Aggregation Of Beta-lactoglobulin Research Articles

Modulating the in vitro gastric digestion of heat-induced beta-lactoglobulin aggregates: Incorporation with polysaccharide

Three heat-induced protein aggregates, beta-lactoglobulin fibrils (BLGF), nanoparticles (BLGN), and worm-like aggregates (BLGW) were chosen to probe the effect of disulfide bond and surface hydrophobicity on their gastric digestion behavior. Furthermore, the effect of polysaccharide (dextran sulfate sodium, DSS) on the digestion behavior of the protein aggregates was investigated. Results showed that disulfide bond had a mild restraint on the digestion extent (maximum up to 4.65%), especially when its content was below 1 mol/mol, while the surface hydrophobicity had a stronger influence (up to 8.96%), and there is definitive positive linear relationship between the surface hydrophobicity and the digestion extent. When incorporated with DSS, both the disulfide bond content and surface hydrophobicity of the aggregates decreased, consequently, and the digestion was impeded, confirming the stronger effect from the surface hydrophobicity. The digestion extent of the heat-induced protein aggregates could be modulated linearly by incorporation of polysaccharide.

The threshold of amyloid aggregation of beta-lactoglobulin: Relevant factor combinations

This study identifies critical factor combinations of pH, temperature, stirring speed, protein and ion concentration that specifically affect the lag-, and growth phase of beta-lactoglobulin amyloid aggregation and provides information on how, when and why certain factor combinations affect the onset of amyloid aggregation.Conditions at the threshold of amyloid formation were chosen to prolong the lag and growth phase for several hours. Temperature was the most important factor in all aggregation stages. Interactions between low pH and temperature in the growth phase were caused by elevated protein denaturation at low pH (DSC). Interactions between stirring speed and temperature in the lag phase were caused by viscosity-dependent shear stress (CFD-simulation).Even if none of the factor combinations could shift the onset of amyloid aggregation, important factor combinations were identified that favour the onset of amyloid aggregation. Therefore, the results of this study could be relevant for industrial production.

Functional ethanol-induced fibrils: Influence of solvents and temperature on amyloid-like aggregation of beta-lactoglobulin

Solvent-induced fibrillar aggregates of beta-lactoglobulin occur only in a certain balance between hydrophobic forces and electrostatic interactions. We hypothesize, that different hydrophobic solvent molecules as well as rising temperatures influence this equilibrium and thus the optimum to produce amyloid aggregates.Dimethyl sulfoxide (DMSO), methanol and ethanol all resulted in polydisperse solutions with worm-like and spherical-aggregates, albeit to different degrees: the volume fraction required for aggregation was DMSO (50%) > methanol (40%) > ethanol (30%) which does not reflect their hydrophobicity. Further solvent addition decreased the fibrillar aggregation again. Increasing the temperature by 10–20 K decreased the solvent concentration needed to induce amyloid-like aggregates.A targeted production of solvent-based amyloid-like aggregates is therefore not only dependent on the hydrophobicity of the solvents, but also on their direct interaction with the protein (denaturation).

Protein oxidation during temperature-induced amyloid aggregation of beta-lactoglobulin

Although the connection between protein oxidation, amyloid aggregation and diseases such as Alzheimer’s is well known there is no information on such effects during preparation of beta-lactoglobulin fibrils. Different morphologies of amyloid aggregates of beta-lactoglobulin were prepared by incubation at pH 2 or pH 3.5 for up to 72 h. After 5 h, amyloid aggregates at pH 2 formed typical fibrils, which consisted of peptides. At pH 3.5, the amyloid aggregates were worm-like and consisted of intact protein. After 72 h, the building blocks at both pH values changed towards smaller peptides. The apparent tyrosine oxidation reached a maximum after 5 h at both pH values, whereas N-formylkynurenine and carbonyls increased continuously during 72 h. In case amyloid structures are used as edible material, the health related effects caused by protein oxidation needs to be considered.

Comparative study on foaming and emulsifying properties of different beta-lactoglobulin aggregates.

