EFFECTS OF HEATING AND PRESSURIZATION ON FOAMING PROPERTIES OF BETA-LACTOGLOBULIN

Jacek Leman, Tomasz Dołgań

ABSTRACT

The effects of heating (80^oC/10 min and 90^oC/2 min) and pressurization (300, 600 and 900 MPa for 10 min and 300 MPa for 10 to 30 min) on foaming properties of beta-lactoglobulin (beta-Lg) obtained from whey retentate in mild conditions were compared in the pH range of 5.0 to 7.0. Generally, heating and pressurization improved the beta-Lg foamability, except that at pH 7.0 (1) heating of the protein worsened (80^oC/10 min) the foaming properties or not changing (90^oC/2 min) the foam capacity - improved the foam stability and (2) pressurization at above 300 MPa drastically worsened the foam stability (600 MPa) or caused that the foam was unstable (900 MPa). The highest foam capacity had beta-Lg pressurized for 10 min at 300 MPa in pH 7.0 and lengthening of the pressurization time up to 30 min resulted in the increase in the foam stability with the foam capacity being practically unchanged. The greatest improvement of beta-Lg foaming properties was f

Key words: beta-lactoglobulin, heating, pressurization, foam capacity, foam stability..

INTRODUCTION

The ability of proteins to form and stabilize foams is of great importance to a variety of foods including bread, cakes, cookies, ice cream and whipped toppings. The foaming properties of proteins, resulting from their ability to form a film on the air-water interface, are dependent on: (1) solubility – enabling rapid diffusion to the interface, (2) amphipathicity - determining enhanced interfacial interactions, (3) conformational flexibility – facilitating unfolding at the interface, (4) interactive segments – enabling secondary interactions in the gaseous, aqueous and interfacial phases, (5) disposition of charged and polar groups – preventing the close approach of bubbles and allowing hydration, and (6) steric effects. The foaming properties of proteins are characterized by determining the foam capacity (foam volume) and foam stability (liquid drainage). The foam capacity is conditioned by the ability of a protein molecule to unfold upon denaturation agents, whereas refolding of denature d protein is important to foam stability [2, 18, 26]. Depending on the degree of protein denaturation and the nature of newly developed protein structure, the foams formed with the protein contribution differ in the surface area of the interfacial film, its mechanical resistance, viscosity, elasticity and ability for water retention.

Beta-lactoglobulin (beta-Lg) is a globular protein with a monomer molecular weight of 18.4 kDa and foaming properties, the secondary structure of which consists predominantly of beta-sheets [3]. In the physiological range of pH (5.5 - 7.5) and moderate ionic strength, beta-Lg is in the form of a dimer, whereas at lower pH (5.2–3.5) it forms tetramers [21]. Each monomer consists of 162 amino acids and contains two intermolecular disulfide bonds and one sulfhydryl group [3]. Beta-Lg has two apolar binding sites and one aromatic binding site [21]. The foaming properties of beta-Lg result from its susceptibility to denaturation and hydrophobicity.

Among denaturation agents (chemical, enzymatic, physical) that destroy the forces stabilizing the protein structure (hydrogen bonds, hydrophobic and sulfhydryl group interactions, van der Waals forces), temperature and high pressure are easiest to apply and legal. Limited heat treatment and pressurization affect the foaming properties of beta-Lg and whey protein concentrates in which it is a major protein [11, 18, 19, 26] . Heating of whey protein concentrate at 70°C for 1 min improved its foaming properties, whereas more severe heating (70 or 90°C for 5 min or longer) resulted in impairement of the foaming properties [26]. The foamability of beta-Lg heated from 20 to 80°C at 2°C/min was by 15% greater than that of non-heated protein [13]. Reduced foamability has been reported for beta-Lg after pressurization of its solutions (0.001 - 0.005 % w/v) at 300 to 900 MPa (Pittia et al., 1996). The foamability of whey protein isolate was improved after pressurization (150 - 450 MPa ) of its 1% w/v solution at pH 7.0, but not at pH 6.0 and 5.0, and the foam stability decreased above 300 MPa [11]. Pressurization at high-protein concentration (2 % w/v) or high-buffer molarity (100 mM) reduced the foaming properties of whey protein isolate [11].

The foaming properties of protein concentrates differ because of the differences in composition and structural states of the protein due to the method of isolation [4, 15]. In the present work we used beta-Lg isolated in mild conditions from a whey retentate to compare the effects of heating (80°C/10 min and 90°C/2 min) and pressurization (300, 600 and 900 MPa/10 min and 300 MPa/10-30 min) in the pH range of 5.0 to 7.0 on the foaming properties of the protein.

