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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.

Soy protein hydrolysates are often supplemented to chemically defined (CD) media in cell cultures, but there is little understanding of the effect of their composition on their functionality (viable cell density, total immunoglobulin (IgG), and specific IgG production). To identify the key parameters (e.g. compound classes) that determine their functionality, hydrolysates were prepared from different starting materials (meal, concentrates, and isolate) and from soybean meal that was heated for different time periods. The functionality of these hydrolysates were compared to those of industrial hydrolysates. From the comparison, it was shown that the variation in industrial and experimental processes of hydrolysate production induced larger variation in the functionality than the variation in starting materials. Moreover, it was observed that the correlations between the functionality and compositional parameters observed in one experiment were absent in the other experiments. During the study, it became apparent that the variations in other factors, like CD media and temperature during culturing also resulted in variation in functionality. The extent of variations in the functionality due to variation in CD media and temperature during culturing was equivalent to the variation caused by varying the hydrolysate composition. The functionality data of the different experiments were fitted with a model that described the relation between specific IgG production and viable cell density. Using the model, the maximum achievable total IgG production could be calculated for a culture condition. This information can provide directions for further optimization of hydrolysates to maximize total IgG production.

The aim of this thesis is to understand the connection between molecular, meso and macroscales of enzymatically cross-linked proteins. It was hypothesised that the techno-functional properties at macroscale, such as bulk rheology and foam stability, are affected by the structure of nanoparticles at mesoscale. The approach was to make α-lactalbumin (α-LA) nanoparticles by using two different enzymes, horseradish peroxidase (HRP) or microbial transglutaminase (mTG), to produce an open and compact mesoscale structure, respectively. In addition to the control over the mesoscale structure, the size of the nanoparticles can be independently controlled by varying the dosage of hydrogen peroxide in the case of HRP and by thermal inactivation in the case of mTG. The other important parameters determining the size are protein concentration and ionic strength. The size (radius of gyration) range that could be achieved by varying the above mentioned control parameters is 20 – 200 nm. The polydispersed nanoparticles were separated by asymmetrical flow field flow fractionation (AF4) and characterised inline with multi angle light scattering (MALS). Polymerization of apo α-LA with HRP and mTG proceeds in a step growth way i.e. first monomers react to form oligomers and the oligomers are cross-linked to form polymers (nanoparticles). Extensive cross-linking of α-LA with HRP gives rise to not only di-tyrosine cross-links, but also tri–octa tyrosine cross-links, which was hitherto unknown. The two different mesoscale structures result in gels of different storage moduli. The storage modulus of gels made by concentrating the α-LA/mTG nanoparticles was around ten times higher than that made with open nanoparticles. The half-life time (t0.5) of the foam made with α-LA nanoparticles was two to six times higher than that of the monomeric α-LA. The higher foam-stability of the α-LA nanoparticles as compared to the monomeric α-LA is due to their higher thickness of the interfacial layer and thin films. In conclusion, it is shown that the techno-functional properties of α-LA are directly correlated to the size and meso-scale structures of the nanoparticles and enzymatic cross-linking is an effective way to control them.

