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Item Aggregation and gelation of bovine b-lactoglobulin, a-lactalbumin and serum albumin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University(Massey University, 1995) Gezimati, JacquelineGelation is one of the most important functional properties of whey proteins in food systems. The properties of whey protein gels are affected by the chemical and physical properties of its protein components, (β- lactoglobulin AB (β-Lg), α-lactalbumin (α-La) and bovine serum albumin (BSA). Heat-induced aggregation and gelation of individual whey proteins, (β-Lg, α-La and BSA and in mixture was studied by dynamic rheology and electrophoresis analysis. The proteins were dispersed in an ionic buffer containing 0.009 M CaCl , 0.012 M NaCl, 0.012 M K HPO and 0.007 M Nacitrate (pH 6.8) which was comparable to the ionic composition of 12% whey protein concentrate solution. Rheological properties of the protein solutions were measured using a Bohlin VOR rheometer after heating to 70, 75 and 80°C, holding at these temperatures for 60 min and after cooling to 25°C. Gel electrophoresis under non-dissociating (Native-PAGE in the absence of dissociating and reducing agents) and dissociating but non-reducing conditions (SDS-PAGE) was used to determine the extents of aggregation in some of the heated protein samples. Gelation temperatures of 10%, w/v, protein solutions were found to be in the range 82.5 - 84°C for β-Lg and 68 - 70°C for BSA while α-La did not gel even at 90°C. Gelation temperatures of protein mixtures containing β- Lg and BSA were dependent on the relative proportion of the two proteins in the mixture. In contrast, the protein mixtures containing β-Lg and α-La gelled at temperatures (~ 83°C) comparable to that of β-Lg alone. Rheological measurements on pure β-Lg and BSA showed that BSA solutions formed self-supporting gels at lower protein concentrations and lower temperatures. Increasing the heating temperature or protein concentration of either β-Lg or BSA resulted in higher values of the storage modulus (G'). It was apparent from the electrophoretic data that protein aggregates were formed as an intermediate prior to the formation of gel net-work. These aggregates appeared to be non-covalently linked initially and became increasingly disulphide-linked during heating. Analysis of mixtures containing β-Lg and BSA during heat treatment showed that at both 70 and 75°C the gelation time decreased with the increasing proportion of BSA. Similarly, the values of G' after 60 min of heating were greater for the gels containing more BSA. G' values of these mixtures were dependent on the heating temperature and the relative proportion of the two proteins. Gel electrophoresis data for a mixture of 5% β-Lg and 5% BSA heated at 70°C showed that prior to gelation most of the BSA had been transformed into aggregates while most of the β-Lg was essentially in the native form. Aggregates of both β-Lg and BSA were formed during heating at 75°C. At both temperatures, gelation commenced after most of the BSA had become covalently cross-linked but before all the β-Lg had become cross-linked. This effect was also apparent for other mixtures. Initially the aggregates appeared to be non-covalently linked and became increasingly disulphide linked with heating. From these results it is apparent that during heating at 70°C, BSA is the main protein forming the gel net-work and some β-Lg aggregates are probably attached to the net-work strand through either hydrophobic interactions or disulphide linkages. During heating at 75°C, two gel net-works are presumed to be formed independently, again with some interactions between the strands of the two net-works. The rheological properties of protein mixtures containing β-Lg and α-La showed that β-Lg was the dominant gelling protein. G' values decreased with increasing relative proportion of α-La in the mixture at both 75 and 80°C. Gelling times increased with increasing proportion of α-La in the mixture at both 75 and 80°C. No aggregate formation was observed during heating of α-La at 75 or 80°C. However, in the presence of β-Lg, α-La aggregated rapidly during heating. This aggregation appears to involve sulphydryl disulphide interchange reactions particulary when the mixtures were heated at 80°C. Almost all the proteins had aggregated through disulphide linkages before any significant increase in G'. It is suggested that during heating and prior to gelation co-polymers of both β-Lg and α-La were formed and this resulted in heterogeneous net-work strands being formed. The results presented in this study suggest that slight differences in the protein composition of WPC are unlikely to affect the gelation properties of WPC. Further studies into the effects of immunoglobulins (Igs) are needed in order to gain further understanding of the contributions of these proteins to rheological properties of WPC gels.Item Whey protein and satiety in humans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Manawatu campus, Palmerston North, New Zealand(Massey University, 2013) Chung Chun Lam, Sylvia Mee SiongProtein is the most satiating macronutrient and there is an effect of dietary protein source, with dairy whey protein being particularly effective in promoting satiety in adult humans. The underlying cause for this remains to be elucidated. The objectives were to confirm that whey protein is more satiating than maltodextrin carbohydrate in adult humans, to understand the potential mediating factors and to investigate which characteristic of whey protein gives rise to its satiating effect. Ad libitum food intake at a subsequent test meal after administration of a preload, subjective feelings of appetite (using visual analogue scales) and plasma concentrations of satiety-related hormones and metabolites were determined. Preload diets enriched with whey protein induced a greater reduction in subsequent food intake and suppression in rated feelings of appetite compared with maltodextrin carbohydrate (p<0.05). The time of consumption of the whey protein preload did not influence the satiety response. Plasma concentrations of pancreatic polypeptide hormone, total amino acids, and the branched-chain amino acids appear to play an important role in mediating the satiating effect of whey protein (sustained increases from 15 to 120 min following preload consumption, p<0.05). To determine the underlying characteristic of whey protein causing the satiating effect, the effects on satiety of whey protein components (glycomacropeptide, alpha-lactalbumin, or beta-lactoglobulin) and a free amino acid mixture simulating the amino acid composition of the whey protein were compared with that of the intact whey protein. The amino acid composition of whey protein per se appears to be important in the regulation of food intake and the induction of satiety. The individual constituent proteins or whey protein itself did not promote higher satiety than that found based on providing the free amino acids. The absorbed amino acid profile would appear to play an important role in mediating the satiating effect of whey protein.Item Studies on heat-induced interactions and gelation of whey protein : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology(Massey University, 1998) Havea, PalatasaThe purpose of this study was to gain greater understanding of the interactions of whey proteins during heat-induced gelation of whey protein concentrate (WPC) solutions. Attention was focused on gaining better knowledge of the relationship between composition of WPC and its ability to form heat-induced gels, and to explore the mechanisms of protein aggregation and gelation in WPC solutions. Interactions of whey proteins (β-lactalbumin, α-lactalbumin and BSA) were studied in three types of commercial WPC (rennet, cheese and acid) solutions, as well as in pure protein model systems, using one-dimensional (1D-) and two-dimensional (2D-) polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography, light scattering and ultracentrifugation. The formation and structure of aggregates and gels were determined by oscillatory rheometry, confocal scanning laser microscopy (CSLM) and transmission electron microscopy (TEM) techniques. Examination of heated (75 °C) rennet WPC solutions at a range of concentrations (10-120 g/kg, pH 6.8) revealed that the extent of protein aggregation and the formation of the intermediate molecular weight products were concentration-dependent. The rates of loss of β-lactoglobulin, α-lactalbumin and BSA during heating increased as the WPC concentration was increased from 10 to 120 g/kg. 2D-PAGE showed that some disulphide-linked β-lactoglobulin dimers were present in heated 10 g/kg solution, but very little was present in heated 120 g/kg solution. SDS was able to dissociate monomeric protein from high molecular weight aggregates in heated 120 g/kg WPC solution but not in 10 g/kg WPC solution. This suggested that in addition to disulphide-linked aggregates, hydrophobic aggregates involving β-lactoglobulin, α-lactalbumin and BSA were formed in heated WPC solutions at high protein concentrations. Examination of the heated acid WPC and cheese WPC solutions (120 g/Kg), using 1D-PAGE and size exclusion chromatography, revealed that the loss of β-lactoglobulin, α-lactalbumin and BSA from the cheese WPC solution was faster than the loss of the same proteins from the acid WPC solutions. It was also found that a considerable proportion of aggregates formed in heated cheese WPC solution was linked by hydrophobic association, whereas the aggregates