Properties Of Β-lactoglobulin Research Articles

Analysis of non-covalent interaction between β-lactoglobulin and hyaluronic acid under ultrasound-assisted treatment: Conformational structures and interfacial properties

Hyaluronic acid (HA) used as a food ingredient is gaining acceptance and popularity. However, the studies available for the effect of HA concentrations on the properties of β-lactoglobulin (β-LG) were limited. In this study, we investigated that the molecular characterization and functional properties of the complex formed by the non-covalent binding of β-LG and HA, as well as the ultrasound-assisted treatment at acidic pH. The optimal pH and ratio of β-LG/HA were set as 7 and 4:1, respectively. The fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking results revealed that the addition of HA and ultrasound induced a decrease in random coil and α-helix and an increase in β-sheet contents in β-LG. By the complexation with HA, the thermal stability, freezing stability, and antioxidant properties of β-LG were all improved under ultrasound treatment. The results of the present study can be useful for the modulation of HA based biopolymer complexes and the exploitation as encapsulating or structuring agents in food industry.

Structure-property relations of β-lactoglobulin/κ-carrageenan mixtures in aqueous foam

There is a great interest in mixed protein/polysaccharide layers at air-water interfaces due to their ability to stabilize aqueous foams. Properties of β-lactoglobulin (BLG)/κ-carrageenan mixtures at different κ-carrageenan concentrations were studied using foam analysis and molecular-level characterizations at the air-water interface. Because air-water interfaces are a major element in aqueous foams, they were investigated with a combination of interface-sensitive methods such as sum-frequency generation (SFG) as well as surface tensiometry. Using SFG spectroscopy we provide new information on the changes in the interfacial charging state and composition when the BLG/κ-carrageenan mixing ratio changes. κ-carrageenan is only weakly surface active as it does not lower the surface tension to a large extend. However, BLG/κ-carrageenan mixtures showed a synergistic effect because the formation of complexes at the air-water interface and in the bulk solution can favorably influence foam stability. BLG (7.5 µM) was mixed with different κ-carrageenan concentrations from 0 to 1 mM while the bulk pH was adjusted to pH values which are below (4.5), slightly above (5.6) and well above (7.0) the isoelectric point of BLG. At pH 4.5, the positive ζ-potential of BLG was drastically reduced to negative values and large aggregates in the bulk formed that showed a reduced surface activity and caused also a reduced net charging state at the air-water interface. The interfacial and bulk properties at pH 4.5, resulted in a poor stability of foams that were likely only stabilized by the weak electrostatic disjoining forces in the foam lamella. At pH 5.6 and 7, cooperative effects of BLG and κ-carrageenan dominate and, in particular, at pH 5.6 give rise to an increase in BLG surface coverage when the κ-carrageenan concentration increases. This resulted in a substantially improved foam stability.

Impact of Saturation of Fatty Acids of Phosphatidylcholine and Oil Phase on Properties of β-Lactoglobulin at the Oil/Water Interface

Oil in water emulsions are commonly stabilized by emulsifying constituents like proteins and/or low molecular weight emulsifiers. The emulsifying constituents can compete or coexist at the interface. Interfacial properties thus depend on molecular structure of the emulsifying constituents and the oil phase and the resulting molecular interactions. The present study systematically analyzed the impact of fatty acid saturation of triacylglycerides and phosphatidylcholine on the interfacial properties of a β-lactoglobulin-stabilized interface. The long-term adsorption behaviour and the viscoelasticity of β-lactoglobulin-films were analyzed with or without addition of phosphatidylcholine via drop tensiometry and dilatational rheology. Results from the present study showed that increasing similarity in fatty acid saturation and thus interaction of phosphatidylcholine and oil phase increased the interfacial tension for the phosphatidylcholine alone or in combination with β-lactoglobulin. The characteristics and stability of interfacial films with β-lactoglobulin-phosphatidylcholine are further affected by interfacial adsorption during changes in interfacial area and crystallization events of low molecular weight emulsifiers. This knowledge gives guidance for improving physical stability of protein-based emulsions in foods and related areas.Graphic abstract

Effect of Oil Hydrophobicity on the Adsorption and Rheology of β-Lactoglobulin at Oil-Water Interfaces.

