailed mechanism by which the aggregation takes place: the size of the critical nucleus increases when decreasing the ionic strength from 50 mM to 13 mM.We have also shown that time-resolved SANS can be used with success in studying protein aggregation and that with enough additional information for the aggregation process one can in practice obtain complete information about the aggregation kinetics of the process.Tapping mode atomic force microscopy results indicate that the fibrils formed at pH 2.0 upon heating at 80°Chave a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. The main difference between the fibrils observed at different ionic strengths is their length and curvature. Fibrils obtained at higher ionic strength are shorter and more curved as opposed to longer and straighter fibrils obtained at lower ionic strengths. In case of higher ionic strength the fibril formation is faster, more fibrils are formed and as a result the mean length of the fibrils is shorter. Fibrils obtained at all ionic strengths exhibit similar type of periodic morphology, which suggests that the detailed mechanism of fibril formation might be independent of the ionic strength, but specific forb-lg.In the case of HEWL we study the effect of pH and temperature on the fibril formation. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism (CD)). The stability of HEWL toward heat treatment is greatly influenced by the pH. The lower the pH, the lower the stability of the protein is. The conditions at pH 2.0 are unique in promoting the fibrilar aggregation of HEWL since heating of solutions at pH 3.0 and 4.0 to temperatures just above the midpoint of the unfolding transition of the molecule does not lead to the appearance of fibrilar aggregates.HEWL fibrils are formed after a lag time that is practically concentration independent. This means that the governing process for the fibril formation is the change in the structure of single protein molecules caused by a prolonged exposure to a temperature close to the midpoint of the unfolding transition. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating.The fibril morphology is complex. The fibrils formed at pH 2.0 are long and straight with a length of the order of 5mm and predominant thickness of about 4 nm and consist of stiff rod-like subunits with length either 124 or 157 nm. On smaller scale the fibrils consist of a coiled structure with a period of ca. 30 nm that gives the appearance of the rod-like subunits probably because of defects occurring every 4 or 5 turns.The fibrils consist mostly of full-length HEWL, although, some fragments due to hydrolysis at pH 2.0 and 57°C are probably incorporated into the fibrils. At any rate the hydrolysis of the protein is not the cause of the aggregation since at pH 3.0 no hydrolysis is detected but fibrils do form.In conclusion we can say that for a full and general description of the processes of fibrilar aggregation of globular proteins the type of specific interaction responsible for the aggregation must be identified. The interacting parts of the protein must also be identified. The last and most difficult task is to characterise the conformation of the protein in solution at conditions suitable for aggregation.

Many food products are emulsions, i.e., a system of two immiscible liquids of which the one is finely dispersed in the other. In many emulsions, a monolayer of protein molecules in the liquid/liquid interface prevents coalescence of the droplets. Therefore, in the formation of a protein-stabilized emulsions the adsorption of proteins at the oil/water interface is an important step. To obtain a better insight in the characteristics of the protein layer that are relevant for stabilizing emulsions we studied protein adsorption using model systems. The proteins we selected were lysozyme anda-lactalbumin. Their adsorption and characteristics in the adsorbed state were investigated at solid/liquid, i.e., silica/water, and liquid/liquid, i.e., oil/water, interfaces.The major interactions determining protein adsorption are (a) electrostatic interaction between the protein molecules and the sorbent surface, (b) changes in the state of hydration of the sorbent surface and the protein molecule, and (c) changes in the three-dimensional structure of the protein molecule. In this thesis, there is an emphasis on electrostatic interactions.To study theinfluence of electrostatic interactions on protein adsorption, lysozyme is chemically modified by adding succinyl groups. The succinyl groups react with lysine and other cationic groups which are converted into anionic groups. Upon succinylation with an excess of succinyl, ten succinyl groups are linked to a lysozyme molecule. As a consequence, the isoelectric point of lysozyme shifts from 11 to 4.5.Besides affecting the charge of the protein, its structural stability decreases.In chapter 2, the influence of succinylation on the structural properties of lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability, as reflected in the denaturation temperature and the Gibbs energy of denaturation, of succinylated lysozyme is lower than that of native lysozyme. It is furthermore remarkable that the change in the heat capacity (DdenC p ) upon thermal denaturation for succinylated lysozyme is much higher than for native lysozyme. This is explained in terms of an extended degree of unfolding of the secondary structure and full exposure to the aqueous environment of the apolar parts of the succinyl groups.In chapter 3, the influence of electrostatic interactions on protein adsorption was studied on solid/liquid interfaces, by comparing the adsorption of lysozyme and succinylated lysozyme at silica surfaces. The succinylation not only affects the charge of the protein, but also its structure stability, as described in chapter 2. Although changes in stability may have an influence on adsorption, our data show that the primary effect of succinylation can be entirely understood in terms of electrostatic interactions. The saturated adsorbed amount as a function of pH has a maximum for both proteins. This maximum coincides with the isoelectric point for succinylated lysozyme and is close to the