Aggregation Of Β-lactoglobulin Research Articles

Properties of β-Lactoglobulin Aggregates and Gels as Affected by Ternary Emulsifier Mixtures of Tween 20, Lecithin, and Sucrose Palmitate.

The influence of sucrose palmitate, Tween 20, and lecithin on the properties of heat-induced aggregates and cold-set gels of β-lactoglobulin was studied based on an experimental mixture design with a fixed total emulsifier concentration. Emulsifiers were added to the protein solution before heating. Aggregate size and absolute values of ζ potential increased with the addition of emulsifiers, among which lecithin had the most pronounced effect. The water retention of the aggregates correlated positively with the aggregate size. Gels had reduced fracture stress and strains with increasing sucrose palmitate and decreasing Tween 20 contents. The fracture properties correlated with the ζ potentials of the aggregates, and larger aggregates led to gels with higher water-holding capacities. The emulsifiers hence influenced the gel properties indirectly via the aggregate properties. The impact of emulsifiers on food structures should therefore be considered when a food product is designed.

Influences of mannosylerythritol lipid-A on the self-assembling structure formation and functional properties of heat-induced β-lactoglobulin aggregates

β-lactoglobulin dominates the behaviors of whey aggregates in food products. Interactions between β-lactoglobulins and surfactants are greatly diverse and have aroused much attention due to their benefits to the food functionality. The influences of the glycolipid biosurfactant mannosylerythritol lipid-A (MEL-A) on the heat-induced β-lactoglobulin aggregates were investigated by measuring the aggregate structure, the binding nature of MEL-A toward β-lactoglobulin, and the interface behaviors as well as the foaming and emulsifying property. The results showed that MEL-A with a concentration above CMC reduced the random β-lactoglobulin aggregation after heat treatment, facilitating the formation of self-assembling core-shell particles composed of a core of β-lactoglobulin aggregates covered by a shell of MEL-A vesicles with diameters less than 2 μm. In the presence of MEL-A, β-lactoglobulin aggregates gave rise to a decreased surface tension at the air-water interface, viscoelasticity and foaming capacity, whereas there was increased foaming stability and emulsifying properties after heat treatment for less than 3 h. Moreover, the excessively heat-induced aggregation was not conducive to the enhancement of both of the functional properties in the current model, while MEL-A effectively prevented the aggregation from proceeding based on the existing molecular interactions. The comprehensive results of infrared spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry confirmed that the interaction forces between MEL-A and β-lactoglobulin were driven by hydrophobic interactions between the hydrophobic groups exposed from the protein and the fatty acid chain or acetyl groups of MEL-A as well as the hydrogen bonding between the mannosyl-D-erythritol group of MEL-A and amino acid.

Molecular chaperone-like properties of sodium caseinate to suppress the pressure-induced aggregation of β-lactoglobulin

ABSTRACTThe objective of this study was to determine whether sodium caseinate can inhibit the aggregation of whey protein induced by pressure treatment. Solutions of β-lactoglobulin (β-Lg, 0.2%, w/v) and mixtures containing 0.2% (w/v) β-Lg and 0–0.5% (w/v) sodium caseinate (NaCas) were pressurized at 400–800 MPa. NaCas suppressed the aggregation of β-Lg induced by pressure treatment, and this function was dependent on the concentration of NaCas. Furthermore, NaCas altered the aggregation process of β-Lg by suppressing the transition of the aggregate from the soluble phase to the insoluble phase and, as a result, the fraction of insoluble aggregates was decreased. During this process, NaCas formed stable complexes with the denatured β-Lg, and the formation of complexes prevented further aggregation of β-Lg. These results indicate that NaCas exhibits a chaperone-like activity under high pressure.

Formation of β-Lactoglobulin Aggregates from Quite, Unfolded Conformations upon Heat Activation.

