Aggregation Of Β-lactoglobulin Research Articles

UVB pretreatment of β-lactoglobulin affects the temperature-induced formation of functional amyloid-like aggregates and promotes oxidative degradation.

Unfolding in combination with or without acid hydrolysis is crucial for the formation of functional amyloid (fibrillar) or amyloid-like (worm-like) β-lactoglobulin (BLG) aggregates, which can be induced through temperature treatment for several hours at pH 2-4. A preceding conformational destabilization of BLG might affect its aggregation. We investigated ultraviolet (UV) B radiation as conformational perturbing treatment to facilitate temperature-induced protein aggregation. 2-h UVB pretreated BLG (UV-BLG) exhibited an accelerated worm-like aggregation at pH 3.5, while at pH 2 the formation of fibrils was decelerated. The UV-induced conformational destabilization lowered the thermal stability and thus facilitates unfolding during thermal treatment. Thereby, the formation of covalent and non-covalent intermolecular interactions was favored, which promoted assembly of intact proteins resulting in worm-like aggregates. The oxidative degradation of UV-BLG was suggested to alter fibrillation-prone protein regions and thereby impede peptide assembly.

Amyloid-like aggregation of recombinant β-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified β-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant β-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.

Structural, Binding and Functional Properties of Milk Protein-Polyphenol Systems: A Review.

Polyphenols (PP) are linked to health benefits (e.g., prevention of cancer, cardiovascular disease and obesity), which are mainly attributed to their antioxidant activity. During digestion, PP are oxidised to a significant degree reducing their bio-functionality. In recent years, the potential of various milk protein systems, including β-casein micelles, β-lactoglobulin aggregates, blood serum albumin aggregates, native casein micelles and re-assembled casein micelles, to bind and protect PP have been investigated. These studies have yet to be systematically reviewed. The functional properties of the milk protein-PP systems depend on the type and concentration of both PP and protein, as well as the structure of the resultant complexes, with environmental and processing factors also having an influence. Milk protein systems protect PP from degradation during digestion, resulting in a higher bioaccessibility and bioavailability, which improve the functional properties of PP upon consumption. This review compares different milk protein systems in terms of physicochemical properties, PP binding performance and ability to enhance the bio-functional properties of PP. The goal is to provide a comprehensive overview on the structural, binding, and functional properties of milk protein-polyphenol systems. It is concluded that milk protein complexes function effectively as delivery systems for PP, protecting PP from oxidation during digestion.

Effect of calcium on the reversible and irreversible thermal denaturation pathway of β-lactoglobulin

Thermal denaturation of β-lactoglobulin (BLG), boosted in the presence of calcium ions, is responsible for the dairy fouling issue in milk processing. However, the reported kinetic model concerning BLG denaturation was generally based on a simplified one-step reaction model where the unfolding of the protein was considered as a kinetically driven reaction. In such a model, the presence of break-slope phenomenon in the Arrhenius plot was justified by the fact that unfolding step constitutes a rate-limited step compared to aggregation at low temperatures. Nevertheless, there is accumulated experimental evidence implying that the unfolding step is much faster than aggregation regardless of the temperature. Therefore, in this paper, it is proposed that the thermal unfolding of BLG is thermodynamically controlled such that a chemical equilibrium between native and unfolded BLG species can be instantaneously established, followed by an irreversible aggregation of unfolded BLG. This novel approach allows to mathematically interprets the break-slope behavior in the Arrhenius plot. The developed model was applied to investigate the effect of Ca 2+ on the unfolding equilibrium and subsequent aggregation process of BLG in whey protein solutions. Ca 2+ was confirmed to have a protective role on the unfolding equilibrium of BLG by increasing the enthalpy Δ H u and decreasing the equilibrium constant K c , while it facilitated the aggregation of BLG by showing a reduction of the corresponding enthalpy and entropy which cannot be seen as reported using one-step reaction model. This approach allows us to be closer to reality when mimicking the heat-induced appearance/disappearance of different BLG species, including native, unfolded and aggregated ones. Accurate population balance of these foulant species during thermal processing could lead to new perspectives on fouling prediction. • Thermal unfolding of β-lactoglobulin can be considered to occur instantaneously. • Unfolding of β-lactoglobulin is thermodynamically driven. • New model mathematically interprets the break-slope phenomenon in Arrhenius plot. • Ionic calcium has a protective role on the unfolding of β-lactoglobulin. • Calcium favors β-lactoglobulin aggregation by showing decreased enthalpy.

Sunset Yellow Dye Induces Amorphous Aggregation in β-Lactoglobulin at Acidic pH: A Multi-Techniques Approach.