isoelectric point for lysozyme. At pH values where the protein is electrostatically repelled by the sorbent, higher ionic strengths increase adsorption, and for electrostatic attraction higher ionic strengths decrease adsorption.In chapter 4, the kinetics of adsorption of lysozyme anda-lactalbumin on silica and hydrophobized silica is investigated. For lysozyme at the hydrophilic interface, the rate of adsorption increases proportionally with increasing protein concentration in solution. The adsorption rate is comparable with the supply rate. It implies that all of the positively charged lysozyme molecules attach at the negatively charged silica surface. Fora-lactalbumin, which is also positively charged, the initial rate of adsorption is a fraction of the supply rate. For both proteins, adsorption saturation increases with increasing supply rate, indicating less spreading of the adsorbed protein molecules when the sorbent surface becomes more rapidly covered by the protein.At the hydrophobic interface, the initial adsorption rate of both proteins is considerably lower than the supply rate, especially for the highest concentration measured. The final adsorbed amount of both proteins is invariant with the supply rate. It suggests that structural rearrangements in the adsorbed protein molecules occur at a shorter time scale than that of the supply.It is furthermore remarkable that for lysozyme at the hydrophobic interface and fora-lactalbumin at both types of interfaces, the adsorption proceeds in a cooperative manner, as is manifested by an increase in the adsorption rate after the first molecules are adsorbed. Besides adsorption, desorption is studied. For lysozyme, it is found that the desorbed amount decreases after longer residence time of the protein at the interface.In chapter 5, the behavior of oil-in-water emulsions stabilized by lysozyme and succinylated lysozyme was compared with surface shear measurements on monolayers of the same proteins at an oil/water interface. Surface shear measurements were performed at different pH values. In the range measured, for both proteins the apparent surface shear viscosity was higher at pH values closer to the isoelectric point. The monolayer of succinylated lysozyme was faster in forming a network, but that of lysozyme had the highest final viscosity reached after 1400 minutes.Oil-in-water emulsions were made and stabilized by lysozyme and succinylated lysozyme. Due to the adsorbed protein layer, these emulsions contain positively and negatively charged oil droplets, respectively. The emulsions were mixed and because of opposite charges on the emulsion droplets, the droplets aggregate. The effect of mixing proportions was investigated and the largest aggregates were found when the mixing ratio deviated from unity.After a long time (more then 24 hours), the surface shear viscosity of lysozyme and succinylated lysozyme still increased, but no difference was found between mixing fresh or aged emulsions. It seems that the formation of a protein network at the interface is not decisive in the protection of the emulsion against coalescence.

Many food products are emulsions, i.e., a system of two immiscible liquids of which the one is finely dispersed in the other. In many emulsions, a monolayer of protein molecules in the liquid/liquid interface prevents coalescence of the droplets. Therefore, in the formation of a protein-stabilized emulsions the adsorption of proteins at the oil/water interface is an important step. To obtain a better insight in the characteristics of the protein layer that are relevant for stabilizing emulsions we studied protein adsorption using model systems. The proteins we selected were lysozyme anda-lactalbumin. Their adsorption and characteristics in the adsorbed state were investigated at solid/liquid, i.e., silica/water, and liquid/liquid, i.e., oil/water, interfaces.The major interactions determining protein adsorption are (a) electrostatic interaction between the protein molecules and the sorbent surface, (b) changes in the state of hydration of the sorbent surface and the protein molecule, and (c) changes in the three-dimensional structure of the protein molecule. In this thesis, there is an emphasis on electrostatic interactions.To study theinfluence of electrostatic interactions on protein adsorption, lysozyme is chemically modified by adding succinyl groups. The succinyl groups react with lysine and other cationic groups which are converted into anionic groups. Upon succinylation with an excess of succinyl, ten succinyl groups are linked to a lysozyme molecule. As a consequence, the isoelectric point of lysozyme shifts from 11 to 4.5.Besides affecting the charge of the protein, its structural stability decreases.In chapter 2, the influence of succinylation on the structural properties of lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability, as reflected in the denaturation temperature and the Gibbs energy of denaturation, of succinylated lysozyme is lower than that of native lysozyme. It is furthermore remarkable that the change in the heat capacity (DdenC p ) upon thermal denaturation for succinylated lysozyme is much higher than for native lysozyme. This is explained in terms of an extended degree of unfolding of the secondary structure and full exposure to the aqueous environment of the apolar parts of the succinyl groups.In chapter 3, the influence of electrostatic interactions on protein adsorption was studied on solid/liquid interfaces, by comparing the adsorption of lysozyme and succinylated lysozyme at silica surfaces. The succinylation not only affects the charge of the protein, but also its structure stability, as described in chapter 2. Although changes in stability may have an influence on adsorption, our data show that the primary effect of succinylation can be entirely understood in terms of electrostatic interactions. The saturated adsorbed amount as a function of pH has a maximum for both proteins. This maximum coincides with the isoelectric point for succinylated lysozyme and is close to the isoelectric point for lysozyme. At pH values where the protein is electrostatically repelled by the sorbent, higher ionic