Multiscale Characterization of Individualized β-Lactoglobulin Microgels Formed upon Heat Treatment under Narrow pH Range Conditions

Abstract

Aqueous dispersions of demineralized beta-lactoglobulin (beta-lg) were held at 85 degrees C for 15 min at a constant protein concentration of 1 wt % in the pH range of 3.0-7.0. This led to denatured protein content ranging from 20% (pH 3.0) to 90% (pH 5.0). The protein aggregates formed were characterized as to their stability to sedimentation (turbidity), morphology, size, surface charge, ANS surface hydrophobicity, and content in accessible thiol groups. Additionally, the changes in secondary structures of the protein upon heating were followed by Fourier transform infrared spectroscopy (FTIR). Stable dispersions (no sedimentation for 10 min) of individualized beta-lg microgels were obtained at specific pH 4.6 and 5.8, corresponding to an aggregation yield of about 80%. The width of the pH region leading to these microgels was 0.3 pH unit below or above the two specific pH values. Microgels were characterized by a spherical shape and remarkably low polydispersity in size (<0.2). Their z-average hydrodynamic diameter determined by dynamic light scattering (DLS) was between 160 and 220 nm, and their zeta-potential was +30 or -40 mV, depending on the initial pH before heating. Microgels obtained at pH 4.6 displayed a lower binding capacity for ANS and a lower content of accessible thiol groups as compared to those obtained at pH 5.8. Both types of microgels might therefore differ in their internal and interfacial structures. Between pH 4.6 and 5.8, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <4.6 or >5.8. Interestingly, DLS experiments showed that before heating, beta-lg was mainly present in an oligomeric state at pH 4.6 and 5.8. This result was confirmed by FTIR measurements indicating the stronger contribution of the 1616-1624 cm(-1) spectral band corresponding to intermolecular beta-sheets in the pH range of 4.0-6.0. Upon heating, FTIR spectroscopy revealed that individualized microgels were obtained under pH conditions where a balance between attractive forces arising from protein unfolding leading to the formation of intermolecular beta-sheets (1616-1624 cm(-1 )band) and the repulsive electrostatic forces due to the initial protein net charge was achieved.

Gelation and thermal characteristics of microwave extracted fish gelatin–natural gum composite gels

“…The turbidity of a mixed biopolymer system is often a consequence of phase separation resulting in the formation of particles that scatter light (Schmitt et al 2009). Turbidity measurements on gelatin-gum GFS can provide information about electrostatic complex formation between the two biopolymers.…”

Production of Whey Protein-Based Aggregates Under Ohmic Heating

“…There is a recognition that β-lg could form fibrils when heated at low pH and low ionic strength; β-lg gels with a fine-stranded microstructure can be made at pH < 4 . It is reported that far below proteins' pI (and also above), aggregates of pure β-lg solutions at 1 % are characterized by a worm-or fibrillar-like structure, instead of a spherical one (Mezzenga and Fischer 2013;Schmitt et al 2009). Several techniques have been reviewed for the production β-lg or WPI fibrils (Loveday et al 2012).…”

“…This interest arises with the possibility of designing whey protein hydrogels' size from micrometers to nanometers, thus allowing their incorporation in foods to impart desirable textural properties. These structures may also be used to protect and improve delivery of value-added bioactive compounds through microencapsulation and nano-encapsulation techniques (Augustin 2003;Bhopatkar et al 2012;Chen and Subirade 2007;Chen et al 2006;Gunasekaran et al 2007;Livney 2010;Nicolai et al 2011;Schmitt et al 2009). However, the mechanism of protein unfolding, aggregation, and gelation is rather complex.…”

Influence of moderate electric fields on gelation of whey protein isolate

“…The process of denaturation of globular whey proteins is assumed to consist of at least two steps: a partial unfolding of the native protein, and a subsequent aggregation of unfolded molecules (Nielsen, Singh, & Latham, 1996). In particular, unfolding of the native conformation of globular whey proteins at neutral pH leads to exposure of free sulfhydryl groups (SH) and hydrophobic amino acid side-chains, normally occluded within bovine serum albumin (BSA) and lg (Kazmierski & Corredig, 2003;Schmitt et al, 2009;Shimada & Cheftel, 1989). With further heat treatment, free SH may rapidly interchange with existing disulfide bonds to generate new inter-and intramolecular disulfide bonds that will engage toward protein aggregation (Fairley, Monahan, German, & Krochta, 1996;Schokker, Singh, Pinder, & Creamer, 2000).…”