Β-lactoglobulin Solutions Research Articles

Accessing the thermal and electric effects in β-lactoglobulin denaturation and interaction with phenolic compounds

Protein-phenolic interactions have been on the spotlight in food research since they can change the structural, functional and nutritional properties of both compounds. Electric field processing technologies, such as ohmic heating (OHM), can contribute to improving food safety, quality and to tailor functionality of foods and ingredients. In this study, the potential of OHM processing in modifying protein-phenolic interactions was accessed. β-lactoglobulin (β-Lg) solutions were processed at 70 °C and 90 °C by conventional heating and OHM (26 V cm−1 at 50 Hz and 20 kHz), followed by their association with different phenolic compounds – i.e., quercetin (Qu), malvidin-3-glucoside (M3G), trans-resveratrol (TR), p-coumaric acid (p-C) and tyrosol (Ty). The protein solutions and protein-phenolic complexes were characterized by UV-spectroscopy, fluorescence spectroscopy, circular dichroism, and antioxidant capacity. Protein-phenolic interactions were confirmed by changes in the UV-absorbance spectra, reduction of the surface hydrophobicity and changes in the secondary structure of the protein. The formation of stable complexes was established through fluorescence quenching analysis and the binding affinities were found to be in the order TR > Qu > p-C > M3G > Ty. Furthermore, the binding parameters and changes of the protein's structural features were impacted by the processing conditions, including those associated with OHM. This study demonstrates for the first time that OHM processing and the control of its variables can potentially be used to tune β-Lg-PhCs interactions.

A model study on the site-specificity of (−)-epicatechin-induced reactions in β-lactoglobulin by high-resolution mass spectrometry in combination with bioinformatics

Polyphenol–protein reactions in model solutions of β-lactoglobulin (β-LG) incubated with (−)-epicatechin at 37 °C and 60 °C were monitored by microLC–timsTOF Pro-MS/MS combined with bioinformatics strategies. The addition of (−)-epicatechin to the model solutions resulted in changes in tryptic peptide profiles. Covalent bond formation between (−)-epicatechin o-quinones and β-LG was identified for the residues S27, S30, K60, C66, K69, and C160, with C160 being the predominant binding site. Furthermore, the incubation of β-LG with (−)-epicatechin significantly promoted oxidation, especially for the residues M7 and M24. The reaction of monomeric (−)-epicatechino-quinone at C160 was also identified in the milk chocolate sample. The adaptation of this study by extending the scope of the reaction products offers significant potential for comprehensive food profiling strategies.

Insight into the binding behavior, structure, and thermal stability properties of β-lactoglobulin/Amoxicillin complex in a neutral environment

Antibiotics are extensively used in agriculture and husbandry, thus threatening human health due to their direct exposure to dairy products. Therefore, it is essential to investigate whether milk proteins are unintended carriers of antibiotics. In this research, therefore, we studied the impact of Amoxicillin on the conformation and structural stability of whey protein β-lactoglobulin by using spectroscopic techniques combined with molecular docking and simulation approaches. Further, the thermal stability of the resultant complex was explored. Amoxicillin was observed to effectively quench β-lactoglobulin's intrinsic fluorescence, suggesting that Amoxicillin was successfully bound to the β-lactoglobulin and a stable complex between them was established. The thermodynamic parameters demonstrated that van der Waals forces and hydrogen bonds dominated the spontaneous bonding process. According to circular dichroism (CD) and Fourier transform infrared (FTIR) analyses, the conformational structure of the β-lactoglobulin was altered upon the complexation with Amoxicillin. Further, this interaction significantly enhanced the thermal stability of β-lactoglobulin compared to sole β-lactoglobulin solutions. Molecular docking theoretically showed the preferred binding locus of Amoxicillin within the β-lactoglobulin structure. Molecular dynamics (MD) simulation results further validated the stability of the β-lactoglobulin-Amoxicillin complex. The findings obtained here are helpful for elucidating the potential of β-lactoglobulin to bind and transport harmful substances into the body, thus opening up new avenues for food security.

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).

