Β-lactoglobulin Gels Research Articles

Characterization of β-lactoglobulin gels induced by high pressure processing

Abstract High pressure processing (HPP) is a non-thermal technology and has been widely used in the food industry. β‑lactoglobulin (β-Lg) is a globular protein and susceptible to pressure. This study investigated the properties of β-Lg gels induced directly by HPP (0.1–600 MPa for 30 min, 25 °C) with different protein concentrations at different pH. Results showed that the lowest protein concentration required to form β-Lg gels was 20% (w/v) at 400 MPa for 30 min at pH 3.0, 5.0 and 7.0, while 14% (w/v) protein was required to form gels at 600 MPa. The gel strength and textural properties increased with the increase of protein concentration and pressure, the gel formed at pH 5.0 had the highest strength. Raman spectra suggested that the content of α-helix decreased and random coil increased of β-Lg in gels with the increase of pressure. β-Lg gels formed more regular and stable network under 600 MPa than that under 400 MPa. Industrial relevance In recent years, high pressure processing (HPP) has been used in the food industry as an innovative technology to improve gel properties of various kinds of proteins, including β‑lactoglobulin, casein, soy protein isolate, surimi, mytolin and so on. It was found that HPP could change the structure of protein, which could cause protein denaturation and aggregation and three-dimensional network structures were formed through disulfide bonds, electrostatic interactions, hydrophobic interaction, hydrogen bonds and others. β‑lactoglobulin is the main component of whey protein, which has been widely used in the food industry as gelling agents. β‑lactoglobulin gels could be used as a wall material and applied to the delivery system of functional components such as riboflavin. The objective of the current study was to investigate the properties of β-Lg gels formed through HPP directly, focusing on the effects of pressure, protein concentration and pH on the rheological properties, texture profile, protein secondary structure and microstructure of β-Lg gels.

On the relationship between the plateau modulus and the threshold frequency in peptide gels.

Relations between static and dynamic viscoelastic responses in gels can be very elucidating and may provide useful tools to study the behavior of bio-materials such as protein hydrogels. An important example comes from the viscoelasticity of semisolid gel-like materials, which is characterized by two regimes: a low-frequency regime, where the storage modulus G'(ω) displays a constant value Geq, and a high-frequency power-law stiffening regime, where G'(ω) ∼ ωn. Recently, by considering Monte Carlo simulations to study the formation of peptides networks, we found an intriguing and somewhat related power-law relationship between the plateau modulus and the threshold frequency, i.e., Geq∼(ω*)Δ with Δ = 2/3. Here we present a simple theoretical approach to describe that relationship and test its validity by using experimental data from a β-lactoglobulin gel. We show that our approach can be used even in the coarsening regime where the fractal model fails. Remarkably, the very same exponent Δ is found to describe the experimental data.

Dissolution of bovine serum albumin hydrogels in alkali

Heat-induced protein gelled deposits, occurring during dairy processing, are industrially cleaned with caustic-based solutions. In order to develop general dissolution mechanisms of protein hydrogels, in analogy to general gelation mechanisms for globular proteins, different protein systems must be characterized. We report the first characterization of the dissolution behaviour of heat-set pure bovine serum albumin (BSA) hydrogels. At low alkali concentrations (<0.2 M NaOH), the formed BSA gels were dissolved as observed for whey protein mixtures or pure β-lactoglobulin gels. The key rate limiting step is suggested to be the destruction of inter-protein non-covalent interactions. At higher NaOH concentrations (>0.2 M) the dissolution rate of BSA gels was fairly constant, unlike seen before. Activation energy estimations of the rate yield very low value in certain conditions, again unlike seen before. It is suggested that mass transfer limitations occur during dissolution due to the large BSA size.

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 High-Pressure Processing on β-lactoglobulin Gel Formation at Low to Medium Temperatures

Effect of High-Pressure Processing on β-lactoglobulin Gel Formation at Low to Medium Temperatures