The adsorption of protein layers at oil-water interfaces is critical to the formation and stability of various emulsions in, for example, technical applications as well as in biological lipid storage. Effects of ionic strength, pH, temperature, and pretreatments of the proteins are well-known. However, the oil phase has been regarded as exchangeable and its role in protein adsorption has been widely ignored. Herein, the influence of systematically selected oil interfaces of high purity on the formation and properties of β-lactoglobulin (β-lg) adsorption layers was evaluated. Droplet profile tensiometry and interfacial rheometry were employed to determine the adsorption kinetics and dilatational and interfacial shear moduli. We show that depending on the molecular size, flexibility, hydrophobicity, polarity, and polarizability of the oils, globular proteins adsorb distinctively. Stronger interactions of polar oils with the hydrophilic exterior of the native β-lg lead to decelerated protein unfolding. This results in lower surface pressures and slower formation of viscoelastic networks. In addition, polar oils interact stronger with the protein network by hydrophilic bonding and thereby act as softening agents. The observed effects of hydrophobic subphases on the adsorbed protein layers provide knowledge, which promotes higher reproducibility in rheological studies and precise tailoring of interfacial films for enhanced formation and stability of emulsions.

Effect of glycation and limited hydrolysis on interfacial and foaming properties of bovine β-lactoglobulin

The effect of limited hydrolysis followed by glycation with galactose, and vice versa, on interfacial and foaming (foamability and foam stability) properties of β-lactoglobulin (β-Lg) at pH 7 and pH 5 has been studied. Hardly any effect of the two treatments, hydrolysis and glycation (HG) or glycation and hydrolysis (GH), on the β-Lg foaming capacity at both pH values was observed. Foam stability, however, was significantly enhanced after both HG and GH at pH 5. Particularly, the system obtained after glycation at 50 °C followed by limited hydrolysis showed an exceptional stability, which might be related to the increase in elastic character and cohesion of the interfacial film, indicated by the increase of the surface dilatational modulus (E) and the decrease of the phase angle (ϕ) over the time. These results indicated that glycation of β-Lg with galactose followed by limited hydrolysis might allow extending the use of this protein as foaming agent, although further research in complex food systems is needed to apply this method in the formulation of acidic foods and beverages.

Emulsifying properties of β-lactoglobulin and Quillaja bark saponin mixtures: Effects of number of homogenization passes, pH, and NaCl concentration

ABSTRACTThe effects of number of homogenization passes, pH, and NaCl concentration on the formation and stability of oil-in-water emulsions comprising a mixture of a biosurfactant (Quillaja bark saponin) and a globular protein (β-lactoglobulin) were investigated. The emulsions were characterized as to visual appearance, droplet size, droplet surface charge, and rheology. The emulsions obtained by different conditions (4, 6, or 8 passes; pH 7, 8, or 9; and 0, 100, or 200 mmol L−1 of NaCl) were polydisperse, presented relatively small average droplet sizes (z-average < 323 nm) as well as negative droplet charge (between –20 and –79.6 mV) in all evaluated conditions. Regardless of the number of homogenization passes, the emulsions exhibited low apparent viscosity and pseudoplastic behavior with small yield stress. Viscoelastic behavior was also observed, thus the emulsions were characterized as weak gels. Four homogenization passes were enough to obtain small droplets in the evaluated conditions. Droplet size was not significantly affected by NaCl concentration and pH (p > 0.05). On the other hand, the absolute ζ-potential values significantly decreased and increased upon increased NaCl content and pH, respectively. Regardless of the tested conditions, all emulsions had good stability against phase separation and droplet aggregation, since no significant changes in average droplet size were observed throughout storage (p > 0.05). In the presence of NaCl, in which droplet charge significantly decreased, emulsion was also stable. Thus, we can conclude that electrostatic repulsion as well as steric repulsion was responsible for stabilization.