Influence of thermally induced structure changes in diluted β-lactoglobulin solutions on their surface activity and behavior in foam fractionation

Surface activity is an intrinsic protein feature, leading to the capability of aqueous protein solutions to form foam. This feature provides opportunities for downstream processing, such as usage of foam fractionation for purification. In order to investigate the impact of the surface activity on the performance of the foam fractionation process, protein solutions with different surface activity were produced by different thermal denaturation of aqueous β-lactoglobulin solutions. The effectiveness of the denaturation procedure was verified with circular dichroic spectroscopy, and the impact on surface activity was determined via dynamic surface tension measurement. The increased surface activity resulted in higher foamate flow rates. Furthermore, the effects could be correlated with secondary structure changes and with the dynamic surface pressure. The new result of this study is that the effect of the denaturation of a protein on foam fractionation depends on the protein concentration. At the lower feed concentration, effects became visible, which could not be observed at the higher concentration.

Surface tension and dilational rheology of mixed β-casein – β-lactoglobulin aqueous solutions at the water/air interface

Bubble profile analysis tensiometry was used to study the surface tension and dilational visco-elasticity of individual and mixed solutions of β-casein (BCS) and β-lactoglobulin (BLG) at various mixing ratios of the components. The results for the individual protein solutions were analysed based on a new theory, which is applicable in the pre-critical and post-critical concentration range of the surface tension isotherm. It is shown that these proteins are capable of displacing each other from the adsorption layer, which affects the surface tension and visco-elasticity modulus of the adsorbed layers. The model developed for protein mixtures agrees well with the experimental data and the calculated dependencies of the surface properties on the solution composition confirm the mutual influence of added proteins.

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.

FTIR Spectroscopic Study of Heat-Induced β-Lactoglobulin Solution under Flow Field

FTIR Spectroscopic Study of Heat-Induced β-Lactoglobulin Solution under Flow Field

Interfacial shear rheology of β-lactoglobulin—Bovine submaxillary mucin layers adsorbed at air/water interface

The interfacial rheological properties of solutions of β-lactoglobulin (BLG), as a model food compound, mixed with bovine submaxillary mucin (BSM), a major salivary protein, have been investigated. Time, frequency, stress sweep and flow measurements have been performed at different pHs (7.4, 5.0 and 3.0), to investigate the air/water interfacial properties. All protein layers (BLG, BSM, and BLG-BSM mixtures) formed an elastic network at the air/water interface with low frequency dependence of the interfacial modulus. The results indicated that BLG moves faster as smaller molecule than mucin, and dominate the surface adsorption and the network formation for the BLG-BSM mixtures. Moreover, BLG-BSM protein mixtures exhibited interfacial properties with lower elastic and viscous moduli than BLG, as a result of competitive displacement of BLG proteins with BSMs from the interface. It is suggested that hydrophobic patches of BSM can be imbedded into the BLG monolayer as driven by a strong hydrophobic interaction with air and disrupt the cohesive assembly of BLG, whereas the hydrophilic (negatively charged) parts of the BSM chain are protruding from the interface towards the bulk water.

Influence of the Protein Particle Morphology and Partitioning on the Behavior of Particle-Stabilized Water-in-Water Emulsions.

Protein fibrils, microgels, and fractal aggregates were produced by heating solutions of β-lactoglobulin (β-lg) under different conditions. The effect of the protein particle morphology on the stability and the structure of water-in-water (W/W) emulsions was studied for mixtures of poly(ethylene oxide) (PEO) and dextran. The protein particles partition to the dextran phase at pH 7.0 where they have a net negative charge, but they prefer the PEO phase at pH 3.0 where they have a net positive charge. The effect of partitioning on the stability and the structure of water-in-water (W/W) emulsions was studied by comparing emulsions at pH 3.0 with those at pH 7.0. The protein particle morphology and preference for one phase or the other are shown to have important consequences for the stability and the structure of the emulsions. Fibrils were found to be the most effective stabilizers at pH 7.0, whereas fractals were most effective at pH 3.0. The average droplet size obtained from confocal scanning laser microscopy was for most systems between 10 and 5 μm but was notably smaller for emulsions with fractals at pH 3.0.