Dissolution and swelling of soy protein isolate hydrogels in alkali

The chemical dissolution of protein hydrogels, in particular the rate limiting mechanism, differs in the only two systems studied, whey and egg white proteins. With the aim to develop mechanisms general to globular protein networks, we study here the dissolution of soy protein isolate (SPI) hydrogels for the first time. SPI gels dissolve in alkali in a similar manner than egg white gels, but much faster, showing traits expected when the cleavage of interprotein disulfide crosslinks is rate limiting. SPI dissolution is reduced when swelling is inhibited by the addition of salts, although less profoundly than in whey protein gels. Equilibrium swelling experiments of SPI gels show a sudden increase at pH ≥ 11.5, which cannot be predicted from the swelling data at lower pH. Dissolution is evident in swollen SPI gels at pH > 12 but only at low salt concentrations. A double dissolution threshold is therefore also observed like in β-lactoglobulin gels, although its origin is different. The pH threshold in whey proteins at pH ∼11.5 is due to the destruction of non-covalent interactions, but in SPI it only causes a large increase in swelling. Dissolution only occurs at higher alkali concentrations where disulfide crosslinks start to be cleaved quickly. The importance of this reaction in SPI gels, where disulfide crosslinks are not extensive in the gel network, is explained by the large size of most soy proteins that will not detach meanwhile a gel network still exists. The size of the initial proteins becomes a new parameter when analyzing the dissolution of different protein systems, in addition to the chemistry involved in the formation of hydrogels.

Gelation of β-lactoglobulin and its fibrils in the presence of transglutaminase

The gelation of β-lactoglobulin (BLG) and its fibrils was investigated in the presence of dithiothreitol (DTT) and transglutaminase (TGase). BLG fibrils were prepared by heating BLG at 80 °C and pH 2 for 10 h and then adjusting to pH 7. The structure of fibrils was observed by atomic force microscopy. The small and large amplitude oscillatory measurements were done to examine the gelation process and to get more insight into gel properties. It was shown that TGase alone could induce gelation for fibrillar BLG although it could not for non-fibrillar BLG. TGase could cross-link BLG fibrils to form a gel at very low protein concentration in comparison with other protein gels, and enhance the network formation of both non-fibrillar and fibrillar BLG in the presence of DTT. The scanning electron microscopic observation revealed that TGase increased the network density for non-fibrillar gels while it made network strands thicker for fibrillar gels.

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.

Interactions and Diffusion in Fine-Stranded β-lactoglobulin Gels Determined via FRAP and Binding

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na2-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na2-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model.

Protein–Peptide Interaction: Study of Heat-Induced Aggregation and Gelation of β-Lactoglobulin in the Presence of Two Peptides from Its Own Hydrolysate

Two peptides, [f135-158] and [f135-162]-SH, were used to study the binding of the peptides to native β-lactolobulin, as well as the subsequent effects on aggregation and gelation of β-lactoglobulin. The binding of the peptide [f135-158] to β-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein-peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135-162]-SH resulted in a 10 times increase in the amount of aggregation of β-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional β-lactoglobulin by [f135-158] resulted in a decrease in gel strength, whereas replacement by peptide [f135-162]-SH increased gel strength.

Rheological and structural properties of β-lactoglobulin and basil seed gum mixture: Effect of heating rate

The effects of heating rate and addition of basil seed gum (BSG) as a novel hydrocolloid on the rheology and microstructure of β-lactoglobulin (BLG) gel were studied using low amplitude dynamic oscillatory shear (SAOS) and confocal laser scanning microscopy (CLSM) measurements. BLG solution and BLG–BSG mixtures at different ratios (20:1, 10:1, 5:1 and 2:1) were heated from 20 to 90°C at four heating rates (0.5, 1, 5 and 10°C/min). Gelation temperature of β-lactoglobulin was reduced by decreasing of heating rate and increasing BSG ratio. The maximum G′ at the end of heating period was reduced greatly by decreasing the BLG–BSG ratio. BLG–BSG mixed systems developed a relatively weak gel, which it enhanced when the ratio was changed from 20:1 to 2:1. Addition of BSG to 10% BLG solution makes a separated phase network in which increasing BSG content induced very fine stranded structure of protein. Therefore, it is possible to achieve distinct structures by mixing different ratios of hydrocolloids at various heating rates.