Pet dogs and cats around the world are commonly fed processed commercial foods throughout their lives. Often heat treatments are used during the processing of these foods to improve nutrient digestibility, shelf life, and food safety. Processing is known to induce the Maillard reaction, in which a reducing sugar binds to a free reactive amino group of an amino acid. In intact proteins, the ε-amino group of lysine is the most abundant free amino group. The reaction reduces the bioavailability of lysine and results in the formation of advanced Maillard reaction products. The aim of this thesis was to determine the occurrence and progression of the Maillard reaction during the manufacturing of pet foods, the subsequent impact on nutritive value of the food, and the bioavailability of Maillard reaction products in cats. In Chapter 2, the scientific literature was reviewed to investigate the current state of knowledge on the Maillard reaction and its potential effect on the nutritive value of pet foods and on pet health. Determination of the difference between total and reactive lysine by chemical methods provides an indication of the Maillard reaction in pet foods. Studies reported that the proportion of reactive lysine is on average 73% (range 39 – 100%) of total lysine, and that foods for growing dogs may be at risk of supplying less lysine than the animals require. The endogenous analogues of Maillard reaction products, advanced glycation end-products, have been associated with age-related diseases in humans, such as diabetes and impaired renal function. In dogs, data indicate higher advanced glycation end-product contents in plasma from dogs suffering from canine diabetes mellitus compared with healthy control animals. In addition, elevated levels of advanced glycation end-products in tissue proteins in dogs were observed for a number of diseases. To date it was unknown to what extent Maillard reaction products were present in pet foods, and whether dietary Maillard reaction products can be associated with the development of diseases such as diabetes and impaired renal function in pet animals. As the Maillard reaction is induced by processing, changing processing conditions should have an influence on the severity of the reaction. However, effects of processing conditions on the difference in total and reactive lysine contents in pet foods were inconsistent and did not always correspond to model systems. Processing temperature was reported to be the most important factor followed by moisture level. In addition, differences between total and reactive lysine were observed in several ingredients commonly used in pet foods. Reviewing the literature indicates that it is unknown to which extent the Maillard reaction occurs and whether Maillard reaction products are present in pet foods. There might be a risk for certain foods not meeting minimal lysine requirements. It is also unknown what the exact effect of processing on the Maillard reaction is in pet foods. The experiment described in Chapter 3 was designed to evaluate whether commercial pet foods meet minimal lysine requirements. Sixty-seven extruded, canned and pelleted commercially available dog and cat foods formulated for growth and maintenance were analysed using conventional amino acid analysis and O-methylisourea as reagent for reactive lysine. Sixty out of the 67 foods in this study, regardless of the type of processing technology used, contained a lower reactive lysine than total lysine content. On average, pelleted and extruded foods contain lower reactive to total lysine ratios compared to canned foods (0.85, 0.89, and 0.93, respectively). All cat foods and foods for adult dogs met minimal lysine requirements. However, eight dry foods for growing dogs contained reactive lysine contents between 96 and 138% of the minimal lysine requirement, indicating that reactive lysine has to be between 62 and 104% digestible to meet minimal requirement. Considering the variability in reactive lysine digestibility, these foods could be at risk of not meeting minimal lysine requirements for growing dogs. In Chapter 4, the foods from Chapter 3 were used to quantitate the Maillard reaction products fructoselysine (FL), carboxymethyllysine (CML), hydroxymethylfurfural (HMF), and the cross-linked amino acid lysinoalanine (LAL) using UPLC-MS. In all foods, Maillard reaction products and LAL were found but in highly variable amounts. Type of processing seems to be a key factor for the concentration of FL, CML and HMF, with on average higher amounts in canned foods than pelleted and extruded foods (on a dry matter basis). The contents of CML and HMF found in commercial pet foods are, on average, within the range reported in processed human food products. Average daily intake (mg/kg body weight0.75) of HMF was 122 times higher for dogs and 38 times higher for cats than the calculated average intake for adult humans. Average daily intake of CML was comparable to the intake of adult humans. As Chapters 3 and 4 indicated that pelleted foods contain more Maillard reaction products than extruded foods, despite the less severe production process, an experiment was designed to gain insight in the effect of steam pelleting on the Maillard reaction in a dog food (Chapter 5). The aim was to examine the effect of conditioning temperature (65 and 90°C) and die hole length (ø 5 × 45, 65, and 80 mm) during pelleting processing of a standard dry dog food on selected indicators of the Maillard reaction (total lysine, reactive lysine, FL, CML, HMF, LAL), browning development and CIE-Lab colour. Steam pelleting did not cause a significant loss of reactive lysine and change of absorbance values. This indicates that the effect of steam pelleting on the nutritive value of the foods is low. However, steam pelleting did increase the content of Maillard reaction products. The formation of the Maillard reaction products was associated with an increase in temperature and die hole length during the steam pelleting process. The unprocessed ingredient mix already contained a larger difference between reactive and total lysine, and contents of Maillard reaction products than was induced during steam pelleting. Therefore, the choice of the ingredients used in this study mainly determines reactive lysine content and Maillard reaction products in the pet food formulation. As it is unknown to which extent extrusion processing influences the Maillard reaction in pet foods, the effect of extrusion processing on selected indicators of the Maillard reaction was determined (Chapter 6). The extrusion parameters temperature (140 and 165°C), moisture content (200 and 300 g/kg) and screw speed (100 and 200 rpm) were applied to two dry dog foods formulated using either intact or hydrolysed proteins. Extrusion processing in general results in a decrease in total and reactive lysine and an increase in FL, CML, HMF and LAL content. However, this effect appeared more pronounced in the diet containing hydrolysed protein. Decreasing temperature and moisture content led to higher total and reactive lysine contents, and less Maillard reaction products in the dog foods. Increasing screw speed had a positive influence on total and reactive lysine, but a negative influence on Maillard reaction products. As was found in Chapter 5, the unprocessed ingredient mixtures in this experiment contained already more Maillard reaction products than was induced during extrusion processing. Whether the Maillard reaction products reported in pet foods are physiologically relevant in pet animals depends on the bioavailability of these components. Therefore, urinary excretion was studied in adult cats fed commercial moist and dry foods containing varying amounts of FL, CML and the amino acid LAL (Chapter 7). A pilot study was first conducted to determine the adaptation time required for stable urinary excretion of the Maillard reaction products when changing diets with contrasting contents of Maillard reaction products. An adaptation time of 1 d was deemed sufficient in adult cats. The short adaptation time indicates an effective urinary excretion of Maillard reaction products. In the main study, six commercially processed dry and six moist diets were fed to 12 adult female cats in two parallel randomized, 36-day, balanced Latin square designs. Urine was collected quantitatively and FL, CML and LAL were analysed in foods and collected urine using HPLC-MS. Daily urinary excretion of FL and CML showed a positive relationship with daily intake in the dry and moist foods. For LAL, no significant relationship was observed. The observed increase in urinary excretion with increasing dietary intake indicates that dietary Maillard reaction products are absorbed from the gastro-intestinal tract of cats and excreted in the urine. Minimum apparent absorption based on urinary excretion (assuming 100% of the excreted component originates from the diet) of FL, CML and LAL was found to range between 8 to 23%, 25 to 73% and 6 to 19%, respectively. Urinary recovery (% ingested) showed a negative relationship with daily intake for FL, CML and LAL in the dry foods and for CML and LAL in the moist foods. The observed decrease in urinary recovery with increasing intake suggests a limiting factor in digestion, absorption, metabolism or urinary excretion. The studies reported in this thesis are one of the first to determine Maillard reaction products in pet foods and the bioavailability of FL, CML and LAL in cats. In addition, the results highlight the importance of reactive lysine measurement in foods for growing dogs used as weaning diets. Contribution of the absorption of dietary Maillard reaction products to the pathogenesis of various health conditions requires further study, as well as the potential role of restriction of dietary Maillard reaction products in prevention and treatment of long-term health implications. Extrusion and pelleting processing do increase the Maillard reaction, however, choice of ingredients appears to have a larger effect on the content of Maillard reaction products and can, therefore, be a useful strategy for pet food manufacturers that want to decrease the content of Maillard reaction products in their pet foods.

