Effect of thermal treatment on interfacial properties of β-lactoglobulin

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Abstract

The changes in the secondary conformation and surface hydrophobicity of β-lactoglobulin subjected to different thermal treatments were characterized at pH values of 7, 5.5 and 4 using circular dichroism (CD) and hydrophobic dye binding. Heating resulted in a decrease in α-helix content with a corresponding increase in random coil at all pH values, this change being more pronounced for small heating times. Heating also resulted in an increase in surface hydrophobicity as a result of partial denaturation, this increase being more pronounced at pH 4. Thermal treatment resulted in a shift of the spread monolayer isotherm at air–water interface to smaller area per molecule due to increased flexibility and more loop formation. Thermal treatment led to an increase in interfacial shear elasticity and viscosity of adsorbed β-lactoglobulin layer at pH 5.5 and 7. Interfacial shear elasticity, shear viscosity, stability of β-lactoglobulin stabilized emulsion and average coalescence time of a single droplet at a planar oil–water interface with adsorbed protein layer exhibited a maximum for protein subjected to 15 min heat treatment at pH 7. At pH 5.5, the interfacial shear rheological properties and average single drop coalescence time were maximum for 15 min heat treatment whereas emulsion stability was maximum for 5 min heat treatment. At pH 7, thermal treatment was found to enhance foam stability. Analysis of thin film drainage indicated that interfacial shear rheological properties do not influence thin film drainage.

Introduction

Proteins, because of their amphiphilic nature, are surface active and are commonly used as food emulsifiers. Upon adsorption at the interface, protein molecules unfold, interact with each other forming a thick viscoelastic layer which provides stability to the emulsion. The physical and chemical properties that govern the emulsifying properties of proteins include size, shape, net charge and distribution of charges, surface hydrophobicity, stability, flexibility, amino acid composition and structure.

The primary structure (sequence), secondary structure (i.e., spatial arrangement of the polypeptide chains stabilized by hydrogen bonding, electrostatic interactions, van der Waals interactions and hydrophobic interactions), tertiary structure (folding of the polypeptide chains that are held together by covalent disulfide linkages) and quaternary structure (spatial arrangement of a protein when it contains more than one polypeptide chain) influence interfacial properties. To elucidate the influence of protein conformation on the surface active properties of proteins, Graham and Phillips [1], [2], [3] compared three structurally very different proteins, lysozyme (globular and rigid), bovine serum albumin (globular and flexible) and β-casein (random coil). It was found that a highly flexible protein such as β-casein adsorbed much faster, unfolded, reoriented and spread at the interface more easily than a highly rigid and compact protein such as lysozyme. It was observed [4] that one of the structural intermediates of bovine serum albumin (BSA) occupied greater area at the interface than either the completely unfolded or the compact native state of BSA. On the other hand, interfacial shear viscosity was found to be much lower for β-casein than for lysozyme or β-lactoglobulin (BLG) [5]. This is indicative of the strong forces acting on adsorbed globular protein molecules at the interface which leads to unfolding of the native tertiary and (some) secondary structure. This, in turn, forms new bonds between various parts of the same and different polypeptide chains. Disulfide bond interchange was found to be induced on adsorption and aging of β-lactoglobulin at the oil–water interface [6]. Emulsion stability was enhanced at higher interfacial shear viscosity [7]. On the other hand, extensive denaturation may result in poor interfacial mechanical properties detrimental to long term stability of the emulsion.

Main forces involved in the stability of protein structure are hydrogen and electrostatic bonds, hydrophobic interactions and disulfide bonds. Proteins have been classified as “soft” when they have low internal stability and consequently can change their conformation upon adsorption, and “hard” if the protein has a high internal stability [8], [9]. Wang and McGuire [10] investigated the surface tension of solutions of mutants of T4 lysozyme of different conformational stability and observed that a less stable protein adsorbs more tightly and occupies more interfacial area than a more stable protein.

Physical (such as temperature, hydrostatic pressure or shear) as well as chemical (such as pH) modifications induce changes in protein structure and hence their interfacial properties. In a series of papers [11], [12], [13], [14], we have examined the adsorption characteristics of native and chemically modified BSA (by alkylation) of higher surface hydrophobicity at air–water interface, as well as the exchange of this protein with lecithin. Differential scanning calorimetric study indicated that the alkylated BSA was less stable than native BSA which was consistent with the observation that the former unfolded more at the air–water interface [13]. Enzymatic [15] and chemical [16] modifications have been shown to result in improved foaming, emulsification and other functional properties. Increase in temperature weakens hydrogen and electrostatic interactions but strengthens hydrophobic interactions (up to 60–70 °C). Amino acid composition affects the thermal stability of proteins. Proteins that contain a greater proportion of hydrophobic amino acid residues tend to be more stable than the more hydrophilic proteins [17]. Heating was found to result in a decrease in α-helix and β-sheet with a corresponding increase in random coil for β-lactoglobulin [18], [19]. Denaturation temperature of β-lactoglobulin was found to be a function of pH [18] increasing from alkaline to acidic pH values. The surface hydrophobicity was found to increase with heating for β-lactoglobulin [20], [21], [22], this effect being pH-dependent. High pressure treatment (400 to 600 MPa) has been found to result in a decrease in surface hydrophobicity of BSA due to aggregation [23] though insignificant change in surface activity of β-casein at air–water interface was observed [24]. Partial denaturation (unfolding) of proteins prior to emulsification usually improves their emulsifying capacity due to an increase in their surface hydrophobicity and molecular flexibility [25], [26], [27]. An inverse correlation of coalescence rate constant and surface hydrophobicity (modified by heating) was found for β-lactoglobulin stabilized peanut oil-in-water emulsion [22].

In this paper, the secondary and tertiary conformations of β-lactoglobulin were modified by thermal treatment and the effect of these modifications on the surface equation of state, interfacial rheology, emulsion and foam stability were investigated.

Section snippets

Physical modification of BLG

Solutions of β-lactoglobulin (Sigma Chemical Co.) in three different buffer systems, which were also purchased from Sigma were made in the pH range of 4 to 7. The pH 4 buffer was prepared with 0.01 M potassium chloride and adjusted with 0.5 M phosphoric acid solution. The pH 5.5 buffer was prepared with 0.01 M solution of ammonium acetate and again adjusted with 0.5 M phosphoric acid solution. The pH 7 buffer was prepared with a mixture of monosodium phosphate and disodium phosphate of molar

Results and discussion

The effect of heating at 80 °C on the CD spectra of β-lactoglobulin at pH 7 is shown in Fig. 1. There is a shift in the spectra, the change being more pronounced for 5 min heating time with smaller change for subsequent heating times. As seen from Table 1, there is a decrease in α-helix from 16 to 12% from native to 5 min heating time with a corresponding increase in random coil content from 38 to 43%. Subsequent heating resulted in negligible change in the secondary structure. Similar behavior

Conclusions

β-lactoglobulin was subjected to different thermal treatments by heating the solution at 80 °C to different times. Heating was found to result in a change in the secondary conformation as indicated by a decrease in α-helix content with a corresponding increase in random coil. As expected, thermal treatment led to an increase in surface hydrophobicity due to exposure of more hydrophobic groups resulting from partial unfolding of the protein molecule. Interestingly, heating resulted in an

Acknowledgment

This work was supported by a grant from Kraft Foods Inc.

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    1

    Current address: Kraft Foods Inc., 801 Waukegan Road, Glenview, IL 60025, USA.

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    Current address: Cadbury-Adams, 182 Tabor Road, Morris Plains, NJ 07950, USA.

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