Surface-Directed Structure Formation of β-Lactoglobulin Inside Droplets

The morphology of β-lactoglobulin structures inside droplets was studied during aggregation and gelation using confocal laser scanning microscopy (CLSM) equipped with a temperature stage and transmission electron microscopy (TEM). The results showed that there is a strong driving force for the protein to move to the interface between oil and water in the droplet, and the β-lactoglobulin formed a dense shell around the droplet built up from the inside of the droplets. Less protein was found inside the droplets. The longer the β-lactoglobulin was allowed to aggregate prior to gel formation, the larger the part of the protein went to the interface, resulting in a thicker shell and very little material being left inside the droplets. The droplets were easily deformed because no network stabilizes them. When 0.5% emulsifier, polyglycerol polyresinoleat (PGPR), was added to the oil phase, the β-lactoglobulin was situated both inside the droplets and at the interface between the droplets and the oil phase; when 2% PGPR was added, the β-lactoglobulin structure was concentrated to the inside of the droplets. The possibility to use the different morphological structures of β-lactoglobulin in droplets to control the diffusion rate through a β-lactoglobulin network was evaluated by fluorescence recovery after photobleaching (FRAP). The results show differences in the diffusion rate due to heterogeneities in the structure: the diffusion of a large water-soluble molecule, FITC-dextran, in a dense particulate gel was 1/4 of the diffusion rate in a more open particulate β-lactoglobulin gel in which the diffusion rate was similar to that in pure water.

Erratum to: Hydrodynamic dispersion in $ \beta$ -lactoglobulin gels measured by PGSE NMR

The displacement scale dependent molecular dynamics of solvent water molecules flowing through β-lactoglobulin gels are measured by pulse gradient spin echo (PGSE) nuclear magnetic resonance (NMR). Gels formed under different p H conditions generate structures which are characterized by magnetic resonance imaging (MRI) and PGSE NMR measured dynamics as homogeneous and heterogeneous. The data presented clearly demonstrate the applicability of the theoretical framework for modeling hydrodynamic dispersion to the analysis of protein gels.

β-Casein aids in the formation of a sodium caprate-induced β-lactoglobulin B gel

The effects of sodium caprate on the gelation of β-lactoglobulin B and a β-lactoglobulin B/β-casein mixture at ambient temperature were investigated using ultrasonic spectroscopy and rheology. A 12% β-lactoglobulin B solution gelled in the presence of 3.6% sodium caprate. Conversely, sodium caprate did not induce the formation of a gel when β-casein was in isolation, regardless of the protein concentration. Although a 6% β-lactoglobulin B/1.8% sodium caprate solution did not form a gel, a gel was formed when 6% β-casein was added to a mixture containing 6% β-lactoglobulin B and 3.6% sodium caprate. This gel showed comparable rheological properties to that of a gel containing 12% β-lactoglobulin B. The results clearly indicated that β-casein aids in the gelation of a β-lactoglobulin B/sodium caprate mixture, when the concentration of β-lactoglobulin B is insufficient to allow for gelation. It appears that β-casein self-aggregation is also inhibited. Therefore, it could be concluded that β-casein can be used as a texture modifier for β-lactoglobulin gelation induced by sodium caprate.

In vitro release of α-tocopherol from emulsion-loaded β-lactoglobulin gels

Cold-set oil-loaded protein gels based on an emulsifying step followed by Ca 2+-induced gelation of pre-denatured β-lactoglobulin (β-LG) have been recently developed. In vitro release and stability of a fat-soluble compound (α-tocopherol) therein were investigated in this work. Release of α-tocopherol was found to be controlled mainly by matrix erosion due to protein degradation. Compound release and matrix erosion were almost complete after incubation under gastric or intestinal conditions for 6.5 h. However, both processes were basically inhibited upon changing the dissolution medium from the gastric to the intestinal type, possibly due to β-LG partial hydrolysis products with greater emulsifying capacity anchoring to the surface of gel oil droplets. The stability of released α-tocopherol was apparently improved by binding to protein and/or hydrolysis products thereof.

Micro-phase separation explains the abrupt structural change of denatured globular protein gels on varying the ionic strength or the pH

Aqueous solutions of globular proteins gel after heat-induced denaturation. The structure of β-lactoglobulin gels was studied as a function of the pH (2–8) and the NaCl concentration (0–1 M) over a wide range of length scales (1 nm-100 µm) using a combination of scattering techniques and microscopy. The gel structure depended on the strength of the electrostatic interaction that was varied by changing the charge density of the proteins (pH) or the screening length (ionic strength). Homogeneous so-called finely stranded gels were formed at strong interaction, while more heterogeneous so-called particulate gels were formed at weak interaction. It is shown that phase separation of growing irreversibly bound protein aggregates explains the abrupt transition between the two structures for small changes of the ionic strength or the pH. Phase separation was limited to the formation of protein rich domains with a radius of several microns that associated into the particulate gels. The structure of homogeneous gels is shown to be determined by the amplitude of the (critical) concentration fluctuations of protein aggregates that were frozen-in by gelation. The characteristic length scales of the homogeneous gels varied between about 20 nm and 1 µm.