MATERIALS AND METHODS

Material
Beta-Lg was obtained from whey after rennet cheese production in a pilot scale. Beta-Lg was isolated from the retentate after the whey ultrafiltration (membrane 10 kDa) and precipitation of alfa-lactalbumin (alfa-La) in a mild conditions (55°C/30 min, pH 3.9) (Fig. 1). The beta-Lg preparation contained 1.3% water and, on dry basis, 85.6% protein, 0.64% non-protein nitrogen, 2.3 % ash, 0.74 % sodium, 0.37 % calcium, 0.09% potassium, and 0.054 % magnesium. Polyacrylamide gel electrophoresis showed the presence of alfa-La in the amount not exceeding 0.2 %. Buffer solutions (50 mmol/dm³): imidazole/HCl, pH 7.0, citrate, pH 6.0 and phosphate, pH 5.0 were prepared using deionized water. Buffer salts were from Sigma Chemical company.

Foaming properties
The foam capacity was determined according to the Puski’s [20] method as follows: 10 cm³ of 5 % w/v water solution of beta-Lg and 40 cm³ of buffer solution of pH 7.0, 6.0 or 5.0 were placed into a 250 cm³ cup of MPW 302 homogenizer (Zakład Mechaniki Precyzyjnej, Warsaw, Poland). The solution was mixed 1 min at 10000 rev/min. The foamy liquid was transferred into a graduated cylinder (250 cm³ ± 2 cm³) and the foam volume was read immediately to express the foam capacity and after 30 min to express the foam stability. The foaming properties were determined for the protein solutions heated 10 min at 80°C or 2 min at 90°C in a sealed glass tubes (14 mm i.d., 16 mm e.d.) or pressurized at 300, 600 and 900 MPa for 10, 20 and 30 min in a teflon tubes using a hydrostatic high-pressure generator Liquid Vessel LV/30/10, Unipress Equipment (Centrum Wysokich Ciśnień PAN, Warsaw, Poland). The foam capacity and foam stability were studied as being affected by: (1) heating of the beta-Lg solutions of pH 5.0, 6.0 and 7.0, (2) pressure at 10 min pressurization of the protein solution of pH 7.0, (3) acidity of the protein solution pressurized 10 min at 300 and 600 MPa. The control sample was non-heated or non-pressurized 5% w/v beta-Lg solution of required pH prepared immediately before the experiments.

Analytical methods
The beta-Lg preparation was analyzed with standard methods for the content of dry matter, total and 12 % TCA-soluble nitrogen (Kjeldahl^’s method), calcium and magnesium (AAS method after wet mineralization in the 3:1 v/v mixture of nitric and perchloric acids), sodium and potassium (flame photometric method after wet mineralization).

Data analysis
The results reported are arithmetic means from the experiments carried out in triplicate and analytical analyses carried out in duplicate. The results for the foam volume measurements differed by not more than 5% between the experiments. Statistical analysis of the results was carried out using Statsoft’s Statistica PL ver. 6.0 software.

RESULTS

Effect of heating
The foam capacity of beta-Lg heated at pH 5.0 and 6.0 was by 5 to 21% greater compared with control (Fig. 2 Aa, Ba). At pH 7.0, the foam capacity of heated beta-Lg decreased by about 15% (80°C/10 min) or did not change (90°C/2 min) (Fig. 2 Ca). Compared with control, the foam stability for beta-Lg heated at pH 5.0 and 6.0 was 1- to 3-fold greater, whereas at pH 7.0 2-fold increase in the foam stability was observed only for beta-Lg heated at 90°C for 2 min (Fig. 3 Aa, Ba, Ca). The greatest, i.e. 3-fold increase in the foam stability was observed for beta-Lg heated at pH 6.0 (Fig. 3 Ba). Statistically significant (p ≥ 0.5) increases in the foam capacity and foam stability were only found for beta-Lg heated at pH 6.0 with statistically insignificant (p ≥ 0.05) differences in the foam capacities and stability of foams for beta-Lg heated at 80°C/10 min and 90°C/2 min.

Effect of pressure
Pressurization of beta-Lg at 300 and 600 MPa and pH 7.0 increased the foam capacity compared with control by about 57 and 22 %, respectively (Fig. 2 Cb). The foam capacity of beta-Lg pressurized at 900 MPa did not differ from that of control (Figure 2 Cc). The foam stability for beta-Lg pressurized at 300 MPa was 11-fold greater compared with control (Fig. 3 Cb). Pressurization at 600 and 900 MPa caused that the formed foams were unstable (Fig. 3 Cb, Cc). Statistically significant (p ≥ 0.05) increases and differences in the foam capacities were found for beta-Lg pressurized at 300 – and 600 MPa/10 min. The increase in the foam stability was statistically significant (p ≥ 0.05) only for beta-Lg pressurized at 300 MPa/10 min.