To understand the differences in peptide composition that result from variations in the conditions of enzymatic hydrolysis of proteins (e.g. substrate concentration) the mechanism of hydrolysis needs to be understood in detail. Therefore, methods and tools were developed to characterize and quantify the peptides formed during enzymatic protein hydrolysis. The information obtained was used to introduce a novel quantitative parameter: the selectivity of the enzyme towards the individual cleavage sites in the substrate, within the given specificity of the enzyme applied. The selectivity describes the rate of hydrolysis of a cleavage site compared to the rate of hydrolysis of all cleavage sites in the parental protein. Large differences in the selectivity of the enzyme towards the cleavage sites after the same type of amino acid residues in a protein were found. For β-lactoglobulin hydrolyzed by Bacillus licheniformis protease the selectivity was found to vary between 0.003 % and 17 % or even 0 for some cleavage sites. The effects of increasing substrate concentration and pH on the hydrolysis were studied. An increase in substrate concentration results in lower kinetics of hydrolysis, related to the available amount of water. This also resulted in significant changes in the enzyme selectivity towards the cleavage sites for which the enzyme has a high selectivity. Changing the pH of hydrolysis resulted in large changes in the kinetics of hydrolysis as well as in the enzyme selectivity. Due to the detailed analysis of the peptide composition, certain a-specific peptides were identified. It was shown that these originate from spontaneous cleavage of formed peptides. The changes in the mechanism of hydrolysis were compared to simulation data. The simulation data were obtained from a stochastic model based on random selection of the substrate and the cleavage site, given the specificity of the enzyme. A quite good agreement was obtained between simulated and experimental data. The parameters and methods developed in this study to describe the mechanism of hydrolysis can potentially be used for more complex systems.