Different conditions in food processing lead to different aggregates of protein. β-Lactoglobulin nanoparticles (BLGNPs) and fibrils (BLGFs) were prepared by adjusting pH and temperature, and thereby their foaming and emulsifying properties were compared to native globular β-lactoglobulin (NGBLG) at pH 7.0 and pH 4.0. A foam analyser, a microparticle size analyser, an interfacial rheometer and an atomic force microscope (AFM) were used to characterise foaming/emulsifying functionalities and interfacial microstructures/mechanical properties. The foam and emulsion stabilities were assessed by measuring the decay of foam height and the variation of emulsion droplet size, and both were found to be in the order of BLGFs > NGBLG > BLGNPs. Foams were more stable at pH 4.0 while emulsions were more stable at pH 7.0. Surface dilatational modulus (E) of NGBLG and its aggregates at pH 4.0 was greater than that at pH 7.0, with BLGFs showing the highest value of E and thus the highest resistance to membrane damage. The emulsion prepared with NGBLG and its aggregates had more negative charges at pH 7.0 than at pH 4.0. The foaming stability seemed to be more controlled by interfacial elasticity while the emulsion stability was more determined by surface charges. AFM analysis demonstrated different microstructures at the air-water interface between pH 4.0 and 7.0 and among the different protein aggregates, which could well explain their foaming and emulsifying properties.

Silver nanoparticle modulates the aggregation of beta-lactoglobulin and induces to form rod-like aggregates

Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ± 3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein β-lactoglobulin (β-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed β-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV–Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed β-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.

Studying a chaperone-like effect of beta-casein on pressure-induced aggregation of beta-lactoglobulin in the presence of alpha-lactalbumin

Protein aggregation can be used to improve functionality in certain food systems, especially in gelled systems. However, in beverages application, this phenomenon is generally undesirable since it is usually related to protein insolubility and turbidity. Nonetheless, some research has demonstrated a molecular chaperone-like property of certain milk proteins that helps avoid protein aggregation. Here, we investigated the effect of beta-casein (β-CN) on pressure-induced aggregation of beta-lactoglobulin (β-lg) in whey solution using various qualitative and quantitative analyses (turbidity, SDS-PAGE, HPSEC and TEM). Protein model solutions containing different ratios of alpha-lactalbumin (α-la), β-lg and β-CN were pressurized by high hydrostatic pressure (HHP). Pressure treatment of β-lg alone generated a highly turbid solution containing large aggregates while the addition of both proteins (α-la and β-CN) at different ratios led to a drastic decrease in turbidity, despite the presence of larger aggregates. In fact, TEM analysis showed larger and amorphous aggregates for β-lg with α-la and β-CN, and globular, denser aggregates for β-lg alone. Further analysis of these aggregates by fractionation (HPSEC) followed by SDS-PAGE showed no β-CN directly involved in β-lg aggregation, suggesting a chaperone-like effect of β-CN under HHP. Our experiments, performed on model dairy solutions, demonstrated that α-la and β-CN inhibits the formation of insoluble aggregates (decreases turbidity) under HHP treatment of β-lg that could be relevant in milk protein fortified beverages.

Metal ions modulate thermal aggregation of beta-lactoglobulin: A joint chemical and physical characterization

Molecular basis of the role played by Cu2+ and Zn2+ ions during the thermal aggregation processes of beta-lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn2+ is able to alter the natural conformational state of BLG, causing a slight unfolding, whereas Cu2+ ions play a role only during the thermal treatment. Thus, Zn2+ ions favor the formation of bigger aggregates and branched fibril-like structures, whereas for Cu2+ ions a greater number of cross-beta structures during thermal incubation and finally, fibrillar structures. The aggregation process occurs in two phases, as suggested by the measurements on the time evolution of the BLG aggregates: the first one is characterized by a partial unfolding of the protein and aggregate growth, forming oligomers and protofibrils, whereas the second one is characterized by further supramolecular assembly, leading to the formation of fibrils.