Effect of pressurization time
Pressurization of b-Lg at 300 MPa for up to 30 min increased the foam capacity and foam stability compared with control (Fig. 4). The foam capacity of beta-Lg pressurized for 10, 20 or 30 min was by about 50 to 60 % greater (Fig. 4 A) and the foams were 11-to 12-fold more stable (Fig. 4 B) compared with controls. The highest foam capacity had beta-Lg pressurized for 10 min (Fig. 4 A) and the most stable foam was formed by beta-Lg pressurized for 30 min (Fig. 4 B). The foam capacities of beta-Lg pressurized for 20 and 30 min were lower by about 6 and 4%, respectively, compared with the foam capacity of beta-Lg pressurized for 10 min. The foam stability of beta-Lg pressurized for 10 and 20 min was by about 8-10% lower than the foam stability of the protein pressurized for 30 min. The increases in the foam capacity and foam stability for pressurized beta-Lg were statistically signifi cant (p ≥ 0.05). Lengthening the pressurization time above 20 min resulted in the statistically significant (p ≥ 0.05) increase in the foam stability and statistically insignificant (p ≥ 0.05) increase in the foam capacity.

Effect of acidity
The foam capacity increased the most (by about 57%) for beta-Lg pressurized at pH 7.0 and 300 MPa (Fig. 2 Cb), and at pH 6.0 and 600 MPa (Fig. 2 Bb). The foam capacity of beta-Lg pressurized at pH 5.0 and 6.0 increased with an increase in pressure from 300 to 600 MPa, as compared with control, whereas at pH 7.0 the increase in pressure decreased the foam capacity (Fig. 2 Cb). The foam stability of pressurized beta-Lg was generally greater than that of controls (Fig. 3 Ab, Bb, Cb), except for beta-Lg pressurized at pH 7.0 and 600 MPa (Fig. 3 Cb). The most stable foam was formed by beta-Lg pressurized at pH 7.0 and 300 MPa (Fig. 3 Cb), but the greatest improvement of the foam stability took place at pH 6.0 and 300 MPa, i.e., 12- and 15-fold, respectively, compared with control (Fig. 3 Bb). The increases in the foam capacity of pressurized beta-Lg were statistically significant (p ≥ 0.05) irrespective of pH. The increases in the foam capacity of heated beta-Lg samples were statistically insignificant (p ≥ 0.05) at pH 5.0 and they were significant (p ≥ 0.05) at pH 6.0. At pH 7.0, the difference in the foam capacities of beta-Lg heated at 80°C/10 min and 90°C/2 min was statistically significant (p ≥ 0.05). The foam stability increased statistically significantly (p ≥ 0.05) for beta-Lg pressurized at 300 MPa/10 min irrespective of pH and for beta-Lg pressurized at 600 MPa/10 min at pH 5.0 and 6.0. Statistically significant increase in the foam stability for heated beta-Lg was found at pH 6.0. At pH 5.0 and 7.0, the foam stability increased statistically significantly (p ≥ 0.05) only for beta-Lg heated at 90°C/2 min.

DISCUSSION

Temperature- or pressure-induced changes in beta-Lg structure are quite well evidenced, but still not recognized in detail [2, 7, 10, 12, 17, 18]. The changes are characterized in terms of partial disintegration of the structure, including dissociation of subunits and unfolding of polypeptide chains, that is followed by refolding of denatured polypeptides with the formation of aggregates. The information about the degrees of dissociation and unfolding is lacking, just as it is unknown the extent of the formation of intermolecular structures and the sequence of those events. What has been agreed is that slight changes that occur upon temperature of 65 to 75 °C and pressure of 50 to 200 MPa are reversible and lead to partial dissociation of beta-Lg polymer to monomers [10, 19]. Reversible changes in the structure are associated with a decrease in alfa-helix and beta-sheet percentages and an increase in beta-structures [8, 16, 17]. In the result of partial dissociation of beta-Lg polymers the area accessible to the solvent increases (so the surface properties of the protein improve) just as does the protein reactivity owing to increased hydrophobicity (because of exposure of hydrophobic fragments) and exposure of sulfhydryl groups [14, 19]. Irreversible changes in the protein structure occur above the given tresholds of temperature (>75°C) and pressure (>200 MPa) when critically denatured beta-Lg monomers form aggregates that are stabilized by covalent (S-S) and non-covalent (hydrophobic interactions, ionic and van der Waals forces) bonds or their combination [7, 9, 10, 14, 17, 24]. The contribution of non-covalent bonds is higher in acid solutions of beta-Lg at pH close to its isoelectric point and in the solutions of high molarity [24]. The covalent bonds (S-S) dominate in neutral medium [6, 17]. Irreversible changes in beta-Lg are associated with the formation of intermolecular beta-structures in the temperature-denatured protein and disordered structures in the pressure-denatured protein [17].

