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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.

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.

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.

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.