Protein aggregation is of two types: (i) amorphous and (ii) amyloid fibril. Several extrinsic factors (temperature, pH, and small ligands) stimulate protein aggregation in vitro. In this study, we have examined the role of sunset yellow (SY) on the β-lactoglobulin (BLG) aggregation at pH 2.0. We have used spectroscopic (turbidity, Rayleigh light scattering (RLS), far-UV CD) and microscopic (transmission electron microscopy [TEM]) techniques to describe the effects of SY on BLG aggregation. Our results showed that BLG aggregation is dependent on SY concentrations. Very low concentrations (0.0–0.07 mM) of SY were unable to induce aggregation, while SY in the concentrations range of 0.1–5.0 mM induces aggregation in BLG. The kinetics of SY-stimulated aggregation is very fast and monomeric form of BLG directly converted into polymeric aggregates. The kinetics results also showed SY-induced BLG aggregation disappeared in the presence of NaCl. The far-UV CD and TEM results indicated the amorphous nature of SY-induced BLG aggregates. We believe that our results clearly suggest that SY dye effectively stimulates BLG aggregation.

Coumarin derivatives inhibit the aggregation of β-lactoglobulin.

The binding of a small molecule to a protein through non-covalent interactions mainly depends on its size and electronic environment. Such binding can change the stability of the three dimensional protein structure which sometimes may destabilize it to accelerate or to inhibit protein aggregation. Coumarin is a widely used fluorescent dye with several biological applications. Different substituents (electron-donating and electron-withdrawing) at different positions of the coumarin moiety can influence its molecular volume, physical and chemical properties. Here we investigate the effect of such substituents of coumarin on the aggregation of a model protein, beta-lactoglobulin (β-lg) through a multi spectroscopic approach. It was observed that coumarin methyl ester with an 8-hydroxyl group can inhibit the β-lg aggregation. This compound can bind the hydrophobic site of beta-lactoglobulin and stabilize a particular protein conformation through the formation of hydrogen bond and hydrophobic interactions. Thus a properly designed compound can inhibit protein–protein interactions through protein–small molecule interactions. Other coumarinoid compounds also are effective in the prevention of thermal aggregation of β-lg.

Insights into the self-aggregating properties of a solvatochromic probe and interaction with β-lactoglobulin

A solvatochromic benzothiazole compound is designed, which exhibits water-induced aggregation and selective detection of β-lactoglobulin at physiological pH.

Heat accelerates degradation of β-lactoglobulin fibrils at neutral pH

Fibrous aggregates of β-lactoglobulin display superior mechanical and interfacial properties compared to the native protein. These properties directly link to the protein morphology and structure. When incorporated into food matrices, during processing protein fibrils are exposed to pH shifts and high temperature conditions, which accelerate their degradation. In the present study, neutralized β-lactoglobulin fibrils were heated at 100 °C and 121 °C for various times to assess their degradation. Fibril morphology, structure, and viscosity in solution were examined by microscopy, scattering, spectroscopy, and rheology. Atomic force microscopy showed the contour length of the protein fibrils decreased gradually with heating at 100 °C and 121 °C, with greater decreases at 121 °C. Increased fibril diameters (∼15–25 nm) were observed at 121 °C for 5–15 min heating and were disrupted upon further heating. Small-angle x-ray scattering indicated an increase in fibril radius with heating at pH 7 followed by a decrease at prolonged heating, whereas fibril length decreased continuously with heating. Thioflavin T fluorescence, circular dichroism and Fourier transform infrared spectroscopy confirmed the conversion of β-sheet to random coils as fibrils were degraded during thermal treatment at pH 7. Surface hydrophobicity of fibrils decreased with increase in heating temperature and time, coinciding with an increase in the content of non-aggregated proteins. Viscosity of fibril solutions increased when fibrils were heated at 100 °C, whereas at 121 °C their viscosity first increased and then decreased. These findings imply heating at 100 °C and 121 °C facilitates degradation and depolymerisation of β-lactoglobulin fibrils with aggregation as an intermediate step.

