Lawrence K. Creamer

Characterization of heat-induced aggregates of β-lactoglobulin, α-lactalbumin and bovine serum albumin in a whey protein concentrate environment

Summary. Bovine b-lactoglobulin (b-lg), a-lactalbumin (a-la) and bovine serum albumin (BSA), dispersed in ultrafiltration permeate, that had been prepared from whey protein concentrate solution (100 g}kg, pH 6‐8), were heated at 75 ∞C. The consequent protein aggregation was studied by one-dimensional (1D) and twodimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g b-lg}kg permeate solution was heated at 75 ∞C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disulphide-bonded dimers, trimers and larger aggregates. When mixtures of b-lg and a-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also observed in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of b-lg, a-la and BSA, and 1:1 disulphide-bonded adducts of a-la and b-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heattreated b-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of a-la. It is likely that when whey protein concentrate is heated under the present conditions, BSA forms disulphide-bonded strands ahead of b-lg and that a-la aggregation with b-lg and with itself is catalysed by the heat-induced unfolded BSA and b-lg.

12‐Bromododecanoic acid binds inside the calyx of bovine β‐lactoglobulin

The X‐ray structure of bovine β‐lactoglobulin with the ligand 12‐bromododecanoic acid as a model for fatty acids has been determined at a resolution of 2.23 Å in the trigonal lattice Z form. The ligand binds inside the calyx, resolving a long‐standing controversy as to where fatty‐acid like ligands bind. The carboxylate head group lies at the surface of the molecule, and the lid to the calyx is open at the pH of crystallization (pH 7.3), consistent with the conformation observed in ligand‐free bovine β‐lactoglobulin in lattice Z at pH 7.1 and pH 8.2.

Electrophoretic characterization of the protein products formed during heat treatment of whey protein concentrate solutions

Whey protein concentrate (WPC) solutions containing 10, 30, 60 and 120 g dry powder/kg were heated at 75°C and whey protein aggregation was studied by following the changes in the distribution of β-lactoglobulin, α-lactalbumin and bovine serum albumin, using one dimensional and two dimensional PAGE. The one dimensional PAGE results showed that a minimal quantity of large aggregates was formed when 10 g WPC/kg solutions were heated at 75°C for up to 16 min whereas appreciable quantities were formed when 30, 60 and 120 g WPC/kg solutions were similarly treated. The two dimensional PAGE analysis showed that some disulphide-linked β-lactoglobulin dimers were present in heated 10 g WPC/kg solution, but very little was present in heated 120 g WPC/kg solution. By contrast, SDS was able to dissociate monomeric protein from high molecular mass aggregates in heated WPC solution of 120 g/kg but not in 10 g WPC/kg solution heated for 30 min. The rates of loss of native-like and SDS-monomeric β-lactoglobulin, α-lactalbumin and bovine serum albumin during heating increased as the WPC concentration was increased from 10 to 120 g/kg. In 120 g WPC/kg solution heated at 75°C, the amounts of SDS-monomeric β-lactoglobulin in each sample were greater than the quantities of native-like protein. However, in WPC solutions of 10, 30 and 60 g/kg, the differences between the amounts of native-like and SDS-monomeric proteins were slight. The loss of the native-like or SDS-monomeric proteins was consistent with a first or second order reaction. In each case, the apparent reaction rate constant appeared to be concentration-dependent, suggesting a change of aggregation mechanism in the more concentrated solutions. Overall, these results indicate that in addition to disulphide-linked aggregates, hydrophobic aggregates involving β-lactoglobulin, α-lactalbumin and bovine serum albumin were formed in heated WPC solution at high protein concentration, as suggested by model studies using binary mixtures of these proteins.

Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.

Denaturation, aggregation and heat stability of milk protein during the manufacture of skim milk powder

The effect of preheat treatment, evaporation and drying in a commercial plant on the denaturation of βlactoglobulin and α-lactalbumin, their incorporation into the casein micelle and the heat stability characteristics of the milks and powders were determined. Preheat treatments between 110 °C for 2 min and 120 °C for 3 min denatured between 80 and 91% of β-lactoglobulin and between 33 and 45% of α-lactalbumin. Evaporation increased the extent of denaturation but spray drying did not increase it further. The incorporation of α-lactalbumin and βlactoglobulin into the micelles was markedly less than the amount that denatured and was not a constant ratio to it. Heat coagulation times at 140 °C of milks, concentrates and powders diluted to the original milk concentration were measured as a function of pH. In general, the greater the collective heat treatment, the shorter the time required to achieve coagulation. Spray drying shifted the peak positions in the pH-heat coagulation time profiles. In contrast, heat coagulation times (measured at 120 °C) of concentrates and powders diluted to 20% total solids content increased with the severity of the preheat treatment. Surprisingly, spray drying markedly increased the heat coagulation times of the diluted concentrates.

Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment

Abstract The interactions that occur when bovine α -lactalbumin ( α -La) and β -lactoglobulin B ( β -Lg) are heated were studied at neutral pH at temperatures between 55°C and 95°C. Samples of the pure proteins were heated separately, in admixture and as mixtures of α -La and preheated β -Lg. The reaction mixtures were analysed by polyacrylamide gel electrophoresis techniques and by near-UV circular dichroism. When holo- α -La was extensively heated by itself at 80°C, it slowly formed non-native monomers and dimers; the apo protein was noticeably more reactive. Mixtures of α -La and β -Lg formed 1:1 aggregates as well as non-native monomers, dimers, etc. of both α -La and β -Lg. The rate of loss of native α -La from the mixture was rapid and comparable with that of β -Lg under the same conditions. However, preheating β -Lg decreased the extent of loss of α -La from the mixtures. It was concluded that the disulphide bond shuffling that occurs during heat treatment to form the non-native species is enhanced by the formation of molten globule intermediates and by thiol catalysis.

The roles of disulphide and non-covalent bonding in the functional properties of heat-induced whey protein gels.

Heat-induced gelation (80 degrees C, 30 min or 85 degrees C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6.9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (approximately equal to 10 nm) than the control gels (approximately equal to 100 nm), whereas the DTT-gels had a more particulate structure (approximately equal to 200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G) values (approximately equal to 13,500 Pa) that were approximately equal to 25 times higher than those of the SDS-gels (approximately equal to 550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 degrees C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

Anomalous behavior of bovine αs1- and β-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Abstract Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of M r and the mobility of the protein through the gel matrix. However, bovine caseins (especially α s1 -casein) have lower mobilities than expected on the basis of their known M r . The binding of SDS to both α s1 -casein ( M r 23,600) and β-casein ( M r 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the α s1 -casein:SDS complex was larger than the β-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both α s1- and β-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13 C NMR results showed that SDS bound to α s1- and β-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of α s1 -casein increased its mobility in SDS-gel electrophoresis, but neither modification affected β-casein mobility. These and other results indicate that the low electrophoretic velocity of α s1 -casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of α s1 -casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the α s1 -casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori , it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the M r of the protein.

Some recent advances in the basic chemistry of milk proteins and lipids

Abstract Sophisticated analytical tools and techniques now are being applied to the study of the major components of milk (protein, fat, lactose and mineral interaction products). Through steady improvement, such tools have moved from the stage of exciting academic breakthroughs to become reliable work-horses. Examples are the application of genetic engineering to study the stability of β-lactoglobulin, GLC-MS to examine the composition of the fat fraction, NMR techniques to study the solution structures of casein peptides, or α-lactalbumin and X-ray crystallography to examine whey protein structure. The future challenge will be to gain understanding of mixed and modified systems involving two or more phases with a combination of dairy and non-dairy ingredients.

Thermal gelation and denaturation of bovine β -lactoglobulins A and B

Heat-induced gelation, an important functional property of β -lactoglobulin, was studied by measuring the rheological properties of both the A and B variants of the protein during and after heat treatment within a range of pH, temperature and concentration. Gel electrophoresis was used to determine the extent of denaturation and disulphide bond crosslinking of some samples. Both variants formed gel networks on heating at temperatures > 75 °C, and under most conditions the storage modulus ( G′ ) of β lactoglobulin A gels was higher than the G′ of β -lactoglobulin B gels, in particular after cooling to 25 °C. A minimum protein concentration of 50 g/1 was required for gel formation at pH 7·0 in 0·1 M-NaCl by both variants at 80 °C. Increasing the protein concentration above 50 g/1 increased G′ , the extent of increase being much greater for the A variant than the B variant. G′ of variant A gels was not much influenced by pH whereas G′ of variant B gels decreased slightly from pH 3 to pH 6 and increased between pH 6 and pH 9. When mixtures of the two variants were gelled G′ increased at the temperature of heating (80 °C) and after cooling (25 °C) as the relative quantity of variant A was increased. Comparisons of the loss of discrete protein bands from electrophoretic gels (native-PAGE, SDS-PAGE and SDS-PAGE of reduced samples) showed that heating β -lactoglobulin solutions of 100 g/1 at pH 7 in 0·1 M-NaCl and at 75, 80 and 85 °C caused a faster loss of both native and SDS-soluble β -lactoglobulin A than of β -lactoglobulin B. It was concluded that the loss of native β -lactoglobulin structure from these solutions during heating was faster than the formation of disulphidelinked aggregates, which was faster than gel formation for both β -lactoglobulin A and β -lactoglobulin B, and that each of these reactions was faster for β -lactoglobulin A than for β -lactoglobulin B. This contrasts with conclusions drawn from some previous studies and may arise from the differences in protein concentration between the present study (∼ 100 g/1) and the previous ones (