Effect of calcium chloride and moderate shear on β-lactoglobulin aggregation in processing-like conditions

The influence of two variables (moderate shear and calcium chloride concentration) on the properties of β-lactoglobulin (β-lg) aggregate suspensions obtained by dynamic heat treatment (85°C for 60s) was studied. Variations in calcium chloride concentration from 5.1 to 7.1mM were imposed, and led to major changes in the aggregate properties. The characterization of the aggregate size was performed using three distinct methods: laser granulometer, dynamic light scattering (DLS) and focus beam reflectance measurement (FBRM). Viscosity and residual native fraction were also determined. Statistical analysis of experimental data underlines the important combined effect of the two variables and paves the way for new ways of controlling the aggregate functionalities. The calcium chloride concentration has an effect on the entire aggregation process, from native β-lg to large aggregates. On the other hand, shear only impacts the formation of the large aggregates.

Influence of pH and ι-Carrageenan Concentration on Physicochemical Properties and Stability of β-Lactoglobulin-Stabilized Oil-in-Water Emulsions

The influence of pH and iota-carrageenan concentration on the properties of beta-lactoglobulin (beta-Lg)-stabilized oil-in-water emulsions was investigated by measuring the particle charge, particle size distribution, and creaming stability. Emulsions containing droplets stabilized by beta-Lg were produced by homogenization, and then, iota-carrageenan was added. At pH 3, the droplet charge did not change for iota-carrageenan concentrations <or=0.1 wt % but decreased rapidly at high concentrations, while the mean particle diameter increased slightly as the iota-carrageenan concentration was increased. These results suggest that the interaction between iota-carrageenan and beta-Lg was weak at pH 3 probably because some sulfate groups were protonated (pK(a) = 2). At pH 4 and pH 5, the droplet charge decreased dramatically as the iota-carrageenan concentration was increased from 0 to 0.15 wt %, but droplet aggregation and creaming occurred in the emulsions, indicating that interfacial complexes between iota-carrageenan and beta-Lg could not stabilize the emulsions, probably due to bridging flocculation. At pH 6, the droplet charge in the primary emulsions was negative and became more negative as the iota-carrageenan concentration was increased. The mean particle diameter was relatively small at all iota-carrageenan concentrations, and emulsions were stable to creaming after 1 week of storage. We propose that carrageenan adsorbed to the droplet surfaces and increased the electrostatic repulsion between droplets. At pH 7 and pH 8, the droplet charge did not change as the iota-carrageenan concentration was increased, but these emulsions became unstable to creaming above a critical carrageenan concentration, which was attributed to depletion flocculation.

Separation and characterization of β-lactoglobulin and α-lactalbumin from whey and whey protein preparations

The properties of β-lactoglobulin (β-Lg) and α-lactalbumin (α-Lac) prepared from commercial samples of liquid whey (LW), whey protein isolate (WPI), or whey protein concentrate (WPC), using the same fractionation process, were studied. The process was based on β-Lg solubility at low pH in the presence of salt, on the weak calcium binding capacity of α-Lac below pH 3.9, and on the ease of chelating the calcium with calcium sequestrants, causing destabilization and precipitation of the calcium-free form of α-Lac. Sodium citrate (NaC) and sodium hexametaphosphate (SHMP) were more effective for the separation of β-Lg and α-Lac from the whey protein sources than the other tested chelating agents. Recoveries of 47–69% of β-Lg originally present in the whey preparations were recorded, with purities ranging from 84% to 95%, and protein contents ranging from 40% to 99%, depending on the source of whey protein and type of chelating agent. The yields of α-Lac in α-Lac fractions obtained without pH adjustment were 23–89%, with purities ranging from 83% to 90%, and protein contents ranging from 65% to 96%; the yields of α-Lac in α-Lac fractions obtained with adjustment to pH 7.5, were 11–43%, with purities ranging from 68% to 73%, and protein contents ranging from 44% to 81%. The ash contents of some protein fractions were relatively high. Glycation was detected in the proteins, with the highest degree of glycation in the proteins from WPC, resulting in higher molecular weights of the proteins from WPC when compared with those from LW and WPI.

