Master of Science / Department of Food Science / Karen A. Schmidt / People who suffer from swallowing disorders are diagnosed with dyphasgia. The beverage for the dyphagia patients should have the apparent viscosity in the range of nectar-like (51 to 350 mPa•s) or honey-like (351 to 1750 mPa•s). Due to the swallowing problems, dysphagia patients usually consume beverages slowly. Thus, the apparent viscosity of beverage for such patients should be high enough to be in the suitable range during the entire time of consumption. Three ratios of α-lactalbumin (α-la)/β-lactoglobulin (β-lg) (3:8, 1:1 and 8:3) were used to prepare the milk systems. These ratio adjusted milk systems were either processed at 70, 80, and 90ºC for 30 min or at 25ºC, and cooled to 25 ± 1ºC. After the process was completed, the milk systems were set quiescently 120 min at 25 ±1ºC. Physical and chemical properties were assessed at various time. For the milk systems at 0 min, the apparent viscosity increased in all 90°C processed-samples, and the increase was in the order of 8:3 (15.96%), 1:1 (6.38%) and 3:8 (2.11%) compared with the 25ºC samples at each ratio. When the milk systems set for 120 min, apparent viscosity increased slightly by 3.7%. The maximum apparent viscosity was 2.18 mPa•s, which was less than nectar-like. Therefore, xanthan gum was added at 0.15 w/w % to enhance rheological properties of the milk systems. α-La/β-lg ratio adjusted milk systems either with or without xanthan gum were prepared, and processed at 90ºC or 25ºC, and cooled to 25 ± 1ºC. Apparent viscosity increased by 48.61 and 89.61% in 3:8 and 8:3 milk systems, respectively for those at 0.15% xanthan gum concentration and processed at 90ºC compared with at 25ºC. Apparent viscosity of 8:3 milk systems at xanthan gum concentration of 0.15% processed at 90°C was 58.7 ± 2.12 mPa•s which was within the nectar-like range. When the samples were set for 120 min, no changes were found in the apparent viscosity of the milk systems. If the rheological properties of the milk systems can be controlled by ingredients interactions, this can be used to develop nutritious products with different forms for dysphagia patients.

In this thesis, mechanisms for enhancing the stability of whey protein emulsions using two approaches were investigated. First, the physicochemical and emulsifying properties of whey protein isolate (WPI), and its two main proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (β-LG), were investigated in response to changes in pH and temperature pre-treatments. Solvent conditions which inhibit protein aggregation, such as pHs away from the isoelectric point, were found to form stable emulsions. In contrast, thermal treatments were found to negatively affect emulsion stability, where the most stable emulsions for WPI, ALA and β-LG were formed at room temperature (i.e. 25°C) at pH 7.0. It was also determined that emulsions formed using WPI, ALA and β-LG were stabilized by electrostatically repulsive forces which prevent flocculation and creaming. Secondly, the use of tailored protein-polysaccharide interactions involving WPI and carrageenan (CG) were explored as a means of enhancing emulsion stability. Carrageenan (CG) partakes in electrostatic attraction with WPI when acidified, leading to the formation of coupled gel networks. CG was selected for its anionic properties and for its well-characterized structure in that kappa-, iota- and lambda-type CG contain 1-, 2- and 3-sulfated groups per disaccharide repeating unit respectively. WPI-CG mixtures formed gel networks once acidified, where WPI-kappa-CG and WPI-iota-CG mixtures formed stiff networks, whereas WPI-lambda-CG formed a weak fluid network. WPI-CG complexes were found to be surface active, causing changes to the interfacial tension and interfacial rheology at pHs corresponding to where electrostatic attraction occurs upon acidification. Electrostatically coupled gel networks were formed in an emulsion, where oil droplets became entrapped within the biopolymer matrix. WPI-CG mixtures were sensitive to WPI-CG mixing ratio as stiffer gels were formed at higher CG content. Furthermore, WPI-iota-CG gels were stiffer than those made with WPI-kappa-CG gels presumably due to the higher number of sulfated groups lending greater opportunities for iota-CG to form bonds with neighboring polymers compared to kappa-CG.