formed in heated acid WPC solutions were linked predominantly by disulphide bonds. TEM and CLSM showed that the aggregates formed in cheese WPC solution were relatively large and "particulated," whereas the aggregates formed in acid WPC solution were small and "fine stranded." The gels formed from the heated cheese WPC solutions had low gel strength and high syneresis, whereas the gels obtained from the acid WPC had high gel strength and good water holding capacity. Results of the dialysis experiments revealed that the differences between the properties of the acid WPC and the cheese WPC gels could be explained largely by their different mineral compositions. Relatively higher concentrations of divalent cations, Ca and Mg, in the cheese WPC was considered to be responsible for high rates of loss of native-like proteins, and the formation of large, hydrophobically-associated and "particulated" aggregates. High concentrations of monovalent cation in the acid WPC solutions probably resulted in slower loss native-like proteins and formation of small and 'fine-stranded" aggregates. Attempt was made to characterise the nature of "insoluble" material in the unheated acid and cheese WPC solutions. Although, both the acid and the cheese WPC solutions contained considerable amounts of "insoluble" material, the amounts in the cheese WPC were greater. This material contained disproportionately higher levels of aggregated BSA and the minor whey proteins; in the cheese WPC it also contained considerable amounts of aggregated β-lactoglobulin and α-lactalbumin as well as phospholipids. The "insoluble" material in acid WPC, had higher casein content. The presence of this material did not appear to affect the gelation characteristics of the cheese WPCs, but had a positive effect on acid WPC gelation. Studies on model systems of pure proteins showed that β-lactoglobulin, α-lactalbumin and BSA interacted to form homogeneous aggregates of each other as well as heterogeneous aggregates. 2D-PAGE clearly showed that when a mixture of these proteins was heated, initially BSA formed aggregates with itself and β-lactoglobulin and α-lactalbumin formed co-aggregates at a later stage of heating. Based on these results, the structure of WPC gel was suggested to be a heterogeneous network formed largely by co-polymers of β-lactoglobulin and α-lactalbumin embedded with "clusters" or "strands" of BSA aggregates. Based on the results of this study, recommendations are made on how this information can be used in the development of new or improved whey products.Item Rheology of whey protein solutions and gels : thesis submitted for the degree of Doctor of Philosophy in Food Technology at Massey University, New Zealand(Massey University, 1993) Tang, QingnongThe use of whey protein products in foods is governed by their nutritional and functional properties. Whey protein products have increasingly been applied in a variety of food systems as functional ingredients. In order to boost applications of whey protein products and to improve, predict and control their functional attributes in food products knowledge is required about how they behave functionally under different conditions, e.g. when product composition, processing history, protein concentration, pH, salt concentration and temperature vary. The flow properties of whey protein concentrate solutions were studied in a Bohlin rheometer. The effects of protein concentration, temperature, pH and salts on the gelation and gel properties of whey protein concentrates and whey protein isolate were also investigated in the same rheometer. Differences in gelation between whey protein concentrates, whey protein isolate, egg white and B-lactoglobulin were studied. Differences between dynamic shear properties determined in a Bohlin rheometer and fracture properties determined in an Instron universal testing machine were also studied. The flow properties of whey protein concentrate solutions changed from Newtonian to pseudoplastic or even thixotropic behaviour, owing to structure formation in the solutions, i.e. to increases in protein intermolecular interactions. Such structure formation resulted from increases in protein concentration, temperature or CaC12 concentration, and from shifting the pH to extreme values. Gelation of whey protein was dependent on protein concentration, gelation temperature, pH, salt content and lactose content. Salt content was the most important factor in determining the gelling properties of various whey protein concentrate products and whey protein isolate. Consistent gelling properties could only be achieved when salt content was carefully controlled. The degree of protein denaturation and lactose content also led to differences in gelling behaviour of whey protein concentrates. Whey protein products, when