Correlation between bulk characteristics of aggregated β-lactoglobulin and its surface and foaming properties

Bovine β-lactoglobulin features a pronounced surface activity, and therefore, is widely applied in order to provide stability to food-related aerated systems. Due to its distinct tertiary and quaternary structure, it can be used both in native state as well as in the form of thermally aggregated particles. In this context, heat treatment (80 °C/90 min) of highly purified solutions of β-lactoglobulin (c = 10.0 g L−1) under variation of solution pH (6.8 or 8.0) and NaCl concentration (0–130 mM) resulted in the formation of soluble aggregates, whose median particle diameters ranged from about 2.5 nm to 1.1 μm. These differences in particle size in combination with differences in particle characteristics (e.g. surface hydrophobicity, zeta potential) had a significant impact on surface properties, i.e. surface tension, dynamics of protein adsorption and interfacial dilatational properties. Thereby, diffusion rates decreased with increasing median particle diameter. Initial protein adsorption was majorly influenced by surface hydrophobicity. However, reverse observations emerged in terms of foam characteristics. Foam stability clearly increased with increasing aggregate size, showing maxima for the largest particles examined. The increasing foam stabilization ability was also reflected in time-resolved bubble properties, e.g. lower mean bubble areas as well as higher bubble counts for larger aggregates. This observation was traced back to the increasing surface hydrophobicity as well as more negative zeta potential of the aggregates with increasing particle diameter. As a result, sterically stabilized and more impermeable surface films in combination with electrostatic repulsive forces led to a reduction in coalescence rate as indicated by a lower coarsening exponent, and thus, a decrease of foam decay.

The effect of aggregation into fractals or microgels on the charge density and the isoionic point of globular proteins

Potentiometric titration curves were obtained for aqueous solutions of native whey protein isolate (WPI) and β-lactoglobulin (β-lg) at different NaCl concentrations. The curves crossed at approximately the same pH, which is considered to be the isoionic point (IIP). Titration with NaCl confirmed that the pH was independent of the salt concentration up to 0.1 M at the IIP, which was 4.9 for WPI and 5.0 for β-lg. Fractal aggregates and microgels of different sizes were formed by heating protein solutions at different pH and different concentrations. The titration curves of the aggregates depended on the type of aggregates, but not on their size. The IIP increased by at most 0.2 pH units after aggregation. For a given pH larger than IIP, the charge density of the proteins (α) was reduced after denaturation and aggregation. The reduction was stronger for microgels than for fractal aggregates. Addition of NaCl or increasing the protein concentration mitigated the effect. Comparison between WPI and β-lg showed that the pH dependence of α was almost the same for pH > 5.0 both for native and aggregated proteins.

Role of ovomucoid in the gelation of a β-lactoglobulin-ovomucoid mixture

The effect of ovomucoid on gelation of β-lactoglobulin—as induced by heating and subsequent cooling—was investigated using a mixture of 5 % (w/v) ovomucoid/5 % (w/v) β-lactoglobulin and pure β-lactoglobulin solutions (5 and 10 % (w/v)) with subsequent analysis by rheological measurement, ultrasonic spectroscopy, scanning electron microscopy, and sodium dodecyl sulfate polyacrylamide electrophoresis. For the three systems, the dynamic modulus of the mixed-protein sample was smaller than that of either of the two pure β-lactoglobulin samples. Although ultrasonic-relative velocity temperature sweeps for all samples showed that the relative velocities decreased with increasing temperature, the gradient values differed. Namely, the decrease for the mixed-protein sample (12 m/s) was intermediate between those of the pure β-lactoglobulin systems. Ultrasonic attenuations of all samples increased with increasing temperature, and the absolute attenuation value of the mixed-protein sample was also intermediate between those of the two pure β-lactoglobulin samples. Electrophoresis performed with or without 2-mercaptoethanol suggested that ovomucoid forms an aggregate with β-lactoglobulin via intermolecular disulfide bonds. Together, these results suggest that ovomucoid has a synergistic effect on β-lactoglobulin gelation despite the great heat stability.

Effect of pH and electrolyte concentration on rising air bubbles in β-lactoglobulin solutions

The local velocity profile (LVP) of a rising bubble can serve as a fingerprint for the dynamic behavior of the adsorption/desorption processes going on at a solution/air interface. The LVP of bubbles in β-lactoglobulin (BLG) solutions proves to be extremely sensitive for the adsorption of BLG at low bulk concentrations. In addition, it can show the impact of pH and ionic strength on the dynamic surface properties of BLG at the solution/air interface. It is observed that the time for establishing an immobile rigid surface layer at the rising bubble surface becomes shorter with increasing pH from 3 to 7. A peculiar behavior is observed at the isoelectric point (IEP) where the LVPs show irregularities. Under dynamic conditions BLG does not exhibit highest surface active at the IEP within the range of BLG concentrations as measured by the rising bubble technique.