strengths increase adsorption, and for electrostatic attraction higher ionic strengths decrease adsorption.In chapter 4, the kinetics of adsorption of lysozyme anda-lactalbumin on silica and hydrophobized silica is investigated. For lysozyme at the hydrophilic interface, the rate of adsorption increases proportionally with increasing protein concentration in solution. The adsorption rate is comparable with the supply rate. It implies that all of the positively charged lysozyme molecules attach at the negatively charged silica surface. Fora-lactalbumin, which is also positively charged, the initial rate of adsorption is a fraction of the supply rate. For both proteins, adsorption saturation increases with increasing supply rate, indicating less spreading of the adsorbed protein molecules when the sorbent surface becomes more rapidly covered by the protein.At the hydrophobic interface, the initial adsorption rate of both proteins is considerably lower than the supply rate, especially for the highest concentration measured. The final adsorbed amount of both proteins is invariant with the supply rate. It suggests that structural rearrangements in the adsorbed protein molecules occur at a shorter time scale than that of the supply.It is furthermore remarkable that for lysozyme at the hydrophobic interface and fora-lactalbumin at both types of interfaces, the adsorption proceeds in a cooperative manner, as is manifested by an increase in the adsorption rate after the first molecules are adsorbed. Besides adsorption, desorption is studied. For lysozyme, it is found that the desorbed amount decreases after longer residence time of the protein at the interface.In chapter 5, the behavior of oil-in-water emulsions stabilized by lysozyme and succinylated lysozyme was compared with surface shear measurements on monolayers of the same proteins at an oil/water interface. Surface shear measurements were performed at different pH values. In the range measured, for both proteins the apparent surface shear viscosity was higher at pH values closer to the isoelectric point. The monolayer of succinylated lysozyme was faster in forming a network, but that of lysozyme had the highest final viscosity reached after 1400 minutes.Oil-in-water emulsions were made and stabilized by lysozyme and succinylated lysozyme. Due to the adsorbed protein layer, these emulsions contain positively and negatively charged oil droplets, respectively. The emulsions were mixed and because of opposite charges on the emulsion droplets, the droplets aggregate. The effect of mixing proportions was investigated and the largest aggregates were found when the mixing ratio deviated from unity.After a long time (more then 24 hours), the surface shear viscosity of lysozyme and succinylated lysozyme still increased, but no difference was found between mixing fresh or aged emulsions. It seems that the formation of a protein network at the interface is not decisive in the protection of the emulsion against coalescence.

Based on the semi-crystalline and biodegradable multiblock-copolymer of poly(p-dioxanon) and poly(?-caprolacton) matrix drug delivery systems (films and particles) are developed for proteins. Protein-loaded microparticles are produced in cylindrical reactors by means of the double emulsification solvent evaporation technique. The loading efficiency of the particles (0,32 - 1,2 wt.-% lysozyme) depends on the experimental parameters (volume of second emulsifier, ethanol as phase mediator in the second emulsifier). Attempts to produce particles by means of the original spray drying method were unsuccesful due to the semi-crystalline property of the polymer. During spraying of the polymer solution, the nozzle is clogged and in the drying chamber, some sticky fibers are formed as well as ruptured spherical particles with a diameter of 10 to 100 µm. SEM shows that a wide size distribution of particles exist. Particles formed by the double emulsification solvent evaporation technique are spherical with a diameter of 0,1 to 2000 µm and a broad size distribution. In most experiments the larger particles (170-1000 µm) represent 80-95% of the total volume. The material properties are examined by analysis of the loaded films. A loading of up to 10 wt.-% protein does not influence the thermal or shape-memory properties of the material. Young's moduli are cut in half (49 MPa) and the buffer or solvent uptake is doubled. There is no difference for the elongation at break for the 5, 10 or 20 wt.-% lysozyme loaded polymer. The strain fixity rate in cyclic thermomechanical tensile tests is in the range of 92% for all polymeric materials, pure or loaded (10 or 20 wt.-% lysozyme). For all samples the strain recovery rate of the first cycle is around 80% and achieves 99% in the following cycles and the transition temperature of the shape memory process is around 31°C. For all samples (0, 5, 10 or 20wt.-% lysozyme) the value of the transition coeffizient ? is (0,26 ( 0,01) 10-1K-1. If the loading is 20 wt.-% lysozyme, the transition is much broader. Hydrolysis of the loaded polymer in hydrochloric acid (pH 1,0), acetate-buffer (pH 4,6) and phosphate-buffer (pH 7,0) proceeds as the hydrolysis of the unloaded polymer with respect to change of chemical composition, thermal properties, mechanical properties and surface topography according to white light interferometry. The hydrolysis of the polymer is enhanced by increased pH value of the solution. Lysozyme released from 10 wt.-% loaded films has a biological activity of 1136 U/mL after 1 d, but the activity is reduced to 9 U/mL after 7 d. Lysozyme released from loaded particles shows an activity of 3 U/mL after 1 d, thus applies to all particles that are produced with different parameter sets. The activity loss of lysozyme is caused by the interactions of the enzyme with methylene chloride under shear stress (homogenizers with 18.000 rpm for 20 s). Investigations show that neither temperature, pressure, or both (to lysozyme powder) nor the vessels, hydrophobic interactions between lysozyme solution and hydrophobic polymer films or shear stress without addition of an organic solvent bate the biological activity of lysozyme.

