Simon M. Loveday

In vitro gastric digestion of heat-induced aggregates of β-lactoglobulin.

In vitro gastric digestion of heat-induced aggregates of β-lactoglobulin (β-LG) in simulated gastric fluid was investigated using sodium dodecyl sulfate-PAGE (under nonreducing and reducing conditions), native PAGE, 2-dimensional electrophoresis, and size exclusion chromatography. Heating at 90°C significantly increased the digestibility of β-LG, with a high initial digestion rate followed by a relatively constant rate of digestion at a high enzyme:substrate (E:S) ratio of 3:1. At a low E:S ratio (1:6), the rate of digestion of β-LG was slower, and intermediate- and low-molecular-weight species could be seen. The high-molecular-weight nonnative aggregates (e.g., pentamers, tetramers, and trimers) were digested relatively rapidly, whereas some of the nonnative dimers were resistant to digestion and others were digested rapidly. The intermediate-molecular-weight species (21 to 23 kDa) were digested slowly. The digestibility of nonnative β-LG aggregates varied significantly depending on the E:S ratio and the types of aggregate. Further investigation is necessary to identify and characterize slowly digested dimers and species of intermediate molecular weight.

Effect of Calcium on the Morphology and Functionality of Whey Protein Nanofibrils

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.

Factors Affecting Rheological Characteristics of Fibril Gels: The Case of β-Lactoglobulin and α-Lactalbumin

Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) have been used to create gels with different rheological characteristics. Values of the gelation time, t(c), the critical gel concentration, c(0), and the equilibrium value of the storage modulus, G, such as at long gelation times, derived from experimental rheological data, are discussed. Fibrils created from beta-lg using solvent incubation and heating result in gels with different rheological properties, probably because of different microstructures and fibril densities. Partial hydrolysis of alpha-la with a serine proteinase from Bacillus licheniformis results in fibrils that are tubes about 20 nm in diameter. Such a fibril gel from a 10% (w/v) alpha-la solution has a higher modulus than a heat-set gel from a 10% (w/w) beta-lg, pH 2.5 solution; it is suggested that one reason for the higher modulus might be the greater stiffness of alpha-la fibrils. However, the gelation times of alpha-la fibrils are longer than those of beta-lg fibrils.

β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils

Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of β-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.

Whey Protein Nanofibrils: The Environment–Morphology–Functionality Relationship in Lyophilization, Rehydration, and Seeding

Amyloid-like fibrils from β-lactoglobulin have potential as efficient thickening and gelling agents for food and biomedical applications, but the link between fibril morphology and bulk viscosity is poorly understood. We examined how lyophilization and rehydration affects the morphology and rheological properties of semiflexible (i.e., straight) and highly flexible (i.e., curly) fibrils, the latter made with 80 mM CaCl(2). Straight fibrils were fractured into short rods by lyophilization and rehydration, whereas curly fibrils sustained little damage. This was reflected in the viscosities of rehydrated fibril dispersions, which were much lower for straight fibrils than for curly fibrils. Lyophilized straight or curly fibrils seeded new fibril growth, but viscosity enhancement due to seeding was negligible. We believe that the increase in fibril concentration caused by seeding was counterbalanced by a decrease in fibril length, reducing the ability of fibrils to form physical entanglement networks.

Formation of β-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conventional heating.

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of β-lactoglobulin (β-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of β-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact β-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of β-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of β-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.

Effect of pH, NaCl, CaCl2 and temperature on self-assembly of β-lactoglobulin into nanofibrils: a central composite design study.

The ability of certain globular proteins to self-assemble into amyloid-like fibrils in vitro opens opportunities for the development of new biomaterials with unique functional properties, like highly efficient gelation and viscosity enhancement. This work explored the individual and interacting effects of pH (1 to 3), NaCl (0-100 mM), CaCl(2) (0-80 mM) and heating temperature (80 to 120 °C) on the kinetics of β-lactoglobulin self-assembly and the morphology of resulting nanofibrils. Statistically significant (p < 0.05) interactions included CaCl(2)*temperature, NaCl*pH, CaCl(2)*pH, temperature*pH and NaCl*CaCl(2). Particularly notable was the very rapid self-assembly at pH 3 and the highly nonlinear effect of pH on self-assembly kinetics. Nanofibril morphologies ranged from long and semiflexible or curled and twisted to short and irregular. There did not seem to be a link between the kinetics of fibril formation and the morphology of fibrils, except at pH 3, where self-assembly was very rapid and fibrils were short and irregular, suggesting haphazard, uncontrolled self-assembly.

Modulating β-lactoglobulin nanofibril self-assembly at pH 2 using glycerol and sorbitol.

β-Lactoglobulin (β-lg) forms fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). When formed at protein concentrations <3%, these fibrils are made up of peptides produced from the acid hydrolysis of the β-lg monomer. The present study investigated the effects of the polyhydroxy alcohols (polyols) glycerol and sorbitol (0-50% w/v) on β-lg self-assembly at pH 2. Glycerol and sorbitol stabilize native protein structure and modulate protein functionality by preferential exclusion. In our study, both polyols decreased the rate of β-lg self-assembly but had no effect on the morphology of fibrils. The mechanism of these effects was studied using circular dichroism spectroscopy and SDS-PAGE. Sorbitol inhibited self-assembly by stabilizing β-lg against unfolding and hydrolysis, resulting in fewer fibrillogenic species, whereas glycerol inhibited nucleation without inhibiting hydrolysis. Both polyols increased the viscosity of the solutions, but viscosity appeared to have little effect on fibril assembly, and we believe that self-assembly was not diffusion-limited under these conditions. This is in agreement with previous reports for other proteins assembling under different conditions. The phenomenon of peptide self-assembly can be decoupled from protein hydrolysis using glycerol.

Water Dynamics in Fresh and Frozen Yeasted Dough

Water is an integral part of wheat flour dough—the amount, physical state, and location of water are crucial to the formation of a dough that will hold gas and produce an open, aerated crumb structure in the final product. This has been understood for centuries by craft bakers, who were highly attuned to the “feel” of dough in their hands. In the 20th century, empirical instruments were invented that simulated part of the breadmaking process, and their limited predictive capacity made them valuable quality control tools. During the latter decades of the 20th century the cost and availability of advanced instrumental methods for characterizing foods improved dramatically, and facilitated a “fundamental science” approach to food research. The physicochemical mechanisms by which water exerts such a strong influence on the character of dough are now better understood. This review contrasts the empirical and fundamental view points, and summarizes recent knowledge about the roles of water in the manufacture of fresh and frozen yeasted dough.

Glycation as a Tool To Probe the Mechanism of β-Lactoglobulin Nanofibril Self-Assembly.

In this study we investigated the effects of different levels of glucosylation and lactosylation on β-Lg self-assembly into nanofibrils at 80 °C and pH 2. Fibrils in heated samples were detected with the thioflavin T assay and transmission electron microscopy, while SDS-PAGE was used to investigate the composition of the heated solutions and fibrils. Glycation had different effects in the nucleation and growth phases. The effect of glycation on the nucleation phase depended on the degree of glycation but not the sugar type, whereas both the type of sugar and the degree of glycation affected the rate of fibril growth. Glycation by either sugar strongly inhibited self-assembly in the growth phase, and lactosylation produced a much stronger effect than glucosylation. We suggest that the varying glycation susceptibility of different lysine residues can explain these observations. The large, polar sugar residues on the glycated fibrillogenic peptides may inhibit fibril assembly by imposing steric restrictions and disrupting hydrophobic interactions.