Disappearance of intermolecular beta-sheets upon adsorption of beta-lactoglobulin aggregates at the oil–water interfaces of emulsions

Native proteins usually undergo structural modification upon adsorption at interface. Heat treatments are commonly applied at the industrial scale and lead to aggregation of proteins. We characterized nanometric aggregates of β-lactoglobulin by infrared spectroscopy in solutions, in hexadecane oil-in-water emulsions and at the air–water interface at low and high (0.1 M) ionic strengths and at pH 7. In solutions, on the contrary to native β-lactoglobulin, all aggregates prepared with or without salt possessed intermolecular β-sheets evidenced by two strong absorption bands at 1614 cm−1 and 1682 cm−1. In emulsions, at low ionic strength, they lose their intermolecular β-sheets once they are adsorbed at the oil–water interface. At high ionic strength, most of aggregates are localized at the interfaces where they lose their intermolecular β-sheets in direct contact with the surface and only partially when they are farther from the interface. The loss of intermolecular β-sheets was similarly observed at the air–liquid interface.

Interlocking of β-carotene in beta-lactoglobulin aggregates produced under high pressure

Vitamin A deficiency is one of the major causes of mortality and morbidity in the developing World. This deficiency can be prevented by alimentary or pharmaceutical supplementation. However, both vitamin A oxidation and isomerization should be prevented, as these phenomenons result in loss of nutritional efficacy. The aim of this study was to investigate the effect of a food protein matrix, β-lactoglobulin (β-Lg) aggregates produced by high pressure (HP), on the stabilization of β-carotene during storage and gastro-duodenal digestion and therefore on its bioavailability. In vitro gastro-duodenal digestion of β-Lg aggregates entrapping β-carotene showed that up to 12% and 33% of total β-carotene was released after peptic and pancreatic digestion, respectively. Overall, our study showed that β-Lg aggregates are efficient for caging and stabilization of β-carotene during storage and digestion. Hence, it may be an interesting approach for the protection and the delivery of vitamin A.

Numerical study of the effect of thiol–disulfide exchange in the cluster phase of β-lactoglobulin aggregation

We report a numerical study of beta-lactoglobulin aggregation using grand canonical Monte Carlo simulations of a simple lattice model in which the proteins are represented by a single lattice point and interact via a sum of a short-ranged attraction, and a long-ranged screened electrostatic repulsion. For certain values of the potential parameters we observe the so-called cluster phase, in which protein aggregates of finite size repel each other. The properties of the cluster phase are dependent on the salt concentration, the charge of the protein, and the strength of the short-ranged attraction. Disulfide bridges are modeled by covalent bonds between the lattice points, and can exchange with free thiols. Allowing the thiol-disulfide exchange leads to a severe lowering in the chemical potential of the cluster transition, or equivalently, a lower monomer density. Moreover, we find that the disulfide bridges (or the free thiol groups) are not uniformly distributed over the aggregate. The free thiol groups are significantly more abundant on the surface than in the core of the aggregate, making the surface more reactive than the inner core. This finding might explain why films made of beta-lactoglobulin by cold gelation, after resolvation, reconstitute finite aggregates rather than a monomer solution.

Analysis by Raman spectroscopy of the conformational structure of whey proteins constituting fouling deposits during the processing in a heat exchanger

Whey protein fouling deposits generated on the hot wall downstream a plate heat exchanger were analyzed by micro Raman spectroscopy (MRS) carried out in the 800–1800 cm −1 range. Deposits were formed using a model beta-lactoglobulin (BLG) fouling solution which was made using a whey protein isolate powder (89 wt.% in BLG) and a known amount of calcium. Thermal denaturation of the fouling solution was also analyzed by MRS as well as isolated BLG aggregates obtained by microfiltration of heated solutions. Specific Raman signatures of aggregates were identified, which were not detected in the Raman spectra of denatured (i.e. unfolded BLG molecule) solutions. MRS analyses at different depths of the deposit reveal a loss of α-helix structures, as observed in denatured BLG solutions, without the detection of aggregate signatures. For the range of calcium content investigated (from 97 to 160 mg l −1), no effect of calcium ions on the molecular conformation of BLG within the deposit was shown. Of great significance, results suggest that, for our set of operating conditions used, the mass distribution of the fouling deposit in a plate heat exchanger is primarily controlled by the distribution of the unfolded protein generated by the denaturation process.

