Hydrostatic pressure effects on the structural properties of condensed whey protein/lactose systems

Abstract

Hydrostatic pressure effects on whey protein/lactose mixtures were recorded with subsequent analysis of their structural, molecular and glass transition properties in comparison to thermal effects at atmospheric pressure. Experimental techniques used were small deformation dynamic oscillation in shear, modulated differential scanning calorimetry, Fourier transform infrared spectroscopy, and theoretical modelling of glass transition phenomena. Levels of solids ranged from 30 to 80% (w/w) in formulations with a protein/co-solute ratio of four-to-one. Addition of lactose protects the secondary conformation of the protein under application of high hydrostatic pressure. Nevertheless, pressurized protein systems are able to form three-dimensional structures due to the reduction in polymeric free volume and the development of an efficient friction coefficient amongst tightly packed particles. Systems can be seen as developing a “molten globular state”, where the structural knots of pressure-treated networks remain in the native conformation but achieve intermolecular cross-linking owing to frictional contact. Furthermore, pressure treated assemblies of condensed whey protein preparations could match the viscoelasticity of the thermally treated counterparts upon cooling below ambient temperatures. That allowed examination of the physical state and morphology of a condensed preparation at 80% solids by the combined framework of reduced variables and free volume theory thus affording derivation of glass transition temperatures for pressurized and atmospheric samples.

Introduction

Whey proteins (WP) are valuable food ingredients that provide essential amino acids and their inherent functional properties of gelation, high solubility, water holding capacity, foaming/emulsification and desirable sensory characteristics enable them to be used in numerous applications (Bouaouina, Desraumax, Loisel, & Legrand, 2006; Dissanayake, Kelly, & Vasiljevic, 2010; Lee, Morr, & Ha, 1992). They are compact globular proteins, with the major constituents being β-lactoglobulin, α-lactalbumin, serum albumin and immunoglobulins (Thompson, Boland, & Singh, 2009). Commercial products of whey protein concentrates (WPC), isolates (WPI) and hydrolysates are widely used by the industry and their incorporation in food systems as functional ingredients is the most practical manner of consumption. As a biofunctional ingredient, WP conveys a number of health benefits including antimicrobial, antihypertensive and antioxidant activity, immune modulation, improved muscle strength, preventing cardiovascular disease, osteoporosis, cancer and hepatitis B, and improving cognitive function or coping ability of highly stressed individuals (Marshall, 2004; Vasiljevic & Shah, 2007). Application of WP in food products, however, is restricted due to their heat-induced destabilisation that may affect their unique physical and physiological functionality as well as the organoleptic attributes (Onwulata, Konstance, & Thomasula, 2004).

As an emerging and non-thermal technology, high pressure processing (HPP) provides new opportunities for applications of food ingredients, with the capacity of maintaining sensory and nutritional quality. High hydrostatic pressure effects on WP in dilute and semi-dilute systems, for instance <10% solids in formulation, have been extensively studied (Considine, Patel, Anema, Singh, & Creamer, 2007; Lopez-Fandino, 2006). In these systems, the extent of pressure induced denaturation on reversible or irreversible conformational changes of WP by altering the equilibrium between protein–protein and protein–solvent interactions have been demonstrated at different levels of pressure and time combinations.

Pressure is a thermodynamic property and process that result in overall reduction in system volume at high levels of pressurisation. The exposure of buried hydrophobic groups to water with resulting “hydration” of these hydrophobic residues or clathrates, the dissociation of chemical species into their respective ions, and the disruption of non-native salt bridges are highly favoured under pressure as they contribute to decreasing volume (Cioni & Strambini, 2002). On thermodynamic grounds, the dominant driving force in high pressure induced protein unfolding is the penetration of water into the protein interior and the resulting “hydration” of hydrophobic side chains (Randolph, Seefeldt, & Carpenter, 2002). Hydrogen bonds are associated with contributing almost a neutral or very little effect to the partial molar volume change and thus both the formation and disruption of hydrogen bonds can be expected during pressure applications (Considine et al., 2007; Randolph et al., 2002).

Turning our attention to proteins in the presence of a co-solute environment, the protective effect of sugars on globular-protein unfolding results in the well known phenomenon of increased heat stability (Dierckx & Huyghebaert, 2002; Dissanayake et al., in press). Addition of sugar raises the chemical potential of the protein to a thermodynamically unfavourable situation. Reduction of the protein/sugar interface is then needed to render the system less unfavourable thermodynamically and, clearly, a drive for compact domains in the protein structure at intermediate to high levels of sugar tend to stabilize the system by decreasing the surface of contact between protein particles and co-solute (Chanasattru, Decker, & McClements, 2008). It has been argued that the prevention of globular proteins from unfolding and enhancement of favourable molecular interactions leading to irreversible denaturation and aggregation could also be the outcome of high pressure processing (He, Azuma, Hagiwari, & Kanno, 2006). In addition, when co-solvent molecules are present, some of the water molecules at the protein surface are replaced by them and thus “unhydrated” areas are considered to be significantly denser in protein and less compressible than protein/water domains.

The co-solute of interest in this work is lactose, a disaccharide of glucose and galactose, which is naturally present in milk. It is a reducing sugar and can contribute to Maillard reaction or freezing point depression in mixture with proteins (Hui, 2006). The relatively low sweetness, crystallisation properties, protein stabilisation, flavour emphasizing ability and enhancement of calcium absorption make lactose an important ingredient for the food industry (Damodaran, Parkin, & Fennema, 2008). Limited information is available on structure–function relationships and interactions in lactose–protein systems under pressure and records of high-pressure effects on condensed WP/lactose systems are non-existent in the literature. This is, therefore, the subject of the present investigation that aims to obtain insights into the behaviour of condensed WP/lactose mixtures upon pressurisation in an effort to support increasing interest for industrial R&D work in novel formulations and non-thermal processing.