Whey is a by-product of cheese manufacture, containing a mixture of proteins of commercial value, each having unique attributes for nutritional, biological and food ingredient applications. A tremendous amount of whey, normally treated as a waste product, is produced worldwide each year. This work describes the cation-exchange adsorption of the two major whey proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) with the purpose of optimising a process for isolating them from whey. Adsorption of pure BLG and ALA was studied onto SP Sepharose FF using 0.1M acetate buffer. Batch experiments were carried out at various pH values for ALA and BLG, and the relevant Langmuir isotherm parameters, dissociation constant, Kd, and maximum binding capacity, qm, were determined. The optimum pH for separation was chosen to be pH 3.7. At pH 3.7, both Kd and qm pertaining to ALA were found to have higher numerical values than those of BLG, implying different characteristics of adsorption of the two proteins on this adsorbent. The Kd for the former protein was almost four times larger than the latter, while qm was 1.3 times higher. Packed-bed column adsorption was performed using a 1-ml column at pH 3.7, flow rate 1 ml/min and initial concentration of 3 mg/ml for BLG and 1.5 mg/ml for ALA both in 0.1M sodium acetate buffer. The t1/2 for the resulting ALA breakthrough was 75% longer than its BLG counterpart. The above results suggest the possibility of the occurrence of competitive adsorption between the proteins when adsorbed simultaneously. In traditional batch uptake experiments, the kinetic rate constants of ALA and BLG in both the single- and two-component systems were determined using the simple kinetic model. The values so obtained implied that BLG was adsorbed faster than ALA. In the confocal laser scanning microscopy experiments, the different behaviour of ALA and BLG in the single-component system with regard to their penetration within the adsorbent beads suggested that the two proteins have different transport mechanisms governing their adsorption. The two-component system results showed that ALA was able to displace BLG in spite of the lower affinity of the former protein to the adsorbent. The packed-bed adsorption and elution of a mixture of ALA and BLG were then investigated under the above conditions but using a 5-ml column. BLG breakthrough occurred first, and its concentration in the outlet exceeded its feed value by 1.6 fold before declining to the feed value, followed by the breakthrough of ALA. ALA displaced and eluted all the BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. The single- and two-component breakthrough curves for ALA and BLG were simulated by the simple kinetic model using the isotherm parameters, but the overshoot phenomenon could only be predicted after correcting these parameters. The evidence of the competitive nature of adsorption observed in binary mixtures was used to develop a facile separation procedure for the two proteins from aqueous solutions of whey concentrate powders. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of the novel separation process, and there was agreement between the experimental and theoretical results.

Le repliement des protéines est un des problèmes centraux de la biologie. Il s'agit de comprendre comment la chaîne polypeptidique d'une protéine se replie pour acquérir sa structure tridimensionnelle biologiquement active. Il a été démontré dans les années 60 que la forme repliée de la protéine est le plus stable d'un point de vue thermodynamique et qu'il est défini par la structure primaire. La réaction de repliement correspond ainsi à la dernière étape de l'utilisation de l'information contenue dans l'ADN. Cependant, Il est possible que les protéines se replient mal et interagissent entre elles pour former des fibres amyloïdes. Ce sont des agrégats structurés impliqués dans plusieurs maladies comme la maladie d'Alzheimer, de Parkinson... <br>Ces phénomènes sont étudiés ici dans le cas de l'alpha-lactalbumine, une protéine du lait qui possède un site de liaison pour le calcium. Le repliement est tout d'abord étudié en présence de métaux se liant au site du calcium. Ces expériences sont couplées à des expériences de dénaturation thermiques pour caractériser le rôle de la fixation des métaux sur les différents états de la protéine et son influence sur la cinétique de repliement.<br>La réaction est ensuite caractérisée en absence d'ion métallique. Elle est alors beaucoup plus lente et complexe. Différentes techniques spectroscopiques sont utilisées. Les résultats obtenus permettent de proposer un schéma réactionnel selon lequel un état précurseur de fibres amyloïdes est transitoirement peuplé. Enfin, pour compléter cette étude, les effets des interactions entre protéines sur la formation de fibres amyloïdes ont été étudiés pour différentes concentrations en sel.

We have developed an analytical technique for single molecule force spectroscopy (SMFS) data that avoids filtering prior to analysis and performs pattern recognition to identify distinct SMFS events. The technique characterizes the signal similarity between all curves in a data set and generates a hierarchical clustering tree, from which clusters can be identified, aligned, and examined to identify key patterns. This procedure was applied to alpha-lactalbumin (aLA) on polystyrene substrates with flat and nanoscale curvature, and bacteriorhodopsin (bR) adsorbed on mica substrates. Cluster patterns identified for the aLA data sets were associated with different higher-order protein-protein interactions. Changes in the frequency of the patterns showed an increase in the monomeric signal from flat to curved substrates. Analysis of the bR data showed a high level of multiple protein SMFS events and allowed for the identification of a set of characteristic three-peak unfolding events.