Role of dietary antioxidant (−)-epicatechin in the development of β-lactoglobulin fibrils

Under specific physico-chemical conditions β-lactoglobulin is seen to form fibrils structurally highly similar to those that are formed by the amyloid-like proteins associated with neurodegenerative disorders, such as Alzheimer and Parkinson diseases. In the present study we provide insights on the possible role that the dietary flavonoid (−)-epicatechin plays on β-lactoglobulin fibril formation. Fibril formation is induced by keeping β-lactoglobulin solutions at pH2.0 and at a temperature of 80°C for 24h. Atomic Force Microscopy measurements suggest that, by adding (−)-epicatechin in the solution, fibrils density is visibly lowered. This last observation is confirmed by Fluorescence Correlation Spectroscopy experiments. With the use of Fourier Transform IR spectroscopy we monitored the relative abundances of the secondary structures components during the heating process. We observed that in the presence of (−)-epicatechin the spectral-weight exchange between different secondary structures is partially inhibited. Molecular Dynamics simulations have been able to provide an atomistic explanation of this experimental observation, showing that (−)-epicatechin interacts with β-lactoglobulin mainly via the residues that, normally in the absence of (−)-epicatechin, are involved in β-sheet formation. Unveiling this molecular mechanism is an important step in the process of identifying suitable molecules apt at finely tuning fibril formation like it is desirable to do in food industry applications.

β-lactoglobulin as food grade surfactant for clove oil-in-water and limonene-in-water emulsion droplets produced by microchannel emulsification

Unheated and heat-aggregated β-lactoglobulin (β-lg) solutions were used to stabilize clove oil-in-water emulsion and limonene-in-water emulsion prepared by microchannel (MC) emulsification. The size of the MC array plate was 15 × 15 mm2 consisting of 100 parallel MCs fabricated on each side of the plate. The channels were 4 μm depth, 71 μm length, and 8.2 μm width, with the terrace length of 29.1 μm. Unheated and heat-aggregated β-lg effectively stabilized clove oil-in-water and limonene-in-water emulsions during production using MC emulsification. The emulsion droplets were steadily produced by the channels. The average diameter of clove oil droplets was around 17 μm, with both unheated and heat-aggregated β-lg, regardless of the concentration of β-lg. The average diameter of the limonene droplets was 18–26 μm depending on the conditions (unheated or heat-aggregated) and the concentration of β-lg. The coefficient of variation for all average droplet diameters was ≤8%, which was an indication of monodisperse droplets. Clove oil-in-water emulsion droplets became polydisperse within 2 h observation, whilst limonene-in-water emulsion droplets remained monodisperse over time. The stability of the oil droplets over time related to the solubility of the oil phases rather than the effects of β-lg as the surfactant. In addition, the droplet sizes at different surfactant concentrations seemed to relate to the viscosity ratio between the dispersed phase and the continuous phase rather than the effects of the surfactant.

β-Lactoglobulin adsorption layers at the water/air surface: 1. Adsorption kinetics and surface pressure isotherm: Effect of pH and ionic strength

