Pure Β-lactoglobulin Research Articles

Production of Ryazhenka, a traditional Ukrainian fermented baked milk, by using electro-activated whey as supplementing ingredient and source of lactulose

In this work, electro-activated whey (EAW) was used as ingredient in the preparation of Ryazhenka, a fermented baked milk, at a supplementation level of 3 and 9%. Fermentation kinetics, viscosity, color, antioxidant capacity, and protein profiles of the final product were studied. Moreover, effect of electro-activation on β-lactoglobulin allergenicity was evaluated by a specific ELISA test. The results showed that addition of electro-activated whey as supplementing ingredient is technologically feasible in the process of Ryazhenka production. The fermentation kinetics showed that this process was in the technologically accepted interval even if the addition of EAW delayed the fermentation phase. The viscosity study showed that addition of EAW permitted to obtain a final product with good followability corresponding to good consistency of drinkable dairy beverages. Moreover, addition of EAW prevented syneresis during the refrigerated storage at 4 °C and enriched the product with lactulose which is a recognized prebiotic. The chromatic evaluation of the final product showed a compatibility of EAW with Ryazhenka which is known for its specific yellowish-brownish color. Addition or EAW significantly improved the antioxidant capacity of the Ryazhenka with values of IC50DPPH = 2.54 ± 0.02 and IC50PFRAP = 244.03 ± 28.83 for the Ryazhenka produced by adding 9% EAW to the milk after the baking process and prior to fermentation. Moreover, by using a β-lactoglobulin specific ELISA test, this study showed a significant decrease of whey and pure β-lactoglobulin allergenicity by 95%. This decrease was also observed in Ryazhenka supplemented with 9% EAW but at lower level. Thus, it can be concluded that electro-activated whey can be considered as a promising functional ingredient containing a prebiotic lactulose as functional ingredient in the dairy industry.

One-step chromatographic method to purify α-lactalbumin from whey for nanotube synthesis purposes

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose™ Fast Flow gel column) was developed to purify α-lactalbumin (α-LA) from whey protein isolate. α-LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure α-LA, which was the main objective of this work, highly pure β-lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained α-LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples’ purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.

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.

Control of thermal fabrication and size of β-lactoglobulin-based microgels and their potential applications

HypothesisFactors influencing fabrication and size of microgels formed from β-lactoglobulin with or without pectin can tune selected attributes for material applications. Protein aggregation was expected to be influenced by pH, added anions, and reducing agents, while ionic strength was expected to be more influenced by electrostatically interacting pectin. ExperimentsTurbidity measurements during thermal aggregation to form microgels were determined for pure β-lactoglobulin as a function of pH, added ionic strength, anion type (chloride, sulfate, and thiocyanate), and reducing agent concentration. β-lactoglobulin and pectin complexation pH values and thermal aggregation were determined by turbidity measurements with added potassium chloride, sulfate, and thiocyanate. Microgel size and morphology were determined by light scattering and atomic force microscopy, respectively. FindingsThermal aggregation of pure β-lactoglobulin increased with decreased pH, reducing conditions, and increased ionic strength with no observed anion effect. β-lactoglobulin microgel radii increased from 86 to 115nm with decreasing pH and increased to 124nm in reducing conditions, while salts promoted agglomeration. Increased ionic strength (0–100mmol/kg) decreased β-lactoglobulin-pectin complexation pH from 5.40 to 5.00, while first increasing and then decreasing thermal aggregation. Thermal aggregation and microgel size were greatest with potassium thiocyanate, followed by potassium chloride and potassium sulfate.

Formation of Non-Native β-Lactoglobulin during Heat-Induced Denaturation

A mechanism describing the denaturation and aggregation behavior during heat-treatment of pure β-lactoglobulin and β-lactoglobulin in whey protein isolate (WPI) under selected conditions (20 to 90 gL−1 in water at pH 7.0, 78 °C) is presented. A combination of reversed-phase and gel permeation chromatography was used to study the disappearance of native β-lactoglobulin and the formation of non-native intermediates in the aggregation process. The mean reaction order for pure β-lactoglobulin and β-lactoglobulin in WPI were the same, 1.4. While the rate of β-lactoglobulin denaturation was greater in WPI there was less aggregation compared to that of pure β-lactoglobulin. More of the β-lactoglobulin in WPI remained in a non-native monomer intermediate state after 30 min of heating. After an initial lag period, during which non-native monomers appeared, aggregates formed and rapidly reached a plateau in terms of their size. These aggregates were visualized using atomic force microscopy. There was no significant effect of protein concentration on either aggregate size or the number of exposed sulfhydryls in the heated solutions.