Proteine übernehmen in nahezu allen biologischen Systemen Schlüsselfunktionen als Katalysatoren von chemischen Prozessen. Ihre Funktionseigenschaften ergeben sich aus der Möglichkeit verschiedenartiger Faltungen in zahlreiche dreidimensionale Strukturen. Mittlerweile ist bekannt, daß bei einer Großzahl an Krankheiten ein Zusammenhang zu einer falsch verlaufenden Faltung von Proteinen besteht. Eines der Proteine von denen bekannt ist, daß Mutationen zu einer durch Amyloidfasern ausgelösten Erkrankung führen können, ist das Lysozym. Die gute Verfügbarkeit großer Mengen dieser Substanz macht es zu einem idealen Modellsystem. In der vorliegenden Arbeit wurden Untersuchungen an Hühnereiweißlysozym durchgeführt, welche die Eigenschaften der Proteinfaltung anhand von spektroskopischen Methoden und thermodynamischen Berechnungen beschreiben sollen. Hierbei standen Versuche zur pH-Abhängigkeit der Wärmekapazität und deren Übergangstemperatur Tm sowie Untersuchungen zur Volumen- und Expansionsänderung während des Denaturierungsprozesses im Vordergrund.

The formation and stabilization of oil-in-water emulsions using well-defined and well-characterized proteins and peptides was studied in order to elucidate the relation between their molecular and functional properties. The emulsions were formed with a high-pressure homogenizer. To study the effect of the homogenizer scale on the emulsion properties, emulsions were prepared with a laboratory and a small industrial homogenizer. The flow in the industrial homogenizer was shown to be turbulent. In the laboratory homogenizer, droplet break-up was found to occur in a bounded laminar type of flow, resulting in a poor operating efficiency. The effect of the flow type on the emulsion properties, however, appeared to be small, if the number of passes through the laboratory homogenizer was sufficiently high.Proteins appeared to have good emulsion forming properties as long as protein aggregation was absent. In those cases, the recoalescence rate during homogenization was found to be similar and only small differences in the droplet size of emulsions could be determined. The surface excess of the emulsion droplets appeared to be governed by the conformational stability and the aggregated state of the proteins. Globular proteins with a high conformational stability yielded relatively low surface excesses, while a flexible random coil protein, likeβ-casein, yielded a relatively high surface excess. Protein aggregation may be due to physicochemical conditions and surface or heat denaturation. If protein aggregates were present, the emulsion droplets were also often aggregated. The droplet size, surface excess, and rate of recoalescence of these aggregated emulsions were usually found to be relatively high.The emulsion forming properties ofβ-casein peptides appeared to be comparable or superior to those of intact proteins. Amphiphilic peptides without the hydrophobic C-terminal domain ofβ-casein yielded a relatively low surface excess, likely due to strong electrostatic interactions between the highly charged groups of the N-terminal end. The surface excess of emulsions made with hydrophobic peptides with a removed N-terminal domain was comparable to those of emulsions made with intactβ-casein. The peptides were due to their relatively small molar mass more readily desorbed from the oil/water interface than intact proteins.The coalescence stability of emulsions made with proteins was high even at low protein concentrations and appeared to be mainly determined by the surface excess of the droplets. The emulsion stabilizing properties ofβ-casein peptides were inferior to those of intact proteins probably due to their relatively low molar mass. Comparison of the stability of emulsions made with amphiphilic peptides with an intact or partially removed N-terminal domain showed that this domain was of great importance for providing stability against coalescence. The coalescence stability of emulsions made with hydrophobic peptides was relatively high, which was attributed to the high surface excess of the droplets. The electrostatic and steric interactions appeared to be of great importance for stabilizing emulsions made with peptides against coalescence as was indicated by the effect of changes in pH and ionic strength on the stability.Keywords: emulsions, formation, stability, molecular properties,β-casein,β-lactoglobulin,α-lactalbumin, lysozyme, ovalbumin, peptides.

Mes activités de recherche se focalisent sur l’étude structurale de la fonction des protéines tant au point de vue expérimental que théorique et plus particulièrement l’aspect de la reconnaissance intermoléculaire. Notre système de référence est celui de la reconnaissance antigène-anticorps. Ce système est le prototype parfait, de part sa fonction, mais également grâce à la dynamique de ce processus lié au concept de maturation. Aucours de ma thèse de doctorat, nous avons mesuré les constantes d’affinité, à l’équilibre et en cinétique, entre des anticorps monoclonaux et un antigène multivalent (virus de la mosaïque du tabac, VMT). De manière inattendue, nos résultats ont démontré une coopérativité négative dans la liaison d’anticorps au VMT. A partir de mon premier stage postdoctoral et jusqu’à aujourd’hui, ma recherche s’est orientée vers l’aspect structuralde la reconnaissance ; incluant le développement de fonctions énergétiques et de techniques d’analyse des structures tridimensionnelles, la construction de modèles moléculaires et l’assemblage de ligand dans leur recepteur.Ce mémoire résume mes cinq dernières années d’activités scientifiques dans le domaine de la modélisation moléculaire, bien que la parution de publications ne reflète guère ce laps de temps. La modélisation moléculaire est une discipline récente qui nécessite, à mon sens, une introduction formelle. Dans ce mémoire on définit la modélisation moléculaire comme l’ensemble des techniques qui permettent d’étudier la fonction d’unemolécule grâce à la connaissance de sa structure tridimensionnelle. Ces techniques incluent la modélisation par homologie, les méthodes de simulations, les méthodes d’assemblage (docking), les méthodes d’étude du repliement ab-initio des protéines. Les approches spectroscopiques comme la résonance magnétique nucléaire (RMN) ou le dichroisme circulaire et la microscopie électronique (ME) sont également inclues. Au sens littéral notre définition intègre également la diffraction des rayons X (RX).L’objectif de ce mémoire est de décrire en détail une de ces techniques : la modélisation par homologie. Ce mémoire focalise principalement sur les protéines bien que la modélisation moléculaire s’applique aussi bien aux acides nucléiques, aux sucres ou aux lipides. En dépit de la jeunesse de cette discipline, il est pratiquement impossible de la couvrir en son intégralité. Malgré une recherche bibliographique approfondie, elle est certainement incomplète due à l’interdisciplinarité de cette technique qui couvre tous les champs de recherches, de la théorie fondamentale à la médecine. Ce mémoire espère présenter l’impact de la modélisation par homologie dans la biologie moderne de la manière la plus juste.Ce mémoire comprend quatre parties. Après une introduction générale, on présentera en détail les diverses approches employées pour la modélisation par homologie. Les contributions personnelles à cette discipline seront enfin exposées. La perspective d’évolution de la modélisation moléculaire sera finalement brièvement discutée.