compared with ·egg white, had a higher gelation temperature, a higher minimum protein concentration for gelation, lower initial gelation rate and lower gel stiffness. The differences in initial gelation rate and gel stiffness could be compensated by adjustment of the salt content of whey protein products. Dynamic viscoelastic measurements on whey protein isolate gels in the region of the sol-gel transition exhibited simple power law relationships between the storage (G' ) and loss (G") moduli and frequency as G' oc ro0·54±0.o2 and G" oc ro051±0.o2 , indicating that the gel in the region of the sol-gel transition could have the geometry of a fractal. The critical exponents calculated from the protein concentration dependence of gelation time and from the site percolation model indicated that the gelation of whey protein is a realization of a percolation process. Compression rigidity modulus (Ec), penetration rigidity (EP), tension rigidity (EJ and storage modulus G' all exhibited a similar pattern of variation with pH. G' , Ec, EP and Ev which were not closely related to the fracture properties and hardness of whey protein concentrate gels, were controlled by electrostatic interactions. The fracture forces and hardness were determined by both disulphide bonds and electrostatic interactions, while fracture strains were mainly controlled by disulphide bonds.Item Whey protein nanofibrils: kinetic, rheological and morphological effects of group IA and IIA cations(International Dairy Journal, 2012) Loveday, SM; Su, Jiahong; Rao, M. Anandha; Anema, Skelte G.; Singh, HarjinderSelf-assembly of whey proteins into amyloid-like fibrils during heating at pH 2 and low ionic strength is sensitive to the presence of NaCl and CaCl2. Our earlier work established that at 10 - 120 mM of these salts speeds up self-assembly and favours short, flexible fibrils over long semiflexible fibrils in a way that depends on cation concentration and cation type. Here we explored how other mono- and divalent salts affected fibril morphology and the rheology of fibril dispersions. Divalent salts (MgCl2, CaCl2, BaCl2) had much stronger effects than monovalent salts (LiCl, NaCl, KCl) on gelation kinetics, and differences between salts of the same type were not large. No marked effects of salt type on fibril morphology were evident, but there were subtle differences in the size and extent of fibril networks with mono- vs. divalent salts, which may explain differences in bulk rheology.Item Investigating astringency mechanism of WPI8855 in acidic condition : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2011) Sun, XiaoliWhey protein isolate is used as a functional ingredient in acidic whey protein beverages, but the associated astringency is a big hurdle to introduce these beverages into the mainstream market. If we can solve the astringency issue, Fonterra would have big advantages over their competitors. Our hypothesis is that whey protein interacts with human saliva proteins and the subsequent precipitation causes astringency. In the present study, ion exchange whey protein isolates (WPI) 8855, and solutions of pure a-lac and ß-lg were used to determine which whey protein fractions are responsible for sedimentation in artificial or human saliva. It has been shown that sedimentation correlates to the level of astringency. Therefore only the level of sedimentation was investigated. The human saliva and artificial saliva were also compared in the astringency titration model in order to determine whether artificial saliva is representative of human saliva. Heat treatment (85°C, 30s) of whey protein solution was performed to mimic commercial beverage manufacture. The heated and non-heated whey protein solutions were titrated with artificial saliva, human saliva or sodium bicarbonate buffer in the range of pH 3 to 6. The sediment was recovered by centrifugation of the titrated samples, and analysed using liquid chromatography-mass spectrometry (LC-MS/MS) or one and two dimensional polyacrylamide gel electrophoresis (PAGE) with amido black and periodic acid Schiff stain. This study showed that ß-lg is the key sedimentation component in heated acidic WPI8855 beverages due to the heat aggregation, pH change through the isoelectric point and interaction with human saliva proteins, including mucin, proline-rich proteins (PRPS) and a-amylase. BSA also interacted with artificial and human saliva, whereas a-lac did not interact with either artificial or human saliva. Heat treatment caused extensive whey protein aggregation and precipitation. Artificial saliva and human saliva behaved differently in this astringency titration model, therefore it is not recommended to use artificial saliva in an in vitro model to predict astringency in vivo. Artificial saliva interacted with