The dynamic surface pressure Π of air bubbles aging in buffered β-lactoglobulin (BLG) solutions containing various protein concentrations CBLG (10−9–10−4M), pH (3–7) and buffer concentrations Cbuff (1, 10 and 100mM) was measured as a function of time t by bubble profile analysis tensiometry. Adsorption kinetics was studied by recording Π(t) data for 80000s and the final Π-values were used to construct the surface pressure isotherm Π(CBLG) for the pH values of 3, 5, 7 (Cbuff=10mM) and 6.3 (in pure water). On the basis of obtained kinetic and concentration dependencies the effect of pH on the protein surface activity was qualitatively analyzed. At constant Cbuff=10mM and relatively low protein concentrations (CBLG<10−8M), BLG exhibits the shortest induction time τind and highest Π-values at pH 7 (negative net charge), in comparison to pH 5 and 3, whereas at sufficiently high protein concentrations (ca. CBLG>10−6M) BLG is the most surface active at pH 5 (negligible net charge in the vicinity of the isoelectric point, pI ≈5.1). At constant Cbuff, BLG solutions with pH 3 show the lowest surface activity. The influence of the ionic strength (buffer concentration) of the solution on the dynamic Π was studied at a selected protein concentration of CBLG=10−5M. The kinetics of adsorption is weakly affected by Cbuff for solutions with pH 5 and significantly enhanced by increasing Cbuff for solutions with pH≠pI. The effect of the chemical nature of the electrolyte was obtained for non-buffered aqueous BLG solutions (natural pH ∼6.3) at different concentrations of NaCl or CaCl2. It is demonstrated that the adsorption kinetics and the surface pressure isotherm of BLG at the water/air surface depend strongly on the protein effective charge, which is dictated by the solution pH and ionic strength.

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).

Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption.

A versatile, convenient, and cost-effective method that can be used for grafting antifouling materials onto different surfaces is highly desirable in many applications. Here, we report the one-step fabrication of antifouling surfaces via the polymerization of dopamine and the simultaneous deposition of anionic hyaluronic acid (HA) on Au substrates. The water contact angle of the Au surfaces decreased from 84.9° to 24.8° after the attachment of a highly uniform polydopamine (PDA)/HA hybrid film. The results of surface plasmon resonance analysis showed that the Au-PDA/HA surfaces adsorbed proteins from solutions of bovine serum albumin, lysozyme, β-lactoglobulin, fibrinogen, and soybean milk in ultralow or low amounts (4.8-31.7 ng/cm(2)). The hydrophilicity and good antifouling performance of the PDA/HA surfaces is attributable to the HA chains that probably attached onto their upper surface via hydrogen bonding between PDA and HA. At the same time, the electrostatic repulsion between PDA and HA probably prevents the aggregation of PDA, resulting in the formation of a highly uniform PDA/HA hybrid film with the HA chains (with a stretched structure) on the upper surface. We also developed a simple method for removing this PDA/HA film and recycling the Au substrates by using an aqueous solution of NaOH as the hydrolyzing agent. The Au surface remained undamaged, and a PDA/HA film could be redeposited on the surface, with the surface exhibiting good antifouling performance even after 10 such cycles. Finally, it was found that this grafting method is applicable to other substrates, including epoxy resins, polystyrene, glass, and steel, owing to the strong adhesion of PDA with these substrates.

Influence of substrate nature and β-lactoglobulin on cleanability after soiling by suspension spraying and drying

Glass and stainless steel (StSteel, AISI304-2R), previously cleaned with ethanol (-Eth) or with ethanol and UV–Ozone treatment (-UVO), were soiled with quartz suspensions in water and in a β-lactoglobulin (β-LGB) solution, and dried. The cleanability (ease of quartz particle detachment) in water was evaluated using a radial-flow cell. The soiling suspension containing β-LGB was used as such or after heating for 4h at 75°C, which provoked coagulation of about 75% of β-LGB. The substrate–solution interfaces were characterized by X-ray photoelectron spectroscopy (XPS) analysis of conditioned substrates and by contact angle measurements. The substrate surfaces are covered by a layer of organic contaminants which are not removed by pre-cleaning or are adsorbed from the surroundings. The presence of β-LGB in the soiling suspension leads to protein adsorption, but a significant amount of contaminants remains at the surface.For three of the substrates tested (Glass-Eth, Glass-UVO, StSteel-UVO) the increase of cleanability when the soiling suspension contained β-LGB may be explained by lower capillary forces acting upon drying. Capillary forces are proportional to the liquid surface tension and depend in a less important way on substrate contact angle. However the order of cleanability observed for the substrates soiled with a suspension of quartz particles in water (Glass-Eth≅Glass-UVO<StSteel-UVO<StSteel-Eth) and the influence of β-LGB on the cleanability of StSteel-Eth may not be explained only by computed capillary forces. The contact angle may exert a direct influence on droplet spreading and particle–substrate contact. The organic contaminants present on the surfaces, which are often neglected by supposing model solid surfaces, may have a significant influence on cleanability through physico-chemical processes which remain to be appreciated.