The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free amino acids, which act as flavour precursors. The ripening of cheese under conditioned storage is time consuming and costly. Addition of specific enzymes to the cheese milk is one of several options to accelerate ripening. A major problem then is that hardly any of these proteases end up in the cheese and most disappear with the whey stream. Entrapment of bacteria and milk fat globules into the casein matrix of the curd is due to their particle character. Immobilisation of proteases onto particles would thus result in retention of these proteins in the curd. Ideally, for reasons of acceptance, these particles should originate from the milk itself or at least be edible.In this study, in a more general approach, soya oil emulsion droplets and casein micelles, being protein aggregates, were tested as the carrier system. Chymosin and lysozyme were taken as the enzymes to be immobilized, because of their relevance to the dairy industry and because they are scientifically well-known. Moreover, their biochemical divergence make them suitable models for study.The literature provides several studies on adsorption of proteins onto interfaces. Few of these proteins are enzymes. In cases where lysozyme was studied, it was included because of its extraordinary properties as a protein and not because of its enzymatic activity. Apart from (phospho)lipases there is scarcely any literature that describes activity of enzymes adsorbed onto the oil/water or air/water interfaces. Proteins tend strongly to accumulate in interfaces and for that reason are said to be very surface active. This adsorption is accompanied by a conformational change of the three-dimensional structure of the protein that results in some unfolding, ranging from almost full stretching of the peptide chain to a more conserved conformation. Hydrophobic residues or patches of the protein, mostly buried inside the molecule, will tend to position themselves next to or even protrude partly into, the hydrophobic phase of an oil/water interface.The extent of conformational change depends on the conformational stability of the protein, which, in turn, depends on pH, temperature, ionic strength etc. Furthermore, the extent of unfolding will be dependent on the surface area available and on the time scale, and hence, on protein concentration. In a static condition, adsorption at the interface will be diffusion driven, whereas during emulsification the time of adsorption will be determined by convection and will be very much shorter. Consequently, conformational changes of proteins during emulsification should be smaller because full surface coverage may be reached before unfolding can occur. In the case of enzymes being adsorbed the emulsification process would therefore offer an opportunity to retain activity.The relation between the surface pressureΠi.e. the extent of surface tension decrease and the amount of protein adsorbed per unit surface area availableΓ, provides a possibility to relate the size of the adsorbed protein molecules, and thereby the extent of unfolding, to the surface load. As mentioned earlier the extent of unfolding should be less at a higherΓvalue. It has been calculated that lysozyme, an enzyme of high conformational stability, hardly unfolds at the air/water interface, even at low surface coverage. For the oil/water interface, however, a considerable increase in the radius of the protein molecules was observed at low surface load. At surface coverage of > 1.5 mg.m -2, the radius remained more or less constant, indicating that substantial unfolding did not occur. Despite this rigidity, the enzyme had lost all of its enzymatic activity in situ and it even remained inactive after desorption.Apparently, conformational changes in the enzyme molecule do not necessarily become manifest in a larger size for the molecule. Chymosin, being an enzyme of smaller conformational stability, naturally lost all of its activity due to adsorption onto the oil/water interface. In experiments with the enzymes coadsorbed simultaneously with bovine serum albumin, or the one after the other, there was no retention of in situ activity. Chymosin also proved to be inactivated at the expanding air/water interface due to air incorporation, if this occurred e.g. during homogenization.During the cheese-making process enzymes like chymosin and lysozyme are retained in the curd. This retention must be due to association of the enzymes with the casein from milk. In order to adsorb the enzymes with retention of activity, the various casein fractions were used to make and stabilize a soya-oil emulsion, and the enzyme was subsequently allowed to associate with the casein. The extent of association of lysozyme with the casein fractions was in the orderα s -casein >β-casein >κ-casein. Only for theκ-casein stabilized emulsion, was lysozyme association dependent on pH within the range of pH 5.2 - 6.4 (greater for a lower pH). Furthermore, the association with the caseins was not dependent on temperature, indicating that hydrophobic interactions were not predominant. The same trends were found with the various caseins in solution, albeit that association withκ-casein hardly occurred. It should be kept in mind that casein adsorbed at an interface will expose other amino acid residues compared to its behaviour when free in solution. For that reason the association behaviour in the two systems may differ.Because the association varies between caseins the extent of association with lysozyme depended on the composition of