Surface adsorption alters the susceptibility of whey proteins to pepsin-digestion

An in vitro digestion model mimicking the gastric phase of the human gastrointestinal tract coupled with SDS–PAGE and MALDI-TOF mass spectroscopy was employed to study the hydrolysis profiles of whey proteins in solution and adsorbed at the oil–water interface. The objective of this work was to understand the differences in hydrolysis behaviour of whey protein isolates once adsorbed at the interface, and comparisons were carried out with pure β-lactoglobulin and α-lactalbumin fractions. In solution, while β-lactoglobulin appeared to be resistant to enzymatic treatment, α-lactalbumin was fully degraded. Adsorption of both proteins at the oil–water interface affected their conformational structure and susceptibility to peptic hydrolysis. Adsorbed β-lactoglobulin was hydrolyzed into small polypeptides and in contrast, the resistance of α-lactalbumin to pepsin increased upon adsorption at the interface. In addition, changes in the particle size distribution of the droplets during pepsin hydrolysis mainly depended on the original protein concentration. The results suggested that exchanges occur at the interface between adsorbed and non-adsorbed protein, that is to say that either some protein desorb from the interface and does not fully recover its structure in solution, or that hydrolysis of the protein at the interface induces further adsorption and hydrolysis of the protein in solution. These mechanisms have important implications in the digestibility of the proteins.

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.

Selective Separation of the Major Whey Proteins Using Ion Exchange Membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate β-lactoglobulin, BSA, and α-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for β-lactoglobulin with less than 1% of the eluate consisting of α-lactalbumin or BSA. The binding capacity of a pure β-lactoglobulin solution was in excess of 1.5 mg/cm2 of membrane. This binding capacity was reduced to approximately 1.2 mg/cm2 when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that α-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by β-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10mM sodium phosphate buffer (pH 6.4). Binding capacities of β-lactoglobulin, α-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.

Light-Scattering Study of the Structure of Aggregates and Gels Formed by Heat-Denatured Whey Protein Isolate and β-Lactoglobulin at Neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.

Effect of high-pressure treatment on the electrospray ionization mass spectrometry (ESI-MS) profiles of whey proteins

The effects of different high-pressure (HP) treatments (pressures ranging between 450 and 650 MPa) on β-lactoglobulin and α-lactalbumin and on two commercial whey protein isolates (WPI) were examined by electrospray ionization mass spectrometry (ESI-MS). HP treatment resulted in substantial changes in the charge-state distribution (CSD) of pure β-lactoglobulin, attributable to exposure of side chains of buried amino acids to solvent, whereas the CSD of pure α-lactalbumin was largely insensitive to HP treatment. The changes in the CSD of these proteins effected by HP treatment differed between the two commercial WPI samples examined, perhaps due to the different production methodologies employed by the two suppliers. These findings illustrate the important role of interactions between the various components in WPI in the response of whey proteins to HP treatment and underscore the difficulty of obtaining consistent rheological and nutraceutical properties by HP treatment of WPI, particularly when different sources of WPI are employed. The information provided by ESI-MS may be useful in monitoring the effects of HP treatment to verify that such consistency is achieved.

Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion method, or proton NMR spectroscopy, reported a wide range of values for the conversion. An alternative method was developed, namely centrifugal filtration, giving a consistent picture of the conversion. The present results help to explain the disparities reported in literature. No fibrils formed upon heating pure α-lactalbumin or pure BSA at pH 2, whereas fibrils formed in pure β-lactoglobulin ( β-lg) and WPI solutions. Experiments indicate that β-lg was the only whey protein involved in fibril formation. In all whey protein samples, hydrolysis occurred during heating at pH 2, as determined by HPLC and SDS-PAGE. When WPI fibrils formed at pH 2 were stored at pH 7 or 10, disulphide bonds were formed in the samples.