The human &alpha;-lactalbumin (&alpha;-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of &alpha;-LA in which all native cysteines have been mutated to alanine (all-Ala &alpha;-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala &alpha;-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the &alpha;- and &beta;-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala &alpha;-LA and that of two other variants ([28-111] &alpha;-LA and 4SS &alpha;-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères. / Skim milk is being used as a protective agent for mammalian semen conservation over half a century. Recently, there has been increased interest in developing extenders free of animal products. However, it is difficult to find suitable component in order to replace milk as an extender, because the mechanisms by which milk protect sperm against cooling and freezing damages during the storage is unknown. The Binder of SPerm (BSP) proteins are the major proteins of bull seminal plasma and they are harmful during sperm storage. In fact, sperm would be in contact with a large quantity of BSP proteins that induce a continuous cholesterol and phospholipids efflux from the sperm membrane during storage. When bull sperm is diluted with an extender containing egg yolk, another compound frequently used in extender, the low-density lipoproteins (LDL) present in the egg yolk prevent the binding of the BSP proteins to the sperm membrane, thus, preventing the lipid efflux from the sperm membrane induced by the BSP proteins. Our hypothesis was that milk proteins would protect sperm during storage by binding BSP proteins. First, we demonstrated by gel filtration that bovine BSP proteins could bind the milk proteins. Skim milk was fractionated into three fractions: F1 (alpha-lactalbumin and beta- lactoglobulin, the major whey proteins and kappa-casein), F2 (mainly caseins and all other milk proteins in small amounts) and F3 (salts, sugars and small peptides). Bovine BSP1 and BSP5 have more affinity for F1 as compared to BSP3 and all the BSP proteins have affinity for F2. We confirmed the interaction between bovine BSP proteins and milk proteins by isothermal titration calorimetry. The binding of BSP1 to casein micelles is characterized by an affinity constant (Ka) of 3.5 × 10^5 M-1 and of a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin (one of the major whey proteins) is characterized by a Ka of 2.4 × 10^5 M-1 and a “n” value of 0.8. These results support our contention that milk can protect sperm by preventing the BSP proteins’ binding to the sperm membrane attributable to a protein : protein interaction, while egg yolk sperm protection is attributable to a protein : lipoprotein interaction. Second, our studies showed that the homologous BSP proteins found in the boar, stallion and ram seminal plasma can bind the milk proteins. These results indicate that the mechanism of sperm protection by milk in these species should be similar to the one in bovine species. Third, we characterized the interaction between bovine BSP1 protein and LDL from hen’s egg yolk. The binding was characterized by a Ka of 3.4 ± 0.4 × 10^6 M-1 and a « n » value of 104 BSP1 per LDL particle. Our results indicated that there is difference between the mechanism of sperm protection by milk and egg yolk. We believe that the results presented in this thesis may help to create new extenders free of animal product for mammal sperm preservation in liquid or frozen state.

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères. / Skim milk is being used as a protective agent for mammalian semen conservation over half a century. Recently, there has been increased interest in developing extenders free of animal products. However, it is difficult to find suitable component in order to replace milk as an extender, because the mechanisms by which milk protect sperm against cooling and freezing damages during the storage is unknown. The Binder of SPerm (BSP) proteins are the major proteins of bull seminal plasma and they are harmful during sperm storage. In fact, sperm would be in contact with a large quantity of BSP proteins that induce a continuous cholesterol and phospholipids efflux from the sperm membrane during storage. When bull sperm is diluted with an extender containing egg yolk, another compound frequently used in extender, the low-density lipoproteins (LDL) present in the egg yolk prevent the binding of the BSP proteins to the sperm membrane, thus, preventing the lipid efflux from the sperm membrane induced by the BSP proteins. Our hypothesis was that milk proteins would protect sperm during storage by binding BSP proteins. First, we demonstrated by gel filtration that bovine BSP proteins could bind the milk proteins. Skim milk was fractionated into three fractions: F1 (alpha-lactalbumin and beta- lactoglobulin, the major whey proteins and kappa-casein), F2 (mainly caseins and all other milk proteins in small amounts) and F3 (salts, sugars and small peptides). Bovine BSP1 and BSP5 have more affinity for F1 as compared to BSP3 and all the BSP proteins have affinity for F2. We confirmed the interaction between bovine BSP proteins and milk proteins by isothermal titration calorimetry. The binding of BSP1 to casein micelles is characterized by an affinity constant (Ka) of 3.5 × 10^5 M-1 and of a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin (one of the major whey proteins) is characterized by a Ka of 2.4 × 10^5 M-1 and a “n” value of 0.8. These results support our contention that milk can protect sperm by preventing the BSP proteins’ binding to the sperm membrane attributable to a protein : protein interaction, while egg yolk sperm protection is attributable to a protein : lipoprotein interaction. Second, our studies showed that the homologous BSP proteins found in the boar, stallion and ram seminal plasma can bind the milk proteins. These results indicate that the mechanism of sperm protection by milk in these species should be similar to the one in bovine species. Third, we characterized the interaction between bovine BSP1 protein and LDL from hen’s egg yolk. The binding was characterized by a Ka of 3.4 ± 0.4 × 10^6 M-1 and a « n » value of 104 BSP1 per LDL particle. Our results indicated that there is difference between the mechanism of sperm protection by milk and egg yolk. We believe that the results presented in this thesis may help to create new extenders free of animal product for mammal sperm preservation in liquid or frozen state.