the casein micelles (aggregates of many casein molecules and calcium phosphate, as occurring especially in milk). As expected, casein micelles containing a higher proportion ofκ-casein associated less with lysozyme. It was found that lysozyme did not lose activity due to association with casein adsorbed on soya oil droplets or free in solution. However, lysozyme activity was markedly reduced when the enzyme was associated with casein micelles. In this system lysozyme also associated with casein in the interior of the casein micelle. The apparent loss of activity was most probably due to internal diffusion limitation. The difference of association for the various systems was also reflected in the free equilibrium concentration at which the surface excess plateau value was reached. In the system of adsorbed caseins this value was reached at a free lysozyme concentration of about 3 _M, whereas for the micellar system this value was about 100 times higher.The association of chymosin with casein has been studied in the same three systems of casein adsorbed onto soya-oil emulsion droplets, caseins in solution and caseins aggregated in casein micelles. It appears that chymosin only associated with adsorbedκ-casein and not with adsorbedαs- orβ-casein. Preceding the association, the caseinomacropeptide part ofκ-casein is split off, followed immediately by the aggregation of the soya-oil emulsion droplets containing the remaining para-κ-casein. This coagulation behaviour is identical to the renneting of milk during the cheese-making process. The association characteristics for chymosin are also comparable. The association was strongly dependent on pH and ionic strength, and on chymosin and casein concentration. Theκ-casein stabilized emulsion has proven to be a good model system for studying chymosin retention in curd. The chymosin associated with para-κ-casein was shown to be still active on addedκ-casein or on a fluorescent small hexapeptide substrate. Consequently, the active centre of the enzyme is presumably not involved in the association with casein.The association of chymosin with caseins free in solution has also been studied. Only in a solution containingκ-casein will addition of chymosin result in protein flocculation and precipitation. This flocculation is due to splitting off the caseino-macropeptide part ofκ-casein and the consecutive aggregation of the fairly hydrophobic and almost electrically neutral para-κ-casein molecules. The precipitated protein fraction also contains associated chymosin, to an extent depending on conditions like pH, ionic strength and casein and chymosin concentrations. In this system time and temperature also affected the extent of chymosin association. The association decreased with increased contact time and was stronger at higher temperatures.The protein content in the supernatant after centrifugation increased not only due to dissociation of chymosin but also due to the presence of casein fragments. Apparently, the dissociation of chymosin was related to its proteolytic action. The dissociation rate increased with decreasing pH where chymosin becomes more active and less specific. The dissociation also increased with temperature for a given time of contact. However, when extrapolated to a contact time of t = 0 (i.e. when dissociation due to proteolysis has not occurred yet) the association was observed to be somewhat stronger for a higher temperature. The effect of temperature on the proteolysis-dependent dissociation, apparently was stronger than its effect on the increase of the association. Since chymosin association depends on mutual association of caseins (see below), it will also depend on the temperature dependence of the latter. Dissociation of chymosin was not found in the system of caseins adsorbed onto emulsion droplets.The addition of small amounts ofαs- orβ-casein strongly decreased the extent of association of chymosin with para-κ-casein. This effect was stronger forαs-casein than forβ-casein. It was also found that the extent of chymosin association (moles of chymosin per mole of para-κ-casein) was larger when the system was diluted or, in other words, when the casein concentration was reduced. Both phenomena can be explained by assuming that competitive association occurs between the caseins and chymosin for interaction with a para-κ-casein molecule. Chymosin is only able to associate with a para-κ-casein molecule when that is not associated with other casein molecules. Thermodynamically speaking, the extent of association of chymosin is determined by the association constants that exist between all caseins under conditions as in the system. These association constants vary with pH, ionic strength, casein concentration and temperature.The model of competitive association is further developed and applied to the association of chymosin with casein micelles of various composition. It follows that chymosin will associate less with casein micelles composed ofαs- andκ-casein than with micelles composed ofβ- andκ-caseins. Again, this behaviour can be explained by competitive association, since different association constants exist for the caseins and chymosin for association with para-κ-casein. The relations for association and dissociation found in this casein micelle system are comparable with those found with caseins in solution. The kinetic model for competitive association is only a crude approximation. It does not provide possibilities of calculating all the association constants occurring in milk from the relations found from retention of chymosin in curd.