Effects of divalent cations on the physical, conformational and immunological properties of bovine allergen β-lactoglobulin aggregates

β-lactoglobulin is a major allergen in whey and always forms aggregates under different conditions. In our study, four kinds of divalent cations (Cu2+, Mg2+, Zn2+ and Ca2+) were used to induce the aggregation of β-lactoglobulin. The molecular interactions and characteristics, thermal stability and conformational structure of aggregates were monitored by silver staining, RP-HPLC, differential scanning calorimetry and spectroscopic methods, respectively. The immunological properties of Cu2+-induced β-lactoglobulin aggregates and dimers were evaluated in a Balb/c mouse model. The results showed that Cu2+ promoted the highest β-lactoglobulin dimer levels compared with other metal ions. The thermal stability of aggregates induced by the four ions was more stable than that of native protein. The specific molecular interaction of aggregates induced by Mg2+, Zn2+ and Ca2+ was disulfide bonding, while Cu2+-induced β-lactoglobulin aggregates were connected by disulfide bonds combined with other bonds. Specially, Cu2+-induced dimer showed decreased allergenicity and a shift from the Th2 response to the Th1 response that balances homeostasis compared with native BLG and Cu2+-induced aggregates. The results may provide a possible method to alleviate the allergenicity of bovine β-lactoglobulin by forming more dimers.

Change in conformational, digestive and immunological characteristics of bovine allergen β-lactoglobulin induced by metal ions in combination with heating

Aggregation of bovine β-lactoglobulin is affected easily by external factors. In this study, effects of metal ions combining with temperature on aggregation of β-lactoglobulin were explored. The conformational characteristics of aggregates were detected by environment scanning electron microscope, CD spectrum and free sulfhydryl group, respectively. Digestive and immunological characteristics were assessed by simulated digestion in vitro and ELISA respectively. The results showed that the morphology of β-lactoglobulin aggregates became more amorphous in Cu2+ and Mg2+ treated samples and more constricted in Zu2+-induced protein. Among them, Cu2+ altered the secondary structure of β-lactoglobulin aggregates and free sulfhydryl content most as well as that in gastric digestion. However, all ion-treated groups had similar digestive stability in intestinal digestion. Specially, Ca2+ and Mg2+ made the antigenicity and potential allergenicity of β-lactoglobulin aggregates decrease, which helps us understand the role of metal ions in immunological characteristics.

Conformational transition and gelation of κ-carrageenan in electrostatic complexation with β-lactoglobulin aggregates

The goal of this study was to evaluate the impact of electrostatic complexation with three different β-lactoglobulin aggregates on the conformational transition and gelation of κ-carrageenan (κ-car). We prepared native granular β-lactoglobulin (NGBLG), nanoparticle β-lactoglobulin (NPBLG), and fibrillary β-lactoglobulin (FBLG), and then assessed their electrostatic complexation with κ-car and the resultant impact on κ-car conformational transition, gelation, and microstructural changes. A quantitative model based on the McGhee-Hippel theory was adopted as a means of describing the impact of electrostatic complexation on the κ-car conformational transition in the presence of these protein aggregates. FBLG resulted in the most significant inhibition of κ-car conformational transition and gelation, whereas NPBLG had the least significant impact on this process. This was attributed to the fact that NPBLG imposed the least steric hindrance of these three aggregates. Together, these data highlight promising approaches to regulating polysaccharide gelation, viscoelasticity, rheological behavior, and conformational transition for use in a range of industrial applications.

Interfacial behaviour of β-lactoglobulin aggregates at the oil-water interface studied using particle tracking and dilatational rheology.

During processing, proteins are easily self-assembled into different aggregates, such as nanoparticles and fibrils. Protein aggregates exhibit a strong interfacial activity due to their morphologies and functional groups on the surface. Their interfacial structure and rheological properties at the oil-water interface have a significant effect on the stability and fat digestion of emulsions in food. In this study, β-lactoglobulin (β-lg) aggregates including β-lg nanoparticles (β-lg NP) and β-lg fibrils (β-lg F) were prepared in solution by controlling the heating temperature and pH, and their surface properties including the electric potential, hydrophobicity, and density of free thiol groups were characterized. The adsorption kinetics, interfacial rheology, and displacement by bile salts (BSs) of native β-lg and its aggregates at the oil (decane)/water interfaces were studied using particle tracking microrheology and dilatational rheology. From the movement of tracer particles at the interface, β-lg NP and β-lg F were found to adsorb faster than native β-lg, and they were found to form interfacial films with a marginally higher elasticity. During the process of protein adsorption, the films of β-lg and its aggregates are not uniform. In the process of protein displacement, β-lg NP has the strongest ability while native β-lg has the weakest ability to resist BS substitution, which is consistent with the results from in vitro digestion experiments. The present study reveals the microrheological behaviour of protein aggregates at the oil-water interface and demonstrates that β-lg thermal aggregates exhibit an excellent emulsification ability and can be used to control fat digestion. The study also illustrates the applicability of microrheological methods to the study of interfacial rheology and its complementarity with dilatational rheological methods.