Shear effects on the rheology of β-lactoglobulin/β-carrageenan mixed gels

Rheological and microstructural effects of preshear treatment on heat-induced pure β-lactoglobulin gels and on mixed gels of 5% β-lactoglobulin and 1% κ-carrageenan have been investigated at pH 5. No salt was used in this work. The shear process with different rate/time combinations was performed just before the gel formation. During the cooling process from 90 to 20 °C, the values of elastic moduli G′ of the pure protein gels showed a mono-continuous profile, whereas the mixed gels showed a bicontinuous profile which indicated the formation of phase-separated gels. The values of G′ of both systems decreased with increase of shear rate and shear duration, and the profiles of G′ could not be modified by shear treatment. The Confocal microscopy revealed that the network structures of the gels have been broken by preshear treatment.

Effect of thickeners on the coalescence of protein-stabilized air bubbles undergoing a pressure drop

The effect of some common food thickeners on the coalescence stability of protein-stabilized air bubbles at a planar air–water interface has been studied, as the bubbles undergo expansion due to a pressure drop. The fraction of bubbles coalescing ( F c) has been measured as a function of bulk protein concentration ( C b) and rate of area expansion. The ranges of protein concentration and rate of area strain studied were 0.01–0.3 wt% and 10 −3 to 2.4 s −1, respectively. Pure β-lactoglobulin, a commercial whey protein isolate (WPI) consisting primarily of β-lactoglobulin, and pure ovalbumin were used. The thickeners used were sucrose (50 wt%), xanthan gum, guar gum, gelatine and pectin. In the absence of thickeners, F c was slightly lower for ovalbumin than for WPI but slightly higher than for pure β-lactoglobulin at the highest C b. It was found that stability with thickeners present was less dependent on expansion rate than for the proteins on their own. The effect of the concentration of thickeners on F c was therefore studied at a fixed area strain-rate of 0.13 s −1. Sucrose had a general stabilizing effect with all the proteins, though this stabilization was less marked at higher protein concentrations and expansion rates. Overall, gelatine had the most marked effect on F c, at concentrations as low as 0.01 wt%. For xanthan and guar gum, effects on F c were significant at concentrations between 0.025 and 0.1 wt%, but were variable, increasing or decreasing the value of F c, depending on the protein and its concentration. Pectin had a less noticeable effect than xanthan or guar gum, and then only at a considerably higher concentration, i.e., 0.25–1 wt%.

Inhibition of heat-induced aggregation of a β-lactoglobulin-stabilized emulsion by very small additions of casein

Heat stability has been studied in model systems of oil-in-water emulsions (3 wt.% total protein, 45 vol.% n-tetradecane, pH 6.8, ionic strength 30–50 mM) with pure β-lactoglobulin (β-lg) as the main emulsifier. The effect of small additions of sodium caseinate, β-casein or α s1-casein prior to emulsion preparation has been investigated. Samples heated for 3 min at 90 °C were monitored with respect to changes in viscosity and particle-size distribution. As expected, the pure β-lg-stabilized emulsions were susceptible to heat-induced changes. But the replacement of just 1% of the β-lg by sodium caseinate (0.03 wt.% caseinate in the total emulsion) led to complete elimination of any heat-induced viscosity or particle size increase. These findings show that a very small proportion of casein can inhibit the susceptibility of a β-lg-based emulsion to heat-induced destabilization. The magnitude of the effect is dependent on the type of casein, with the order of effectiveness being β-casein > sodium caseinate > α s1-casein. This work has potential implications for the development of milk protein-stabilized emulsions of improved shelf life.

Interactions of polysaccharides with β-lactoglobulin spread monolayers at the air–water interface

In the present work we have studied the static (film structure and elasticity) and dynamic characteristics (surface dilatational properties) of β-lactoglobulin (βLG) monolayers spread at the air–water interface in the presence of polysaccharides in the aqueous phase, at 20 °C and at pH 7. The measurements were performed on a fully automated Wilhelmy-type film balance. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied-kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). Xanthan gum (XG) and λ-carrageenan (λC) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface active and non-surface active polysaccharides on static—when the polysaccharide was added in the subphase the π- A isotherms shifted to higher surface pressure values as the time increased-and dynamic—the presence of polysaccharide in the aqueous phase decreased the surface dilatational modulus of a pure β-lactoglobulin monolayer-characteristics of β-lactoglobulin monolayers. To explain the observed effects three phenomena were taken into account: (i) the ability of the polysaccharide to adsorb at the interface by it-self and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide, depending on the protein-polysaccharide system.