The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free amino acids, which act as flavour precursors. The ripening of cheese under conditioned storage is time consuming and costly. Addition of specific enzymes to the cheese milk is one of several options to accelerate ripening. A major problem then is that hardly any of these proteases end up in the cheese and most disappear with the whey stream. Entrapment of bacteria and milk fat globules into the casein matrix of the curd is due to their particle character. Immobilisation of proteases onto particles would thus result in retention of these proteins in the curd. Ideally, for reasons of acceptance, these particles should originate from the milk itself or at least be edible.In this study, in a more general approach, soya oil emulsion droplets and casein micelles, being protein aggregates, were tested as the carrier system. Chymosin and lysozyme were taken as the enzymes to be immobilized, because of their relevance to the dairy industry and because they are scientifically well-known. Moreover, their biochemical divergence make them suitable models for study.The literature provides several studies on adsorption of proteins onto interfaces. Few of these proteins are enzymes. In cases where lysozyme was studied, it was included because of its extraordinary properties as a protein and not because of its enzymatic activity. Apart from (phospho)lipases there is scarcely any literature that describes activity of enzymes adsorbed onto the oil/water or air/water interfaces. Proteins tend strongly to accumulate in interfaces and for that reason are said to be very surface active. This adsorption is accompanied by a conformational change of the three-dimensional structure of the protein that results in some unfolding, ranging from almost full stretching of the peptide chain to a more conserved conformation. Hydrophobic residues or patches of the protein, mostly buried inside the molecule, will tend to position themselves next to or even protrude partly into, the hydrophobic phase of an oil/water interface.The extent of conformational change depends on the conformational stability of the protein, which, in turn, depends on pH, temperature, ionic strength etc. Furthermore, the extent of unfolding will be dependent on the surface area available and on the time scale, and hence, on protein concentration. In a static condition, adsorption at the interface will be diffusion driven, whereas during emulsification the time of adsorption will be determined by convection and will be very much shorter. Consequently, conformational changes of proteins during emulsification should be smaller because full surface coverage may be reached before unfolding can occur. In the case of enzymes being adsorbed the emulsification process would therefore offer an opportunity to retain activity.The relation between the surface pressureΠi.e. the extent of surface tension decrease and the amount of protein adsorbed per unit surface area availableΓ, provides a possibility to relate the size of the adsorbed protein molecules, and thereby the extent of unfolding, to the surface load. As mentioned earlier the extent of unfolding should be less at a higherΓvalue. It has been calculated that lysozyme, an enzyme of high conformational stability, hardly unfolds at the air/water interface, even at low surface coverage. For the oil/water interface, however, a considerable increase in the radius of the protein molecules was observed at low surface load. At surface coverage of > 1.5 mg.m -2, the radius remained more or less constant, indicating that substantial unfolding did not occur. Despite this rigidity, the enzyme had lost all of its enzymatic activity in situ and it even remained inactive after desorption.Apparently, conformational changes in the enzyme molecule do not necessarily become manifest in a larger size for the molecule. Chymosin, being an enzyme of smaller conformational stability, naturally lost all of its activity due to adsorption onto the oil/water interface. In experiments with the enzymes coadsorbed simultaneously with bovine serum albumin, or the one after the other, there was no retention of in situ activity. Chymosin also proved to be inactivated at the expanding air/water interface due to air incorporation, if this occurred e.g. during homogenization.During the cheese-making process enzymes like chymosin and lysozyme are retained in the curd. This retention must be due to association of the enzymes with the casein from milk. In order to adsorb the enzymes with retention of activity, the various casein fractions were used to make and stabilize a soya-oil emulsion, and the enzyme was subsequently allowed to associate with the casein. The extent of association of lysozyme with the casein fractions was in the orderα s -casein >β-casein >κ-casein. Only for theκ-casein stabilized emulsion, was lysozyme association dependent on pH within the range of pH 5.2 - 6.4 (greater for a lower pH). Furthermore, the association with the caseins was not dependent on temperature, indicating that hydrophobic interactions were not predominant. The same trends were found with the various caseins in solution, albeit that association withκ-casein hardly occurred. It should be kept in mind that casein adsorbed at an interface will expose other amino acid residues compared to its behaviour when free in solution. For that reason the association behaviour in the two systems may differ.Because the association varies between caseins the extent of association with lysozyme depended on the composition of the casein micelles (aggregates of many casein molecules and calcium phosphate, as occurring especially in milk). As expected, casein micelles containing a higher proportion ofκ-casein associated less with lysozyme. It was found that lysozyme did not lose activity due to association with casein adsorbed on soya oil droplets or free in solution. However, lysozyme activity was markedly reduced when the enzyme was associated with casein micelles. In this system lysozyme also associated with casein in the interior of the casein micelle. The apparent loss of activity was most probably due to internal diffusion limitation. The difference of association for the various systems was also reflected in the free equilibrium concentration at which the surface excess plateau value was reached. In the system of adsorbed caseins this value was reached at a free lysozyme concentration of about 3 _M, whereas for the micellar system this value was about 100 times higher.The association of chymosin with casein has been studied in the same three systems of casein adsorbed onto soya-oil emulsion droplets, caseins in solution and caseins aggregated in casein micelles. It appears that chymosin only associated with adsorbedκ-casein and not with adsorbedαs- orβ-casein. Preceding the association, the caseinomacropeptide part ofκ-casein is split off, followed immediately by the aggregation of the soya-oil emulsion droplets containing the remaining para-κ-casein. This coagulation behaviour is identical to the renneting of milk during the cheese-making process. The association characteristics for chymosin are also comparable. The association was strongly dependent on pH and ionic strength, and on chymosin and casein concentration. Theκ-casein stabilized emulsion has proven to be a good model