Fibril assembly in whey protein mixtures

The objective of this thesis was to study fibril assembly in mixtures of whey proteins. The effect of the composition of the protein mixture on the structures and the resulting phase behaviour was investigated. The current work has shown that beta-lactoglobulin is responsible for the fibril assembly in whey protein mixtures upon heating at pH 2 and low ionic strength. To determine the efficiency of fibril formation, a method was developed to measure the conversion of whey protein monomers into fibrils. This method was used to study the effect of heating time, protein concentration, and stirring and seeding on the formation of fibrils in whey protein isolate solutions. The results have provided a better understanding of the mechanism of fibril formation and a model for fibril formation is proposed, including an activation, nucleation, growth, and termination step. Based on the method to measure the conversion two fast assays were found to determine the fibril concentration quantitatively. These two fast assays can be used for studying the kinetics of protein assembly in general, once they have been calibrated for a certain protein

Thioflavin T and Birefringence Assays to Determine the Conversion of Proteins into Fibrils

The conversion of protein monomers into fibrils can be determined using the centrifugal filtration method. The results of this method were used to calibrate steady-shear birefringence and Thioflavin T fluorescence measurements. For both measurements, a linear correlation with the fibril concentration was extracted, resulting in two fast assays to determine the fibril concentration quantitatively. From birefringence measurements and the conversion determined using the centrifugal filtration method, we were able to calculate more precise values for the birefringence per unit length of the fibrils (M) and the flexibility of the fibrils (ß

Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion method, or proton NMR spectroscopy, reported a wide range of values for the conversion. An alternative method was developed, namely centrifugal filtration, giving a consistent picture of the conversion. The present results help to explain the disparities reported in literature. No fibrils formed upon heating pure ¿-lactalbumin or pure BSA at pH 2, whereas fibrils formed in pure ß-lactoglobulin (ß-lg) and WPI solutions. Experiments indicate that ß-lg was the only whey protein involved in fibril formation. In all whey protein samples, hydrolysis occurred during heating at pH 2, as determined by HPLC and SDS-PAGE. When WPI fibrils formed at pH 2 were stored at pH 7 or 10, disulphide bonds were formed in the samples. Keywords: Heat-induced aggregation; Whey proteins; Fibrils; Conversion; HPLC; SDS-PAG

Thioflavin T and Birefringence Assays to Determine the Conversion of Proteins into Fibrils

The conversion of protein monomers into fibrils can be determined using the centrifugal filtration method. The results of this method were used to calibrate steady-shear birefringence and Thioflavin T fluorescence measurements. For both measurements, a linear correlation with the fibril concentration was extracted, resulting in two fast assays to determine the fibril concentration quantitatively. From birefringence measurements and the conversion determined using the centrifugal filtration method, we were able to calculate more precise values for the birefringence per unit length of the fibrils (M) and the flexibility of the fibrils (ß

Effect of Stirring and Seeding on Whey Protein Fibril Formation

The effect of stirring and seeding on the formation of fibrils in whey protein isolate (WPI) solutions was studied. More fibrils of a similar length are formed when WPI is stirred during heating at pH 2 and 80 C compared to samples that were heated at rest. Addition of seeds did not show an additional effect compared to samples that were stirred. We propose a model for fibril formation, including an activation, nucleation, growth, and termination step. The activation and nucleation steps are the rate-determining steps. Fibril growth is relatively fast but terminates after prolonged heating. Two processes that possibly induce termination of fibril growth are hydrolysis of nonassembled monomers and inactivation of the growth ends of the fibrils. Stirring may break up immature fibrils, thus producing more active fibrils. Stirring also seems to accelerate the kinetics of fibril formation, resulting in an increase of the number of fibrils forme

Ca2+-Induced Cold Set Gelation of Whey Protein Isolate Fibrils

In this paper we describe the rheological behaviour of Ca2+-induced cold-set gels of whey protein mixtures. Coldset gels are important applications for products with a low thermal stability. In previous work [1], we determined the state diagram for whey protein mixtures that were heated for 10 h at pH 2 at 80°C. Under these conditions, the major whey protein, ß-lactoglobulin (ß-lg), forms fibrils. When whey protein mixtures are heated at protein concentrations in the liquid solution regime of the state diagram, cold-set gels can be formed by adding Ca2+ ions at pH 7. We studied the rheological behaviour of cold-set gels for various sample compositions for whey protein mixtures. When keeping the total whey protein concentration constant, the elastic modulus, G¿, for the cold-set gels decreased for increasing a-lactalbumin and bovine serum albumin ratios, because less material (blg fibrils) was available to form a gel network. In the cold-set gels the interactions between the ß-lg fibrils induced by the calcium ions are dominant. The ß-lg fibrils are forming the cold-set gel network and therefore determine the gel strength. a-Lactalbumin and bovine serum albumin are not incorporated in the stress-bearing structure of the gel

Method for improving the functional properties of a globular protein, protein thus prepared, use thereof and products containing the protein

The invention relates to a method for improving the functional properties of globular proteins, comprising the steps of providing a solution of one or more globular proteins, in which solution the protein(s) is / are at least partially aggregated in fibrils; and performing one or more of the following steps in random order: increasing the pH; increasing the salt concentration; concentrating the solution; and changing the solvent quality of the solution. Preferably, the solution of the on or more glubular protein is provided by heating at a low pH or the addition of a denaturing agent. The invention also relates to the protein additive thus obtained, to the use thereof for food and non-food applications and to the food and non-food products containing the protein additive

Mesoscopic structure and viscoelastic properties of ß-lactoglobulin gels at low pH and low ionic strength

Abstract In this paper, we have investigated the mesoscopic structure and rheological properties of heat-set -lactoglobulin gels at low pH and low ionic strength. We have determined the scaling of the elastic or storage modulus of the gel with protein concentration. Four batches with a pH of 2 and an ionic strength of 13 mM showed a wide range of scaling behavior. In two of the batches, two concentration regimes with distinctive exponents could be detected. We constructed the phase diagram for this system as a function of pH and ionic strength, and were able to show that the wide spread in scaling behavior between the batches is the result of differences in the mescopic structure of the gels. These differences in mesoscopic structure were the result of small variations in pH and ionic strength between the batches. Our experiments show that when characterizing gel properties of -lactoglobulin solutions at very acidic conditions and low ionic strength, extreme care has to be taken to stabilize the pH of these systems. Small variations in pH have a drastic effect on the structure of the gels, and on the macroscopic viscoelastic properties

Mesoscopic structure and viscoelastic properties of ß-lactoglobulin gels at low pH and low ionic strength

Abstract In this paper, we have investigated the mesoscopic structure and rheological properties of heat-set -lactoglobulin gels at low pH and low ionic strength. We have determined the scaling of the elastic or storage modulus of the gel with protein concentration. Four batches with a pH of 2 and an ionic strength of 13 mM showed a wide range of scaling behavior. In two of the batches, two concentration regimes with distinctive exponents could be detected. We constructed the phase diagram for this system as a function of pH and ionic strength, and were able to show that the wide spread in scaling behavior between the batches is the result of differences in the mescopic structure of the gels. These differences in mesoscopic structure were the result of small variations in pH and ionic strength between the batches. Our experiments show that when characterizing gel properties of -lactoglobulin solutions at very acidic conditions and low ionic strength, extreme care has to be taken to stabilize the pH of these systems. Small variations in pH have a drastic effect on the structure of the gels, and on the macroscopic viscoelastic properties