Effect of salts on the alkaline degradation of β-lactoglobulin gels and aggregates: Existence of a dissolution threshold

The effect of NaCl and CaCl 2 on the alkaline degradation of β-lactoglobulin gels and aggregates, and particularly on the onset of dissolution, is studied. For gels, measurements of solubility in 0.063–0.5 M NaOH at 20 °C show the existence of a practical dissolution threshold in NaCl concentration, lying between 0.24 and 0.47 M. For aggregates, destruction of soluble β-lactoglobulin in alkali, followed by size exclusion chromatography, yields similar results. Furthermore, during dissolution of a gel in alkali at high NaCl concentrations, the protein aggregates released are very large ( e.g. ∼40% are larger than 200 kDa). CaCl 2 is found to cause similar inhibition of dissolution to NaCl, but at concentrations about 30× lower (∼10 mM). The threshold is hypothesised to arise from a combination of physical entanglements caused by the high protein concentration under conditions where little swelling occurs, and hydrophobic/electrostatic interactions between aggregates favoured by the high concentration of salts.

In situ microstructure evaluation during gelation of β-lactoglobulin

In situ microstructure evolution of heat-induced β-lactoglobulin (BLG) gels were investigated using confocal laser scanning microscopy (CLSM) at various gel preparation conditions: pH (2, 5, and 7), protein concentration (5%, 10%, and 15%), and salt concentration (NaCl 0, 0.1, and 0.3 M). Temperature-induced CLSM micrographs were used to measure several morphological features of the evolving protein clusters. The cluster area, perimeter, and circularity served as useful microstructural indices of BLG gels. Varying gelation conditions yielded BLG gels of different microstructures during heating. At pH 2 and 7, average area, and perimeter of protein clusters increased with protein content. Larger electrostatic repulsion provided microstructure with stranded clusters, whereas less electrostatic repulsion resulted in particulate clusters. The cluster size and shape were greatly affected by gelation pH; at pH 2 and 7, the average area, and perimeter increased with increasing salt content which provided particulate features to the gel microstructure. The combined effect of protein and salt content and pH is critical in imparting the gel its overall microstructure.

Diffusion of NaOH into a protein gel

The diffusivity of NaOH in a layer of protein gel on an inert surface is measured by combining experimental study of the reactive dissolution of the gel with simulation of transport and reaction within the gel. Pure β-lactoglobulin gels, formed under three different gelation conditions, were used as a model system. The alkaline solutions cause the gel to swell, and destroy the interprotein interactions in the gel matrix. The swelling and cleavage reactions depend on the local concentration of hydroxide and so are sensitive to the rate of transport of hydroxide through the gel. Experiments were performed to determine the effect of the NaOH concentration on the hydroxide penetration thickness and on the velocity of the penetration front marked by phenolthalein indicator. Simple simulations and a more advanced semi-theoretical analysis were performed, with proteins being treated as ideal polyelectrolyte polymers. Both yielded good agreement with the experimental results. The effective diffusivity of NaOH in the protein gels was found to be similar to that in water. The analytical procedure can be extended in principle to any protein gel.

The pH Threshold in the Dissolution of β-Lactoglobulin Gels and Aggregates in Alkali

The existence of a practical minimum pH for the dissolution of heat-induced whey gels in alkaline solutions has been studied using beta-lactoglobulin (betaLg) as a model protein. A sharp transition in solubility was observed between pH 11 and 12; this transition shifts to higher pHs for gels formed at higher temperatures and for longer gelling times. The breakdown reactions of heat-induced aggregates in alkali were monitored with size exclusion chromatography. The destruction of large aggregates was faster at higher pH and also showed a transition between pH 11 and 12. Using tryptophan fluorescence and near- and far-UV circular dichroism, this transition was assigned to the base-induced denaturation observed in solutions of aggregates (pK 11.53). It is suggested that the high protein repulsion caused by the large number of charges at pH > 11.5 drives the unfolding of the protein and the disruption of the intermolecular noncovalent bonds. Concentrated urea and GuHCl were found to be less effective than a pH 12 solution in destroying large aggregates. Aggregates formed for a long time (80 degrees C for 24 h) contained a larger number of intermolecular disulfide bonds that hinder the dissolution process. Gels formed at low temperatures (65 degrees C for 60 min), with fewer intermolecular noncovalent bonds, showed a similar solubility-pH profile to that observed for the base-induced denaturation of unheated beta-lactoglobulin (betaLg) (pK 10.63).