In presence of calcium ions, β-lactoglobulin (BLG) unfolds and subsequently aggregates after heating. This process has important pharmaceutical and agroalimentary applications. Nowadays, the molecular mechanism of unfolding and BLG aggregation, and the role of calcium in the mechanism, is poorly understood. Actually, in most studies, data have been acquired at room temperature, after heating and after aggregation, which makes it difficult to establish a clear causal-temporal relation between calcium binding, heat, and aggregation. Thus, the goal of the present study is to get accurate, nanoscale data about the molecular events leading to BLG unfolding and calcium-dependent aggregation. The molecular transformation of BLG during heating has been investigated, using the NMR pulse field gradient technique, operating in a high field (900 MHz). Thanks to this technique, the molecular conformation of newly formed unfolded BLG molecules can be distinguished in a large pool of native ones. The present work shows that BLG at neutral pH at 65 °C displays fast, cooperative-like unfolding, in which no long-lived intermediary state (as a molten globule one) is detected, before aggregation. These data also indicate that calcium ions bind unfolded BLG in specific sites which might be a necessary feature to form the aggregate. Finally, these data also provide an NMR-based methodology to monitor the rate of protein unfolding using NMR.

Microwave induced conformational change and aggregation of bovine β-lactoglobulin: An insight to non-thermal effect

Microwave induced conformational change and aggregation of bovine β-lactoglobulin: An insight to non-thermal effect

Maillard induced aggregation of individual milk proteins and interactions involved.

The aggregation of α-lactalbumin, β-lactoglobulin and β-casein after heating in dry state was studied in absence and presence of saccharides. In absence of saccharides, differences were observed in the extent of aggregation. Differences between the proteins were mostly due to differences in covalent aggregation. The presence of glucose during the heat treatment of milk proteins significantly increased the extent of aggregation, and decreased differences between proteins. α-Lactalbumin was selected as a model protein for the study of cross-links formed after heat treatment. In the presence of saccharides, these cross-links were found to consist of 36% of disulphide bridges (compared to >75% in the absence of glucose), followed by other cross-links such as lanthionine. Larger saccharides led to a decrease in Maillard induced aggregation; maltotriose actually even inhibited the formation of α-lactalbumin aggregates.

Heat-induced amyloid-like aggregation of β-lactoglobulin regulated by glycation: A comparison of five kinds of reducing saccharides

The Maillard reaction occurs extensively in food industry and can change the aggregation behavior of dietary proteins. In this work, we reported the influence of glycation on the heat-induced amyloid-like aggregation behavior of β-lactoglobulin (β-Lg). Glycation was shown to accelerate the unfolding behavior of β-Lg and thereby increased fibrillation rate during initial heating according to 1-anilinonaphthalene-8-sulfonate and thioflavin T assays. Prolongation of fibrils was found to be limited by glycation derived from glucose, fructose and lactose, possibly due to the decrease in β-sheet structure of β-Lg based on circular dichroism spectral results and transmission electron microscopy images. β-Lg incubated with maltodextrin appeared to associate into globular micelles. Both covalent (disulfide bond and crosslinking structure) and non-covalent changes (steric hindrance and hydrogen bonds) induced by glycation are proposed to account for these fibrillation kinetic and conformational changes of β-Lg aggregates. According to these findings, glycation are promising methods to regulate both fibrillation kinetics and fibril conformation in the food industry.

Application of ultrasound pretreatment and glycation in regulating the heat-induced amyloid-like aggregation of β-lactoglobulin

Ultrasonication and glycation are widely used in the food industry. In this study, we evaluated the independent and combined effects of ultrasound pretreatment (UP) and glycation on the heat-induced amyloid-like aggregation of β-lactoglobulin (β-Lg) by investigating both the fibrillation kinetics and aggregate conformations. Both UP and glycation respectively were found to accelerate the fibrillation process by promoting the denaturation and unfolding behavior of β-Lg according to both the thioflavine-T binding assay. Glycation reduced the fibrillation capacity of β-Lg by reducing the content of β-sheet secondary structures in β-Lg on the basis on circular dichroism spectral measurements. Based on the results of transmission electron microscopy, glycation derived from glucose and lactose suppressed the elongation of fibrils, and β-Lg incubated with maltodextrin was shown to form spherical particle micelles. When applied jointly, UP was found to limit the glycation process by promoting aggregation during the initial heating, which may bury a portion of the glycation sites. According to these findings, UP and glycation are promising methods that can be used independently or jointly to regulate both fibrillation kinetics and fibril conformation in the food industry.

Resveratrol Induces the Conversion from Amyloid to Amorphous Aggregation of β-lactoglobulin>.

