Cold-set thickening mechanism of β-lactoglobulin at low pH: Concentration effects
Introduction
Beyond conveying nutritional benefits, whey proteins are also used as emulsifiers and foaming agents in food systems (Kinsella & Whitehead, 1998). There is an interest in developing protein based thickening agents for nutritional considerations (Hudson, Daubert, & Foegeding, 2000). Thickening functionality of whey proteins is typically achieved with a thermal treatment. However, there has been an interest recently in formation of cold gelling whey protein ingredients for potential application in food systems where heat may not be desirable. Typically in cold-gelation studies, whey proteins are heated at neutral pH and low ionic strength, above their denaturation temperature and below their critical concentration for gelation, to obtain solutions of soluble protein aggregates. These solutions are then cooled, and cold gelation is induced with salt addition (Barbut and Foegeding, 1993, Hongsprabhas and Barbut, 1997, Bryant and McClements, 2000) or by lowering the pH (Alting et al., 2000, Alting et al., 2002, Alting et al., 2003, Alting et al., 2004). In a different approach, β-lactoglobulin solutions were heated at pH 2.0, and then following cooling, the pH was adjusted to 7 or 8 and finally CaCl2 was applied for network formation (Veerman, Baptist, Sagis, & van der Linden, 2003). This process resulted in a critical concentration for gelation that was an order of magnitude lower than traditional cold-gelation methods. Cold gelling ingredients at a slightly alkaline pH have also been described in powdered form which gels upon redispersion in a salt containing solution (Thomsen, 1994).
In previous studies, Hudson et al. (2000) developed a procedure allowing for the production of a cold-thickening whey protein ingredient without any addition of salt or heat. This procedure involved pH adjustment to 3.35, thermal gelation, freeze drying, and finally grinding to a powder (Fig. 1). The modified powders (Hudson et al., 2000) impart instant thickening capability upon reconstitution in water. Originally, this procedure was applied to whey protein isolate, but has also been shown to work with whey protein concentrates (Resch and Daubert, 2002, Resch et al., 2004). The modified powders possess desirable functionality over a wide range of pH and thermal preparation conditions (Hudson & Daubert, 2002); however, the thickening mechanism remains unknown.
Mechanisms of whey protein, specifically β-lactoglobulin, aggregation have been extensively studied at pH 7 and pH 2, while limited studies have addressed aggregation mechanisms between pH 3 and pH 4 (Renard and Lefebvre, 1992, Sagis et al., 2002, Veerman et al., 2002, Veerman et al., 2003, Schokker et al., 2000, Aymard et al., 1996, Langton and Hermansson, 1992, Elshereef et al., 2006, Aymard et al., 1999, Sakurai et al., 2001, Castelletto and Hamley, 2007, Wada et al., 2006, Renard et al., 1998, Ikeda and Morris, 2002, Arnaudov et al., 2003, Kavanagh et al., 2000).
Depending on the pH and ionic strength, different types of aggregates are formed by β-lactoglobulin which includes fibrils, flexible strands, branched and random aggregates (Bolder, Hendrickx, Sagis, & van der Linden, 2006). Large random aggregates are formed at pH values close to the isoelectric point and at high ionic strength conditions (Aymard et al., 1996, Langton and Hermansson, 1992, Sagis et al., 2002). At pH values far from the isoelectric point and at low ionic strength, strand-like aggregates are formed (Aymard et al., 1999, Kavanagh et al., 2000, Langton and Hermansson, 1992, Stading and Hermansson, 1990). These strand-like aggregates can be rigid (fibrils), semi-flexible, or branched (Sagis et al., 2002).
At pH 2.0 and low ionic strength, long rigid strands (fibrils) are formed by β-lactoglobulin after heating (Veerman et al., 2002, Veerman et al., 2003, Ikeda and Morris, 2002, Kavanagh et al., 2000). At pH 2, β-lactoglobulin is highly charged (+20), forming fibrillar aggregates with diameter equivalent to that of a monomer (2–4 nm) (Veerman et al., 2002, Veerman et al., 2003, Ikeda and Morris, 2002, Kavanagh et al., 2000). Flexibility of these fibrils was shown to vary with ionic strength. Persistence length (length over which correlation in the direction of tangent is lost or simply the length that persists in a particular direction) is frequently used to quantify the flexibility/rigidity of polymer chains and biomacromolecules. Flexibility of fibrils increased (indicated by a decrease in persistence length) and contour length (curvilinear length of fibrils) decreased with increasing ionic strength (Veerman et al., 2002, Aymard et al., 1999, Kavanagh et al., 2000).
The motivation for this study was to investigate basic mechanisms behind thickening functionality of cold-thickening whey protein ingredients at low pH in hopes of achieving capability to mechanistically tailor functional attributes of modified whey protein ingredients. The objective of this study was to investigate concentration effects on the aggregation mechanism of β-lactoglobulin at pH 3.35 and thickening functionality of modified whey protein powders. In this study, concentration effects were investigated using capillary viscometry, rotational viscometry, transmission electron microscopy (TEM), and high performance liquid chromatography-multi-angle laser light scattering (HPLC-MALS).
Section snippets
Protein material
β-lactoglobulin (BioPureR, ∼94% pure, total protein ∼ 98% dry basis) was donated by Davisco Foods Inc.
Solution preparation
β-Lactoglobulin solutions of different concentrations (2–9% w/w) were prepared by dissolving β-lactoglobulin in deionized (DI) water by continuous stirring at room temperature for 1–2 h. Sodium azide (0.02%) was added to all samples to prevent microbial growth. Thereafter, solutions were adjusted to pH 3.35 using 6 N HCl. Following pH adjustment, solutions were heated at 85 °C for 3 h in a
Concentration and dilution effects on heated solutions
β-Lactoglobulin solutions at low concentrations (2–7% w/w) heated for 3 h at pH 3.35 behaved like a Newtonian fluid (n ∼ 1.0). However, pseudoplasticity increased rapidly with increasing concentration above 7% as determined by the values of power-law indices (n ∼ 0.97) at 8% w/w and 0.2 at 9% w/w (data not shown to emphasize differences observed at low concentrations since viscosity of this solution was about two logs higher than 8%) (Fig. 3A). These results expectedly showed that solutions
Conclusions
Concentration dependent differences were observed in thickening functionality of modified ingredient made from β-lactoglobulin, and a critical concentration was identified below which no significant thickening functionality (cold gelling) could be achieved in β-lactoglobulin dispersions. It was found that at pH 3.35 flexible fibrillar networks were formed with diameter of strands ∼ 5 nm and persistence length of about 35 nm. Differences in network structures below and above a critical
Acknowledgements
We would like to thank Davisco Foods Inc. for generously donating β-lactoglobulin for this study. We would also like to thank Dr. Debra Clare for her technical assistance with SDS-PAGE, Sharon Ramsey with Rheology, Dr. Bongkosh Vardhanabhuti with HPLC-MALS, Penny Amato with freeze drying and Abbey Woods with TEM. Finally, we would like to thank Dairy Management Incorporation and Southeast Dairy Foods Research Center for funding this project.
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