Fractionation of whey proteins by bipolar membrane electroacidification

Bipolar membrane electroacidification (BMEA) is a technology offering the coupled effect of demineralization and acidification by using the properties of bipolar membranes to dissociate water molecules at their interfaces and of cation exchange membranes (CEM) to demineralize by migration of low molecular weight ionic species. The aim of the present study was to demonstrate the feasability of BMEA to fractionate whey proteins and to study the effect of protein concentration on its performance in comparison with chemical acidification. These results demonstrated the feasability of BMEA for whey protein separation and the influence of the initial protein concentration on the purity and yield of the separated fraction. At 5% whey protein isolate (WPI) initial concentration, this technology allows the separation of 98% pure β-lactoglobulin (β-lg) fraction with a 44.0% recovery yield. At 10% WPI initial concentration, BMEA allows the production of a β-lg-enriched fraction containing 97.3 of β-lg and 2.7% α-lactalbumin (α-la), for a 98% total protein purity.

Coalescence stability of gas bubbles subjected to rapid pressure change at a planar air/water interface

We report the effects of rapid and slow bubble expansion, due to a reduction in air pressure, on bubble coalescence stability at a planar air–water interface. Bubbles stabilised by pure β-lactoglobulin (BLG), a commercial whey protein isolate (WPI) consisting primarily of BLG, and the non-ionic surfactant Tween 20, were examined. A semi-automatic image analysis routine has been developed to size and count touching bubbles in close-packed arrays. It was found that the bubble size distribution does not change significantly after expansion and coalescence, except for the lowest bulk protein/surfactant concentration ( C b). Bubbles stabilized by BLG were increasingly more stable against rapid expansion with increasing C b, compared with bubbles stabilised by WPI, suggesting significant differences between the adsorbed protein films. For both proteins the fraction of coalescence, ( F c) increased with increasing expansion rate, until a plateau in F c was reached. For Tween 20, F c decreased with increasing number of injected stable bubbles per unit area of planar interface. For bubbles formed in mixtures of BLG and Tween 20 (at high expansion rate) F c increased with increasing molar ratio of Tween 20:BLG. For proteins F c is probably determined by both adsorption kinetics and the interfacial rheology of the adsorbed films, whilst other factors, such has thin film hydrodynamics, bubble size, bubble packing and the relative mobility of the bubbles at the interface, probably have additional, but more minor influence on overall bubble stability.

Disproportionation of clustered protein-stabilized bubbles at planar air–water interfaces

The rate of shrinkage of air bubbles, of initial radius from 50 to 200 μm, injected beneath a planar air–water interface has been measured. Bubbles were stabilized in solutions of 0.05 wt% gelatin or pure β-lactoglobulin. It has been observed that small size differences between two closely spaced or touching bubbles result in markedly divergent rates of shrinkage for the two. By studying a number of different initial bubble configurations, it is demonstrated that the overall change in bubble size distribution is strongly dependent on local, interbubble gas diffusion. In this respect, the strong tendency for the gelatin-stabilized bubbles to aggregate and shrink, while remaining in contact, produced patterns of disproportionation significantly different from those observed with β-lactoglobulin. In β-lactoglobulin solutions, it was usually found that bubbles initially in contact shrank away from each other with time, becoming increasingly isolated as a result. A theoretical approach that can exactly incorporate the perturbation of local diffusion fluxes due to the proximity of two bubbles is presented. This enables one to map a “stability diagram” that delineates regions where the larger bubble of a pair will either shrink or grow, according to the relative size of the bubbles and their separation. Theoretical calculations show that it is possible for a bubble to exhibit more complex shrinkage behavior than is predicted by a mean field approach or the Lifshitz, Slyozov, and Wagner (LSW) theory of Ostwald ripening for dilute systems. The inclusion of dilatational elasticity in the theoretical model introduces additional complications, which are also briefly discussed.

Isolation of α-lactalbumin, β-lactoglobulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of α-lactalbumin, β-lactoglobulin, and bovine serum albumin from cow’s milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and β-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which β-lactoglobulin was separated from α-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure β-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of α-lactalbumin, β-lactoglobulin, and bovine serum albumin thus purified.