Kinetics of Formation and Physicochemical Characterization of Thermally‐Induced β‐Lactoglobulin Aggregates

The kinetics of heat denaturation and aggregation for beta-lactoglobulin dispersions (5% w/v) were studied at 3 pHs (6, 6.4, and 6.8) and at a heating temperature of 80 degrees C. Protein aggregates were characterized for hydrodynamic diameter, microstructure, and molecular weight by means of dynamic light scattering, transmission electron microscopy, and polyacrylamide gel electrophoresis, respectively. Concentration of native beta-lactoglobulin decreased with holding time and with a decrease in the pH. Apparent rate constants were calculated for beta-lactoglobulin denaturation applying the general kinetic equation solved for a reaction order of 1.5. Values of the apparent reaction rate constant k = 7.5, 6.3 and 5.6 x 10(-3) s(-1) were found for pH 6, 6.4, and 6.8, respectively. Decreasing the pH of the dispersions produced higher aggregate sizes. After a holding time of 900 s, average hydrodynamic diameters for beta-lactoglobulin aggregates at pH 6, 6.4, and 6.8 were 96, 49, and 42 nm, respectively. These results were confirmed by transmission electron microscopy images, where a shift in the size and morphology of aggregates was found, from large and spherical at pH 6 to smaller and linear aggregates at pH 6.8. beta-Lactoglobulin formed disulfide-linked intermediates (dimers, trimers, tetramers) and so on) which then formed high molecular weight aggregates. From the results obtained by DLS, TEM, and SDS-PAGE a mechanism for beta-lactoglobulin aggregation was proposed. This study shows that heat treatment can be used to produce protein aggregates with different sizes and morphologies to be utilized as ingredients in foods.

The Role of Small Oligomers on an Amyloidogenic Free Energy Landscape

We combine atomic force microscopy particle size distribution measurements with earlier measurements on 1-anilino-8-naphthalene sulfonate, thioflavin T and dynamic light scattering to develop a quantitative kinetic model for the aggregation of beta-lactoglobulin into amyloid. We directly compare our simulations to the population distributions provided by dynamic light scattering and atomic force microscopy. We combine species in the simulation according to structural type to compare with the fluorescence fingerprint results. The kinetic model of amyloidogenesis leads to an aggregation free energy landscape. We define the roles of and propose a classification scheme for different oligomeric species based on their location on the aggregation free energy landscape. We relate the different types of oligomers to the amyloid cascade hypothesis and the toxic oligomer hypothesis for amyloid-related diseases. We discuss existing kinetic mechanisms in terms of the different types of oligomers. We provide a possible resolution to the toxic oligomer-amyloid coincidence.

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer’s patches

The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy. Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed. Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis. These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

The Effect of Molecular Chaperone, Alpha-Crystallin, on the Heat-Induced Aggregation of Beta-lactoglobulin

Aggregation of beta-lactoglobulin occurs mainly via intermolecular disulphide bond exchange. Upon heating, beta-lactoglobulin aggregated which increased with increasing pH. The presence of DTT led to more rapid aggregation and precipitation of beta-lactoglobulin. Alpha-Crystallin prevented the aggregation of heat-stressed beta-lactoglobulin and was a more efficient chaperone at higher pH values. In the presence of DTT, however, alpha-crystallin was a less efficient chaperone due to faster aggregation of heated and reduced beta-lactoglobulin.