Separation of aggregated β-lactoglobulin with optimised yield in a decanter centrifuge

The interest in individual whey protein fractions, especially the main proteins α-lactalbumin and β-lactoglobulin, has been growing due to their unique nutritional value and their exceptional technofunctional properties. Various fractionation and purification methods have been published. However, none of them achieved highest purity, maximal recovery, and transferability to industrial scale. This study used thermal treatment for selective aggregation of β-lactoglobulin, which was subsequently separated in a pilot-scale decanter centrifuge from the α-lactalbumin-enriched centrate. The aim was to investigate if the centrifugal separation can be improved by pH-induced flocculation of the aggregates compared with non-flocculated suspensions at pH 7.0. With various analytical methods, it was demonstrated that the strongest flocculation occurred at pH 4.4 and facilitated separation of aggregate fragments sized below 20 μm. Finally, separation efficiencies of more than 99% were achieved by aggregate flocculation in the pilot-scale decanter even at low centrifugal forces.

Electrostatic complexation of β-lactoglobulin aggregates with κ-carrageenan and the resulting emulsifying and foaming properties

The electrostatic complexation of protein and polysaccharide and the functional properties of the complexes are significantly affected by the structure of protein aggregates and are important in the development of new food ingredients. In this work, natural globular β-lactoglobulin (NGBLG), β-lactoglobulin nanoparticles (BLGNP), and β-lactoglobulin fibrils (BLGF) were prepared and complexed with κ-carrageenan (κ-car). Phase diagrams of the NGBLG-, BLGNP-, and BLGF-κ-car systems were established and divided into 4 regions: mixed soluble polymers (I), intramolecular soluble complex (II), intermolecular soluble complex (III), and intermolecular insoluble complex (IV). Aggregation shifted the boundaries of regions III and IV of BLGF- or BLGNP-κ-car to lower pH and higher protein aggregates/κ-car weight ratio (r), especially for BLGF-κ-car. The emulsifying and foaming properties of the 3 mixed systems were investigated in regions I and II. Complexes in region II had significantly better emulsifying properties than the corresponding mixtures in region I and the pure protein aggregates. Interestingly, phase separation resulted in different effects on the foaming properties of the 3 BLG-κ-car complexes, in which BLGF-κ-car complexation in region II decreased the foaming properties in region I but the complexation of NGBLG-κ-car and BLGNP-κ-car in region II increased the foaming properties. The BLGF-κ-car complex in regions I and II provided the best emulsifying and foaming properties. Interfacial data both on oil-water and air-water interfaces overall explained the emulsifying and foaming properties of the complexes.

Amyloid aggregation of spin-labeled β-lactoglobulin. Part I: Influence of spin labeling on amyloid aggregation

Site-directed spin labeling (SDSL) of the natural food protein β-lactoglobulin (β-lg) was established with the aim of characterizing amyloid aggregation while explicitly avoiding the usual manipulation of primary protein structure. For its successful application, spin labels must not alter secondary protein structure or the formation of β-lg amyloid aggregates. The two spin labels—the MTSSL (flexible S–S binding) and Iodoacetamido-proxyl spin label (IPSL) (more rigid C–S binding)—were used for amyloid aggregation at pH 2 and pH 3.5. At pH 3.5, IPSL caused minor changes in the secondary protein structure, where it reduced intra- and intermolecular β-sheets as determined by ATR-FTIR. Analysis of the extent of amyloid aggregation using thioflavin T fluorescence indicated that the spin probes interfered with the binding of the fluorescent probes to the β-sheets. Non-amyloid and amyloid fractions were obtained from the amyloid aggregated system by ultrafiltration (300 kDa), which also proved to be equivalent and independent of the spin labeling process. Atomic force microscopy and size-exclusion chromatography results suggest the same building blocks and morphologies between unlabeled and spin-labeled proteins; therefore, substantial changes in the behavior of β-lg during amyloid aggregation can be excluded. Ultimately, 10–17% of the β-lg molecules were labeled so that the SDSL approach can be used to label the natural food protein at these low concentrations without affecting protein conformation or the formation of amyloid aggregates, which may subsequently provide deep insights into the aggregation mechanism of food proteins under processing conditions.