Protein aggregation is a wide-ranging phenomenon. Protein aggregation mainly includes two types: one is amorphous aggregation, the other is amyloid aggregation. In particular, amyloid aggregation in vivo can cause several fatal diseases. We have investigated the influence of resveratrol on the amyloid aggregation of β-lactoglobulin. The results demonstrated resveratrol inhibited the amyloid aggregation and enhanced the amorphous aggregation of β- lactoglobulin. The main objective of this study was to investigate the effects of resveratrol on the amyloid aggregation of β-lactoglobulin. β-lactoglobulin was incubated at pH 2.0 and 70 °C. ThT fluorescence, Congo red and transmission electron microscopy were used to monitor the formation of amyloid aggregates. In addition, resveratrol was added intoβ-lactoglobulin solutions. Intrinsic fluorescence, Circular dichroism and 1-Anilinonaphthalene-8-sulfonic Acid (ANS) fluorescence were used to investigate conformational and hydrophobic changes. Furthermore, we also studied the effect of resveratrol on the amyloid aggregation of β-lactoglobulin by using ThT fluorescence, Congo red and transmission electron microscopy at acidic pH and high temperature. ThT fluorescence, Congo red and transmission electron microscopy analysis showed that β-lactoglobulin could form the amyloid fibrils when it was incubated under acidic pH and high temperature conditions. At the same time, the analysis also demonstrated resveratrol inhibited the formation of amyloid aggregates and enhanced the formation amorphous aggregates. Intrinsic fluorescence, Circular dichroism and 1-Anilinonaphthalene-8-sulfonic Acid (ANS) fluorescence analysis indicated that resveratrol could alter the conformation and increased the hydrophobicity of β- lactoglobulin. Our results indicated that resveratrol could effectively inhibit the formation of amyloid aggregates and enhance the formation of amorphous aggregates of β-lactoglobulin. Thus, resveratrol could be a potential inhibitor for preventing the formation of amyloid aggregates.

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol−1 and 8.93kcalmol−1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~77Å and ~117Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69kcalmol−1 and 9.09kcalmol−1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.

Role of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) in β-lactoglobulin aggregation

Protein aggregation is a prevalent phenomenon. It is important to study protein aggregation under different solution conditions. In this study, using 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence spectra, we investigated the critical micelle concentration (CMC) of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). We also studied the effects of DOTAP on amyloid aggregation of β-lactoglobulin using intrinsic fluorescence spectra, circular dichroism spectra, thioflavin T fluorescence, a Congo red binding assay and transmission electron microscopy. We observed that DOTAP had a dual role on β-lactoglobulin amyloid aggregation. DOTAP inhibited the amyloid aggregation below the CMC, while it had the opposite effect above the CMC. Moreover, the results of transmission electron microscopy showed that spherical aggregates were formed above the CMC. These results led us to conclude that cationic lipids could be used as modulators of protein self-assembly.

Mechanism of co-aggregation in a protein mixture with small additives

Co-aggregation plays an important role in processing protein-rich food materials under heterogeneous conditions. The main cause of co-aggregation is an electrostatic attraction between oppositely charged molecules. This study investigated thermal aggregation of β-lactoglobulin (BLG) (pI=5.1) and lysozyme (LYZ) (pI=10.7) as a model for the heterogeneous conditions of a protein solution. BLG and LYZ were more aggregated in the mixture than in the single solutions. Co-aggregation of the BLG–LYZ mixture was not observed below 60°C at which temperature BLG and LYZ retained their native structures. Adding sugars, salts, or amino acids to the BLG–LYZ mixture during the heat treatment revealed the co-aggregation process as follows. (i) All additives tested suppressed both the nucleation and growth of aggregates. (ii) Salts affected nucleation stage to the same degree, except arginine hydrochloride (Arg). (iii) Arg specifically suppressed both nucleation and growth of aggregates. These results indicate that co-aggregation in a protein mixture is more sensitive to the partial unfolding of proteins than that in a single protein solution, due to the presence of electrostatic attraction between different molecules. These results provide new insight into protein aggregation as well as the molecular mechanism of additives under heterogeneous conditions.

Effect of pH on the reaction mechanism of thermal denaturation and aggregation of bovine β-lactoglobulin

New insights in the aggregation mechanisms of the whey protein β-lactoglobulin were gained by detailed investigation of the denaturation behavior (80–120 °C) at variable pH values (5.1, 6.8, 8.0). Denaturation followed a 1.5 order kinetic and reaction velocity was depending on pH and temperature. Characterization of aggregates formed at 80 °C was correlated to the denaturation progress and comprised particle size measurement, quantification of free thiol groups and analysis of quaternary structures as well as stabilizing forces by various types of gel electrophoresis. Due to low electrostatic repulsion at pH 5.1, a fast and steady aggregate growth was observed, where intermolecular disulfide bonds gain more importance with increasing degree of denaturation. Contrary to that, denaturation at pH 6.8 and 8.0 yielded lower reaction rate constants and a steady increase of the amount of exposed thiol groups, resulting in non-native oligomers which were mainly non-covalently connected to small aggregates.