In last two decades, enzymatic cross-linking of proteins has a growing interest in food technology for better tailoring protein functionality. However, the relation between physical and functional properties of enzymatically cross-linked proteins has been hardly addressed so far. The aim of this thesis was to elucidate the effect of enzymatic protein cross- linking on the physical and functional properties of protein nanoparticles at multiple lengthscale. In the first part of this thesis, as a model system, the enzymatic cross-linking of globular whey protein apo-α-lactalbumin (α-LA) by horseradish peroxidase (HRP) was discussed in details. In comparison with HRP, in the second part of the thesis, we also addressed to what extent both laccase (LC) (from trametes versicolor) and tyrosinase (TYR) (from agaricus bisporus) differ in catalyzing oxidative cross-linking of α-LA. Both HRP and LC were capable of creating self-similar large α-LA nanoparticles that have an open architecture at similar lengthscales whereas TYR led to the formation of α-LA oligomers only. All HRP-, LC-nanoparticles and TYR-oligomers exhibited a high extent of secondary structure content preserved whereas their almost all tertiary structure was lost upon enzymatic cross-linking. HRP-catalyzed cross-linking of α-LA resulted in more hydrophilic nanoparticles than LC-cross-linked α-LA nanoparticles. Whereas both HRP- and LC-nanoparticles exhibited very high colloidal and thermal stability against protein aggregation at pH 5.8 and 7.0, HRP- nanoparticles were more stable than LC-nanoparticles upon heating and in the presence of dithiothreitol (DTT). This suggests that, unlike HRP- nanoparticles, not only the dityrosine bonds but also disulfide cross-linking stabilizes LC-nanoparticles. Dilute dispersions of HRP-nanoparticles exhibited a high viscosity and a hydrophilic nature. As these dispersions were concentrated, they jammed above their critical overlapping concentration and thus created physical transparent protein hydrogels at relatively low protein concentration (4% w/v). These properties of HRP- nanoparticle dispersions offer high thickening properties that are comparable with polysaccharide in food applications as protein-based thickeners.

In this thesis, the effect of molecular properties on the aggregation and flocculation behaviour is studied. The aggregation behaviour was thought to be mainly affected by the structural stability of the protein. A decreased structural stability results in unfolded proteins which are more prone to aggregation. The flocculation behaviour was shown to be affected by the adsorbed amount at saturation and the adsorption rate. These parameters have been combined in a surface coverage model, which describes the stabilization of emulsions away from the iso‑electric point (pI) to be affected by excess protein in the continuous phase. In addition, a model was proposed for the prediction of the adsorbed amount at saturation. This is influenced by the protein charge and radius and system conditions (i.e. pH and ionic strength). The adsorption rate, which is a measure for the affinity of the protein towards the adsorption to the interface, was shown to increase with increasing relative exposed hydrophobicity and a decrease of the electrostatic repulsion (i.e. decrease of ionic strength or the protein charge). Close to the pI, the applicability of protein-stabilized emulsions is limited. Hence, a steric interaction was introduced to stabilize the emulsion. It was shown that glycation of the protein with a trisaccharide was sufficient to sterically stabilize the emulsions against pH-induced flocculation.