Amyloid aggregation of spin-labeled β-lactoglobulin. Part II: Identification of spin-labeled protein and peptide sequences after amyloid aggregation

Site-directed spin labeling (SDSL) of natural β-lactoglobulin (β-lg) was established. Combined electron paramagnetic resonance (EPR) and mass spectrometric analysis following tryptic digestion demonstrated that spin labels bind site-specifically but are not directed to all five cysteine residues to various preferred and reproducible extents. MTSSL and iodoacetamido-proxyl spin label (IPSL) were 80 and 60% reliably bound to the H strand, respectively, and combined in one spectral component and buried in the protein core. After heat incubation at pH 2 and fractionation, all labeled side chains (peptides) were part of the amyloid and non-amyloid fractions, even if they could not detect amyloid structures. It was assumed that the IPSL-labeled side chains of peptides with Cys160 from random coil were incorporated into small non-amyloid aggregates in non-polar environments. After heating at pH 3.5, a rearrangement of the previous α-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of β-sheets to the water/DMSO-environment. β-sheets were likely densely packed by the accumulation of intermolecular β-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary β-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a one-dimensional arrangement of the entire protein aggregates was similar to a string of pearls. Thus, SDSL of proteins containing several cysteine residues can be used to gain deep insights into the aggregation mechanism of proteins under food processing conditions.

Kinetics of denaturation and aggregation of highly concentrated β-Lactoglobulin under defined thermomechanical treatment

The influence of thermomechanical treatment (temperature 60 °C – 100 °C and shear rate 0.05 s−1 – 50 s−1) on the denaturation and aggregation of β-Lactoglobulin at high concentrations (50 % – 70 %) was investigated. While denaturation and aggregation reaction rate decreased at higher concentrations, a combination of heat and mechanical treatment resulted in an increase in reaction rates and consequently, in higher degrees of non-disulfide covalent bond aggregation. The kinetic parameters were determined by non-linear regression. Denaturation (combined effect of unfolding and aggregation) and covalent bond aggregation were found to follow 2.15 and 1.7 order kinetics, respectively. At the investigated conditions, the activation energy calculated for the denaturation and covalent bond aggregation reaction ranged from 25 to 283 kJ mol−1. Although the activation energy for the denaturation and aggregation reactions decreases with increasing shear rate, it was found that the mechanical treatment mainly affected the reactions at high temperatures, probably due to the increased molecular motion.

Heat-induced amyloid-like aggregation of β-lactoglobulin affected by glycation by α-dicarbonyl compounds in a model study.

α-Dicarbonyl compounds are widely generated in the Maillard reaction, caramelization and oil oxidation during heat treatment. These compounds can readily react with lysine and arginine residues of a protein, whereas the influence of these compounds on protein structure and quality has seldom been revealed. This study compared influence of glycation by glucose and α-dicarbonyl compounds on amyloid-like aggregation of β-lactoglobulin (β-LG), both fibrillation kinetics and conformation of aggregates were studied. Compared with glycation by glucose, the glycation by α-dicarbonyl compounds resulted in faster reduction of free amino group, sulfydryl group, and the relative content of β-sheet secondary structure, according to the ultraviolet (UV) spectra or circular dichroism (CD) spectra results. Based on the analysis of fibrillation kinetics using thioflavin T (ThT) binding assay, the glycation by α-dicarbonyls were more efficient in suppressing the growth of fibrillar aggregates. In addition, glycation by α-dicarbonyl resulted in amorphous oligomers, which were compared with the amyloid-like aggregates in control and glucose-glycated samples, based on the transmission electron microscopy (TEM) observation. Glycation by α-dicarbonyl compounds induced larger decline in the β-sheet structure of β-LG than glycation by glucose, and thus largely suppressed the amyloid-like aggregation of β-LG and changed the morphology of aggregates. © 2019 Society of Chemical Industry.

The effect of alkyl ammonium ionic liquids on thermal denaturation aggregation of β-lactoglobulin

Differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) measurements, and scanning and transmission electron microscope (SEM and TEM) of β-lactoglobulin (β-LG) are performed in alkyl (methyl, ethyl, and propyl) ammonium nitrate ionic liquids (RAN-ILs) – water mixtures at 25–100 °C. The addition of these RANs decreased the thermal denaturation temperature of β-LG, and the effect is larger with an increase of the hydrophobic part of the cation of the ILs. In ethyl ammonium nitrate (EAN) and propyl ammonium nitrate (PAN) mixtures, the fractal aggregate structure of β-LG was found after the thermal denaturation occurred. On the other hand, in methyl ammonium nitrate (MAN) mixture, an amorphous aggregate was formed as well as in NaCl and NaNO3 solutions. The difference of the effect between MAN and EAN (and PAN) could arise from the balance of hydrophobic/hydrophilic parts of the cation of RAN. These results are similar to those of alcohol-water mixtures. The nanoheterogeneous structure of the RAN-water mixture which consists of the hydrophobic and hydrophilic parts might affect the aggregation structure of β-LG.

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.