whey protein and caused additional precipitation compared to titration with sodium bicarbonate, whereas human saliva was able to hinder some whey protein sedimentation caused by titration with sodium bicarbonate. If astringency is caused by the amount of precipitation of protein, heat treatment would be a major factor in the astringency of whey proteins.Item β-Lactoglobulin nanofibrils: Effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions(Elsevier Ltd, 2012-05) Loveday SM; Wang XL; Rao MA; Anema SG; Singh HAlmost all published studies of heat-induced b-lactoglobulin self-assembly into amyloid-like fibrils at low pH and low ionic strength have involved heating at 80 C, and the effect of heating temperature on self-assembly has received little attention. Here we heated b-lactoglobulin at pH 2 and 75 C, 80 C, 90 C, 100 C, 110 C or 120 C and investigated the kinetics of self-assembly (using Thioflavin T fluorescence), the morphology of fibrils, and the rheological properties of fibril dispersions. Self-assembly occurred at all temperatures tested. Thioflavin T fluorescence increased sigmoidally at all temperatures, however it decreased sharply with >3.3 h heating at 110 C and with >5 h heating at 120 C. The sharp decreases were attributed partly to local gelation, but destruction of fibrils may have occurred at 120 C. Thioflavin T fluorescence results indicated that maximal rates of fibril formation increased with increasing temperature, especially above 100 C, but fibril yield (maximum Thioflavin T fluorescence) was not affected by temperature. At 100 C and 110 C, fibrils were slightly shorter than at 80 C, but otherwise they looked very similar. Fibrils made by heating at 120 C for 1 h were also similar, but heating at 120 C for 8 h gave predominantly short fibrils, apparently the products of larger fibrils fragmenting. Heating at 100 C gave consistently higher viscosity than at 80 C, and heating for >2 h at 120 C decreased viscosity, which may have been linked with fibril fragmentation seen in micrographs.Item Immunomodulatory properties of bovine whey proteins and whey protein concentrates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science and Immunology at Massey University, Palmerston North, New Zealand(Massey University, 2004) Low, Pauline Ping LinIn recent years, partly due to advances in protein separation technology, many studies have focused on the immunomodulatory activity of bovine milk and colostral protein components. Individual milk proteins have been purified for the purpose of studying their physical properties and physiological functions. Today there is substantial evidence to indicate that the major components of bovine milk, such as whey protein and several highly purified whey protein isolates, can regulate immune function in heterologous species. Intense research has focused on identifying biologically active components within bovine milk whey, as well as characterising the mode by which mammalian immune function is modulated by these components. However, information regarding the effect of bovine whey proteins on immune responses to orally and parentally-administered antigens is currently conflicting and far from exhaustive. Consequently, this thesis sought not only to investigate the immunomodulatory ability of previously untested bovine whey products on general immunoresponses but also to investigate the ability of bovine whey proteins to modulate murine immune responses to vaccines currently in routine medical use. Initially, individual whey proteins (α-lactalbumin, β-lactoglobulin and lactoferrin) were screened for their in vitro effects on lymphoid cell function and phagocytic function. These in vitro studies found that the individual whey protein components had a positive immunomodulatory effect, providing evidence that these components have the potential to enhance immune function, and investigation into their immunomodulatory capabilities in an in vivo murine model was consequently undertaken. The results of the in vivo studies demonstrated that the dietary whey protein isolates and whey protein concentrate tested in this study could not only enhance two important indices of ex vivo lymphoid and non-lymphoid cell function (lymphocyte proliferation and phagocytic function) but could enhance mucosal and systemic antibody responses to orally and systemically administered human vaccines. The demonstrated benefits to the immune system of dietary whey proteins in the murine model could result in the production of immune-boosting, nutritionally and physiologically advantageous food supplements suitable for human consumption. Of particular relevancy to modern human health is the use of whey proteins as dietary adjuvants or immunopotentiators to increase immune responses to commonly administered vaccines.