Evaluation of structural characteristics determining surface and foaming properties of β-lactoglobulin aggregates

Due to its structural versatility and remarkable surface activity, β-lactoglobulin (β-Lg) is a prominent food structuring agent in both its native state and in the form of heat-induced aggregates. In this study, we considered the properties of soluble aggregates of pure β-Lg having a median diameter of 50nm. The behavior of the aggregates at the air/water interface has been probed as a function of pH, with the aim of evaluating the controversial role of surface charge in connection with the functionality of particle-stabilized foams. Based on these new experimental results, it would appear that charge shielding around the isoelectric point (pI) leads to significantly increased foam stability as compared to samples having a significant positive or negative zeta potential. Against expectations, however, maximum foam stability was not linked to minimum foam drainage. High foamability and foam stability have been found to correlate with maxima in the rates of initial surface pressure increase and with interfacial dilatational properties. Evidence for efficient stabilization of the air/water interface was also reflected in a small average bubble size and a low rate of mean bubble area increase for aggregates with pH∼pI. Information from additional comparative experiments on systems with native β-Lg enabled an evaluation of the significance of surface hydrophobicity and aggregate particle size in relation to the foaming properties.

Coupling population balance model and residence time distribution for pilot-scale modelling of β-lactoglobulin aggregation process

This study presents a hybrid modelling approach for the prediction of the thermally-induced aggregation of whey protein solutions in a continuous process. Modelling a continuous process needs to account the fluid flow phenomena because the different fluid parcels are not subjected to the same time/temperature/shear history. The modelling approach developed combines a residence time distribution (RTD) model and a colloidal aggregation model based on Population Balance Equations (PBE). This combined model has been compared, on one side, to a Population Balance Model assuming plug flow, and on the other side, to experimental data. A 6% β-lactoglobulin solution with different CaCl2 concentrations (5.0, 6.0, 6.4, 6.6 and 7.0 mM) was used for experimental investigations. Experimental heat treatments were carried out at 87 °C with a holding time of 49 s using tubular heat exchanger and holder. The integration of residence time distribution appears to be very important when the product is subject to large transformations (formation of large aggregates > 10 μm). Comparison of predictions obtained from both models to the experimental data shows that the coupled PBE/RTD model is able to predict the characteristics of the suspensions (aggregate size and viscosity) and successfully integrates the impact of calcium upon aggregation process, while the model assuming plug flow is suitable only when aggregation is low (for low calcium concentrations).

Insight into the co-solvent induced conformational changes and aggregation of bovine β-lactoglobulin

Many proteins form ordered irreversible aggregates called amyloid fibrils which are responsible for several neurodegenerative diseases. β-lactoglobulin (β-lg), an important globular milk protein, self-assembles to form amyloid-like fibrils on heating at low pH. The present study investigated the effects of two commonly used organic solvents acetonitrile (MeCN) and antimicrobial preservative benzyl alcohol (BA) on the conformation and self-assembly of β-lg at ambient condition. Both MeCN and BA induced a concentration-dependent conformational change showing exposure of hydrophobic patches, loss of tertiary structure and higher α-helical structure at moderate concentrations. In the presence of 50–80% (v/v) MeCN and 1.5–3% (v/v) BA further structural transitions from α-helical to non-native β-sheet structure were observed with a molten globule-like intermediate at 70% MeCN. These non-native β-sheet structures have high tendency to form aggregates. The formation of β-lg self-assembly was confirmed by Thioflavin T studies, Congo red assay, Rayleigh scattering and dynamic light scattering analysis. Transmission electron microscopy studies showed amyloid fibril formation in both MeCN and BA. Our results showed that BA enhances the unfolding and self-assembly of β-lg at much lower concentration than MeCN. Thus solvent composition forces the protein to achieve the non-native structures which are responsible for protein aggregation.

Investigating the effect of structural transition on aggregation of β-lactoglobulin.