In this thesis, the mild isolation of an algae soluble protein isolate (ASPI) and the characterisation of its techno-functional properties are described. The ASPI was isolated from the green microalgae Tetraselmis sp. by beadmilling and subsequent anion exchange adsorption. The isolate obtained contained 59 ± 7% (w/w) protein and 20 ± 6% (w/w) carbohydrates, the latter composed for approximately one fourth of uronic acids (4.8 ± 0.4% [w/w]). In the pH range 5.5 – 6.5, in which currently used legumin seed protein isolates (e.g. soy) show low solubility, ASPI retained high solubility independent of ionic strength. In the soluble pH range, the foam stability of ASPI is superior to the foam stabilities of whey protein isolate (WPI) and egg white albumin (EWA). At pH 7, ASPI stabilized foams are 1.7 times more stable than WPI stabilized foams. Further fractionation of APSI results in foams even 3 times more stable than WPI stabilized foams. In addition, emulsions stabilised with ASPI are stable against droplet aggregation around pH 5 at low ionic strength, while emulsions stabilised by WPI are not stable at this pH. The stability of ASPI emulsions at this pH is attributed to the co-adsorption of the charged polysaccharide fraction present in ASPI. The role of the charged polysaccharides on stabilisation of the emulsions was confirmed by fractionating ASPI into protein-rich and charged polysaccharide-rich fractions. The combination of charged polysaccharides and proteins in ASPI results in good techno-functional properties that are between that of pure proteins and that of the naturally occurring protein-polysaccharide hybrid gum arabic (GA). It is concluded that ASPI represents an attractive substitute for currently used high-value food protein isolates. Due to the combination of the positive interfacial properties of its protein fraction with the broad pH stability of its charged polysaccharide fraction, ASPI possesses the positive attributes of two types of techno-functional ingredients.

In this thesis the effects of peptides, or protein hydrolysates on the heat-induced aggregation and gelation of (concentrated) protein systems were studied. First, it was investigated if specific peptides could influence the heat-induced denaturation and aggregation of intact proteins solutions, and which peptide properties dominated the different interactions. Next, the effects of the peptides on the heat-induced gelation of intact proteins as a model for a potential high protein food system were studied. It was found that certain peptides in the hydrolysate show binding to native proteins, and some additional peptides bind to unfolded proteins. Since the same peptides were shown to bind to not only β-lactoglobulin, but also other to proteins, it is concluded that the binding does not depend on specific molecular details of the protein. The hydrophobicity and charge were found to be important in determining the binding and the effect on aggregation. With the hydrolysates, as well as with two synthesized peptides (modelled on those found in the hydrolysate) it was confirmed that the addition of these binding peptides has significant effects on the heat-induced aggregation. In the gelation experiments performed in this study a dominant effect was found for peptides containing free SH groups. While it is expected that the changes in aggregation behaviour, induced by the binding of non-cysteine-containing peptides also affects the gel properties, this was not found with the techniques used. Finally, disulfide-containing peptides were found to reduce the presence of sulfurous volatiles formed after heating of β-lactoglobulin, WPI and lysozyme. Since only certain peptides exhibit binding to intact proteins, it is expected that control over the hydrolysis process and, thereby the concentrations of such specific peptides, can be used to produce hydrolysates with specific functionalities in this respect.

The research presented in this thesis aimed at controlling the horseradish peroxidase-catalyzed cross-linking of bovine α lactalbumin and the implications of this cross-linking for the foam stabilizing properties. Attention is also given to microreactors and their potential to control the enzymatic cross-linking of proteins. The proportion of cross-linked α lactalbumin dimers, oligomers and polymers could be directed by variations in ionic strength, pH, H2O2, and temperature. Covalent α lactalbumin dimers were proteolytic digested. FTMS analysis of the peptide mixture resulted in the unambiguous identification of a Tyr18 Tyr50 dityrosine cross-link. Structural modeling of the α lactalbumin dimer indicated that favorite electrostatics direct the selectivity of the cross-linking reaction and, hence, the formation of an intermolecular cross-link. The formation of the Tyr18-Tyr50 cross-link suggests that further cross-linking of α lactalbumin dimers enables the formation of linear polymers. A microreactor system was set up to obtain control over the reaction conditions to cross-link proteins. The enzymatic cross-linking of α lactalbumin was analyzed as a function of enzyme and substrate(s) feed. The increase in absorption at 318 nm due to dityrosine formation was found to be directly correlated to the decrease in monomeric α lactalbumin and was shown to be a good tool to monitor the cross-linking reaction. The α lactalbumin oligomers produced were investigated for their foam stabilizing properties. Cross-linked α lactalbumin oligomers did not stabilize foams, whereas α lactalbumin polymers acted as an anti-foam, destabilizing other protein films.