Effects of Salt Concentration on Formation and Dissociation of β-Lactoglobulin/Pectin Complexes

The formation and dissociation of beta-lactoglobulin/pectin complexes at various sodium chloride concentrations (CNaCl) have been studied by turbidimetric titration. An increase of CNaCl up to 0.1 M shifts the critical pHphi1, which designates the formation of beta-lactoglobulin/pectin coacervates, to higher pH values, whereas further increase of CNaCl from 0.1 to 0.8 M decreases pHphi1 values. These salt effects can be explained in terms of a salt-enhanced effect at lower salt concentrations or a salt-reduced effect at higher salt concentrations, respectively. On the other hand, the value of pHphi2, which corresponds to the dissociation of beta-lactoglobulin/pectin coacervates, tends to have smaller pH values when CNaCl increases from 0.1 to 0.3 M. No observable pHphi2 values are found at CNaCl higher than 0.3 M. The disappearance of pHphi2 is mainly attributed to the strong self-aggregation capability of beta-lactoglobulin at higher CNaCl. The aggregation of beta-lactoglobulin at high CNaCl is reversible, as suggested by the atomic force microscopy results.

Characterization of β-Lactoglobulin A Gelation in the Presence of Sodium Caprate by Ultrasound Spectroscopy and Electron Microscopy

The effect of sodium caprate (a fatty acid salt) on the formation of beta-lactoglobulin A gels was studied at constant temperature (30 or 35 degrees C) using ultrasonic spectroscopy. During incubation at these temperatures, ultrasonic attenuation increased with the addition of sodium caprate, and reached a plateau after 5-7 h of incubation. Comparing beta-lactoglobulin A with and without sodium caprate, a decrease in net ultrasonic velocity was observed. These results suggested that aggregation occurred during incubation with sodium caprate, and the sample showed an increase in compressibility. Transmission electron microscopy with negative staining showed the formation of filamentous aggregates of beta-lactoglobulin A at around 3-4.5 h of incubation with sodium caprate. These results demonstrated that sodium caprate induced the formation of structures with unique gel properties compared to those formed by heating beta-lactoglobulin in the presence of NaCl alone.

Effects of Temperature, pH, and Salt Concentration on β-Lactoglobulin Deposition Kinetics Studied by Optical Waveguide Lightmode Spectroscopy

Deposition kinetics of beta-lactoglobulin at a solid-liquid interface was studied with optical waveguide lightmode spectroscopy (OWLS) over a range of temperatures between 61 and 83 degrees C. A new temperature-controlled cell for OWLS measurements allows fast, on-line monitoring of the deposit formation at elevated temperatures. Primary protein layers were deposited at 25 degrees C in order to precondition and stabilize the waveguide surface. Sustained deposition lasting from a few minutes (around 80 degrees C) to hours (below 70 degrees C) resulted in multilayer deposits up to several tens of nanometers thick. The measured deposition rates were strongly influenced by temperature, pH, and NaCl concentration. Deposition rates decreased with increasing pH from 5.5. to 7.4, in a trend similar to that for noncovalent aggregation of beta-lactoglobulin in solution. Activation energies for deposition rates decreased with increasing pH, from 340 kJ/mol at pH 5.5 to 230 kJ/mol at pH 7.4 and were similar to the activation energies for denaturation of beta-lactoglobulin in solution.

Observation of salt-induced β-lactoglobulin aggregation using sedimentation field-flow fractionation

Sedimentation field-flow fractionation (SdFFF) was applied in order to characterize particle sizes of beta-lactoglobulin aggregates induced by Ca2+ or Zn2+. Aggregation induced by Zn2+ was faster than that induced by Ca2+. Effects of Zn2+ and beta-lactoglobulin concentrations, as well as contact time, on the aggregation of beta-lactoglobulin were examined. All factors exhibited a combined effect on the size of aggregates, whereby larger aggregates were obtained at increased concentrations of Zn2+ and beta-lactoglobulin. At fixed concentrations of 2% (w/v) beta-lactoglobulin and 10 mM Zn2+, the particle size of the aggregates increased from 0.19 microm (at 15 min) to 0.38 microm (at 2880 min). Further, a hyphenated technique of SdFFF and inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to examine whether intermolecular ionic bridges take part in salt-induced beta-lactoglobulin aggregation. With SdFFF-ICP-OES, protein-cation-protein cross-linkages were observed for beta-lactoglobulin aggregation induced by Zn2+, but not for that induced by Ca2+.