system for studying chymosin retention in curd. The chymosin associated with para-κ-casein was shown to be still active on addedκ-casein or on a fluorescent small hexapeptide substrate. Consequently, the active centre of the enzyme is presumably not involved in the association with casein.The association of chymosin with caseins free in solution has also been studied. Only in a solution containingκ-casein will addition of chymosin result in protein flocculation and precipitation. This flocculation is due to splitting off the caseino-macropeptide part ofκ-casein and the consecutive aggregation of the fairly hydrophobic and almost electrically neutral para-κ-casein molecules. The precipitated protein fraction also contains associated chymosin, to an extent depending on conditions like pH, ionic strength and casein and chymosin concentrations. In this system time and temperature also affected the extent of chymosin association. The association decreased with increased contact time and was stronger at higher temperatures.The protein content in the supernatant after centrifugation increased not only due to dissociation of chymosin but also due to the presence of casein fragments. Apparently, the dissociation of chymosin was related to its proteolytic action. The dissociation rate increased with decreasing pH where chymosin becomes more active and less specific. The dissociation also increased with temperature for a given time of contact. However, when extrapolated to a contact time of t = 0 (i.e. when dissociation due to proteolysis has not occurred yet) the association was observed to be somewhat stronger for a higher temperature. The effect of temperature on the proteolysis-dependent dissociation, apparently was stronger than its effect on the increase of the association. Since chymosin association depends on mutual association of caseins (see below), it will also depend on the temperature dependence of the latter. Dissociation of chymosin was not found in the system of caseins adsorbed onto emulsion droplets.The addition of small amounts ofαs- orβ-casein strongly decreased the extent of association of chymosin with para-κ-casein. This effect was stronger forαs-casein than forβ-casein. It was also found that the extent of chymosin association (moles of chymosin per mole of para-κ-casein) was larger when the system was diluted or, in other words, when the casein concentration was reduced. Both phenomena can be explained by assuming that competitive association occurs between the caseins and chymosin for interaction with a para-κ-casein molecule. Chymosin is only able to associate with a para-κ-casein molecule when that is not associated with other casein molecules. Thermodynamically speaking, the extent of association of chymosin is determined by the association constants that exist between all caseins under conditions as in the system. These association constants vary with pH, ionic strength, casein concentration and temperature.The model of competitive association is further developed and applied to the association of chymosin with casein micelles of various composition. It follows that chymosin will associate less with casein micelles composed ofαs- andκ-casein than with micelles composed ofβ- andκ-caseins. Again, this behaviour can be explained by competitive association, since different association constants exist for the caseins and chymosin for association with para-κ-casein. The relations for association and dissociation found in this casein micelle system are comparable with those found with caseins in solution. The kinetic model for competitive association is only a crude approximation. It does not provide possibilities of calculating all the association constants occurring in milk from the relations found from retention of chymosin in curd.

Investigations were done on the influence of Lysozyme from hen-eggwhite on the heat resistance of bacterial spores in foodstuffs and the shelflife of various products e.g. rissole. The decrease of the heat resistance of bacterial spores is dependent on the bacterial species itself and the environment of the spores (buffer, pH, foodstuff etc.). Therefore this effect could not be utilized in all foodstuffs. The prolonging of shelflife was possible in combination with a cold-storage at 6xC.

By using enzymes for biopreservation, the thermal damage and the addition of preservatives to food can be avoided or reduced. Enzymatically catalysed reactions exhibit several advantages compared to currently used preservation methods, for example low incubation temperature and minimal interfering or side reactions. Another important point is that the velocity of the reaction is dependent on external parameters, and therefore the desired effect in the product can be influenced by controlling the reaction conditions. The example of milk demonstrates that nature has already developed some partly enzymatic mechanisms which show antibacterial effects. The lactoperoxidase-system, lactoferrin, glucoseoxidase and lysozyme are good examples which have been discussed in detail in this paper. For the food industry, several reasons exist for choosing glucoseoxidase as an additive (mostly in combination with other enzymes) as it can be deduced from the substrates used and from the products formed in this enzymatic reaction. The essential objectives are the removal of oxygen or glucose from the food, the production of the toxic peroxide, and the lowering of the pH-value in the medium. Another important group of antimicrobially active enzymes are those with cell wall disrupting specificities, such as lysozyme, glucanase and chitinase. Our lab has obtained a considerable amount of results and experience in the area of enzymatic biopreservation using lysozyme. In vitro experiments demonstrated that the heat resistance of spores of the genus Bacillus can be significantly decreased by pretreatment with lysozyme. The shelf life of salads with a mild acidification can be significantly prolonged, as was shown in various addition experiments under praxis relevant conditions.

Background: Application of natural compounds, including essential oils (EOs) and lysozyme is an effective method against growth of bacterial pathogens in foods. Objective: Determination of minimum inhibitory concentration (MIC) of lysozyme and Zataria multiflora Boiss. essential oil on L. monocytogenes. Methods: In this study different concentrations of lysozyme and Zataria multiflora Boiss. EOwere used alone and in combination on BHI broth to determine MIC of Zataria multiflora Boiss. EO and lysozyme with macro dilution and micro dilution methods and effect of sub inhibitory concentrations of them on bacterial growth curve of L. monocytogenes. Results: The minimum inhibitory concentration (MIC) of Z. multiflora Boiss EO was estimated ٪0.04 using macro and microdilution. lysozyme at the highest concentration (1000 μg/ ml) was not effective in inhibition of bacterial growth and no MIC value obtained. Combination of EO and Lysozyme decreased the MIC value to %0.02 and 250μg/ mlfor Z. multifloraBoiss. EO and lysozyme, respectively. The results of growth curve analysis showed that combination was effective in increasing the lag phase. Conclusion: Z. multiflora Boiss and lysozyme showed to be effective against bacterial growth and its potential application in food systems may be suggested.