Effects of medium and chemical modification on thermal characteristics of Β-lactoglobulin

The thermal properties of Β-lactoglobulin (Β-LG) were studied by differential scanning calorimetry (DSC) under different medium conditions.pH, neutral salts, protein perturbants, and polyols all affected the DSC characteristics of Β-LG. Acylation with fatty acids also changed the thermal properties, particularly peak width at half-height. The results suggest that the structural stability of Β-LG is controlled by non-covalent forces, particularly electrostatic and hydrophobic interactions. Disulfide bonds did not contribute to the thermal response of Β-LG. Fatty N-acyl-amino acids caused marked increases in thermal stability and decreases in denaturation enthalpy, and additional peaks were observed in the presence of some palmitoyl derivatives.

Reductive Methylation of β-Lactoglobulin

The effect of reductive methylation on the properties of β-lactoglobulin was studied with preparations in which 6, 32, or 80% of the amino groups were methylated. Content of sulfhydryl groups was not changed and little difference in electrophoretic mobility of the protein was evident on polyacrylamide gel electrophoresis. Thin-layer isoelectric focusing from pH 4 to 6 indicated that protein in which 80% of the amino groups were modified showed an increase in isoelectric point of no more than .1 pH unit for each electrophoretic component, compared with untreated β-lactoglobulin (A plus B variants). Reductive methylation with [carbon-14] formaldehyde at low reagent concentration yielded a product which contained approximately one mole of [carbon-14] methyl groups for each mole of β-lactoglobulin. Tracer [carbon-14]β-lactoglobulin added to skim milk exhibited a heat-denaturation curve similar to that described for unmodified β-lactoglobulin. Such a radio-actively labeled derivative will be useful for studying protein interactions in milk.

The detection and occurrence of bovine β-lactoglobulin C

A rapid simple method of starch-gel electrophoresis for the detection of bovine whey protein types is described. Only small samples of skim milk are required. No prior separation of the whey proteins is needed. Excellent resolution and reliable results are obtained providing careful attention is given to the starch composition and concentration of the pH 8.5, NaOH-H 3BO 3, gel buffer. This method has revealed the existence of a new β-lactoglobulin variant which has been designated Type C. The incidence of β-lactoglobulin C has been examined in five breeds and it has been found only in the Jersey. The significance of this finding is discussed. The isolation and properties of β-lactoglobulin C are described in the following paper.

Application of Polarization of Fluorescence Technique to Protein Studies. I. The Rotatory Properties of β-lactoglobulin

Summary A study was conducted using the polarization of fluorescence method to characterize β -lactoglobulin. The method involved forming a fluorescent dye conjugate of the protein molecule with a dye molecule (1-dimethylaminonaphthalene-5-sulfonyl chloride) which imparted fluorescent properties to the conjugate. Only minor alteration in the physical properties of the protein was produced by the conjugation process. Curves of reciprocal polarization vs. T/η for β -lactoglobulin yielded an average slope of 1.02×10 −4 at 0.02 ionic strength and 0.72×10 −4 at 0.10 ionic strength. The apparent molecular volume of a spherical protein molecule, having the same relaxation time as the slightly elongated β -lactoglobulin molecule, was 98,310 cm3 at 0.10 ionic strength and 64,580cm 3 at ionic strength 0.02. The mean harmonic relaxation time for β -lactoglobulin was 6.85×10 −8 sec at 0.02 ionic strength and 10.91×10 −8 sec at 0.10 ionic strength. Addition of sucrose to protein solutions to increase the viscosity and, thereby, reduce the rate of rotatory diffusion of the fluorescent β -lactoglobulin molecule, resulted in substantial reduction in the apparent molecular volume.

The determination of certain properties of β-lactoglobulin and its lactose derivatives

In an attempt to elucidate further the browning reaction in milk, two purified components, crystalline β-lactoglobulin and lactose, have been reacted in solution. This reaction set in immediately at 53°C., as shown by the rapid formation of fluorescent products. The increase in optical density, depicting further steps in the browning reaction, showed a short lag period and then rapid development. With these changes in physical properties were associated a decrease in free amino and basic groups and a resistance to enzymic digestion, particularly in regard to the release of lysine. In order to ascertain the possible role of cysteine in this reaction, the thiol groups were determined. Native β-lactoglobulin was found to contain 0.50 sulfhydryl groups/10 4 g. or 2 groups/mole. A reduction of these groups was observed in the control experiment and no further destruction was noted upon browning.