The extent of temperature- and pressure-induced changes in beta-Lg is dependent on the temperature and pressure ranges, time of heating or pressurization, native structure of the protein and its concentration as well as on environmental (solvent, pH, ionic strength) and methodological factors [10 ,12, 17, 18].

In our study, heating and pressurization of beta-Lg changed the foaming properties - pressurization more than heating. Generally, both heating and pressurization improved more or less the foamability of beta-Lg; only heating of the protein in neutral solution impaired (80°C/10 min) the foaming properties or not changing (90°C/2 min) the foam capacity improved the foam stability, and pressurization above 300 MPa for 10 min resulted in unstable foams.

The highest foam capacity had beta-Lg pressurized for 10 min at 300 MPa in neutral solution and lengthening of the pressurization time at that pressure up to 30 min resulted in an increase in the foam stability with practically unchanged foam capacity. The greatest improvement of beta-Lg foamability was found for its slightly acid solution (pH 6.0) after 10 min pressurization at 600 MPa and long- (10 min) or short (2 min) heating at 80 and 90°C, respectively. Regarding the improvement of beta-Lg foamability, the same effects had then pressurization at 300 MPa/10 min at pH 7.0 as pressurization at 600 MPa/10 min at pH 6.0. In acid solution (pH 5.0), heated and pressurized beta-Lg had the lowest foam capacity and foam stability at the reverse relationship between them, i.e., the improvement of the foam capacity was associated with the impairement of the foam stability. The improvement of the foam capacity of beta-Lg in this solution was greater after 10 min pressurization at 600 MPa and 10 min heating at 80°C, but the foam stability was worse than after 10 min pressurization at 300 MPa and 2 min heating at 90°C. The stability of beta-Lg foams increased to a greater degree than the foam capacity both upon heating and pressurizatiom.

It should be then assumed that denaturation caused by heating and pressurization at the optimum conditions promoted depolymerization and denaturation of beta-Lg and the formation of thick interfacial film owing to adsorption of quickly diffusing molecules of depolymerized and denatured protein molecules and their refolding through numerous interactions, in the results of which viscoelastic film was formed at the interface that prevented the liquid leakage. Unstable foams and impaired foam capacity would prove much more intensive denaturation of beta-Lg associated with aggregation in the result of which the rate of the protein diffusion to the interface decreased as did the ability for interactions because of greater molecular weight, decreased hydrophobicity and solubility. Domination of aggregation over dissociation and unfolding of polypeptide chains might have taken place in the case of pressurization of neutral beta-Lg solution at above 300 MPa since pressure-induced denaturation of bet a-Lg is more extensive in the neutral medium than acid medium [5] and in the cases of heating and pressurization of acid (pH 5.0) solution of beta-Lg, in which the processes of aggregation are additionally stimulated due to reduced electrostatic repulsion [19, 23]. Instability of foams formed by beta-Lg pressurized at above 300 MPa in the neutral solution parrallels with the structure differences in gels made by pressure and heating [22, 25]. Pressure induces lower gel rigidity and elasticity than heating, suggesting weaker intermolecular or interparticular forces. Thus, depletion of protein able to adsorb at the air-water interface because of heavy aggregation or too extensive denaturation causing a harmful loss of the secondary structure resulted in insufficient thickness of the foam films and their poor surface rheology properties (lessened viscoelasticity and resistance to shear during foaming) and, as a consequence, the foams collapsed.

CONCLUSIONS

1. Heating (80°C/10 min or 90°C/2 min) and pressurization (300-900 MPa/10-30 min) influenced the foamability of beta-Lg to a different degree (improving it in most cases), with pressurization being generally more effective and the foam stability being more affected than the foam capacity.

2. The effects of heating and pressurization on beta-Lg foamability were pH-dependent; at pH 7.0, pressurization improved the foam capacity in contrast to heating, but the foam stability decreased dramatically upon pressure above 300 MPa; at pH 6.0, the improvement of the foamability could probably be tailored by increasing the intensity of pressure over 600 MPa, and at pH 5.0, the reverse relationship between the foam capacity and foam stability might be a matter of a compromise at optimization.

3. The intensity of pressure was of greater importance to beta-Lg foamability than the time of pressurization.

4. Long heating at lower temperature (80°C/10 min) of acid solutions of beta-Lg was more effective for its foam capacity, but not for the foam stability, than short heating at higher temperature (90°C/2 min).

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