β-lactoglobulin (BLG), the major bovine whey protein, is a well-characterized globular protein. It is a model protein for studying the structural transition and aggregation. BLG unfolds and aggregates through chemical and physical processes. It is a predominantly β-sheet protein but, the non-native α-helical intermediate accumulates in its folding pathway. The present study aims to understand more about which stage of the protein folding is prone to aggregation. The intermediate states were trapped by TFE and their aggregation and structural changes evaluated, for this purpose. The experiments were carried out at various pH values, ionic strengths, protein concentrations and heating times by turbidity measurements, circular dichroism and fluorescence spectroscopy. Furthermore, the aggregated species at various molecular weights were detected by SDS-PAGE. Only a small change was observed in the secondary and tertiary structures of the protein at 10% TFE, but a further increase of TFE concentration results in induction of new α-helical structure and disruption of the rigid tertiary structure. The turbidity measurement indicated that the aggregation of BLG reaches a maximum level at 10% TFE on all experimental conditions and from this point forward, it decreases with increasing the amount of TFE. In conclusion, the results showed that the α-helical state is resistant to aggregation, in spite that its tertiary structure is partially unfolded. BLG becomes prone to aggregation, when its non-native α-helical structure converts to the β-sheet structure.

The Combined Effect of High Hydrostatic Pressure and Calcium Salts on the Stability, Solubility and Gel Formation of β-Lactoglobulin.

Stability, aggregation and gelation of β-Lactoglobulin are affected by high pressure and salts of the Hofmeister series. Little is known about their combined effects on structure formation processes of β-Lactoglobulin, mainly because many salts of the series are not suitable for use in food. Here, we investigate the effect of calcium salts on the strength of pressure-induced gels, inspired by the fact that high pressure and salts change the water structure in a similar way. We find that the larger the applied pressures, the higher the strength of the gels. In addition to pressure, there is a significant influence by the type of anions and the amount of added calcium salts. Gel strength increases in the order CaCl2 < Ca (NO3)2 < CaI2. This trend correlates with the position of the salts in the Hofmeister series. The results are explained by analogy with the thermal aggregate formation by taking reaction rates for unfolding and aggregation, as well as specific/non-specific salts effect into consideration.

Ferric Ions Inhibit the Amyloid Fibrillation of β-Lactoglobulin at High Temperature.

The energetics of amyloid fibrillar aggregation of β-lactoglobulin (βLG) following incubation at high temperature and acid pH was studied by differential scanning calorimetry in the presence of Cu(2+) or Fe(3+) cations, and without any metal. Cu(2+) and metal-free protein solutions showed a distinct exothermic response that disappeared almost completely when the Fe(3+) molar concentration was ten times greater than the βLG concentration. Thioflavin T fluorescence studies in solution and atomic force microscopy analysis of the deposit left on flat mica substrates by heat-incubated βLG solutions correlated the absence of exothermic response of Fe(3+)-βLG solutions with a lack of fibril production. In contrast, abundant fibril deposits were observed for Cu(2+)-βLG solutions, with a rich polymorphism of multistrand fibrillar structures. Electron paramagnetic resonance revealed that Fe(3+) permanently binds to βLG in the aggregate state whereas Cu(2+) plays a catalytic role without binding to the protein. We propose that Fe(3+) inhibits fibril production after binding to a key region of the protein sequence, possibly interfering with the nucleation step of the fibrillation process and opening a nonfibrillar aggregation pathway. These findings suggest that transition metal ions can be utilized to effectively modulate protein self-assembly into a variety of structures with distinct morphologies at the nanoscale level.

Influence of denaturation and aggregation of β-lactoglobulin on its tryptic hydrolysis and the release of functional peptides

Whereas previous studies showed that thermal pre-treatment of whey proteins promote their enzymatic hydrolysis, to date no correlation between the conformation of denatured protein and the release of individual peptides has been considered. Hence, in this study total denaturation of β-lactoglobulin was performed at defined pH-values to enable the generation of different denatured particles. The denatured proteins were used as substrate for tryptic hydrolysis and the hydrolysis progress was characterised by the degree of hydrolysis (DH) and the release of functional peptides, detected using LC–ESI-TOF/MS. Denaturation and subsequent aggregation of β-lactoglobulin, induced by thermal treatment at pH 5.1, altered the DH slightly, whereas the release of investigated peptides was significantly decreased. Contrary, denaturation at pH 6.8 and 8.0 led to formation of non-native monomers and reduced the DH to 75%, but showed promoting as well as reducing effects on the release of peptides, depending on their location within the protein.