Modification of whey protein functionality

Several attempts have been already taken to modify whey proteins and thereby improve their functionality. In reality, for the successful utilization of functional properties of whey proteins in different food systems, they should be recovered in their native, undenatured state and subsequently incorporated in heterogeneous food matrixes. Although chemical and enzymatic modifications of whey proteins are widely applied, they have certain limitations (Panyam and Kilara, 1996; Spellman et al., 2005). The application of ultrasound for short duration of time to breakdown protein aggregates formed due to preheating treatments, and to prevent reformation of aggregates and the consequent viscosity increase are also in current interest of research (Ashokkumar et al., 2009). In addition, the use of mechanical forces under isothermal and/or isobaric conditions has a significant potential to modify whey proteins (Manski et. al., 2007; Iordache and Jelen, 2003). One of such popular approaches is extrusion, where proteins are thermally denatured under high pressure conditions technically known as thermoplastic melt. The denatured proteins become fibre form as a result of the process and sudden release

of pressure evaporates water and expands the product. The extent of expansion can be controlled by adjusting the pressure and temperature (Damodaran et al., 2008). This method has been employed for years and used to produce low- moisture food products such as snacks and breakfast cereals. Modern developed extruders are used for the protein texturisation at relatively high pressures and temperatures and at moderate mechanical shear rates resulting in reversible and non-reversible effects to proteins which ultimately have an effect on the functionality. Although, extrusion helps to increase the nutritional value of food products, the structural collapse and poor expansion are the main drawbacks (Onwulata and Tomasula, 2004). The high hydrostatic pressure is an alternative method to process dairy products without the adverse effects of thermal denaturation (Patel et al., 2005). However, the static high pressure induced changes of proteins in bovine milk are still not fully exploited and it is also in the experimental level (Lopez-Fandino, 2006; Huppertz, et al., 2006).

2.7.1 Microparticulation of whey proteins

Microparticulation of WP is a robust technique that can be applied to produce novel ingredients with modulated functionalities. In this process, WP are subjected to dynamic high pressure shearing which can be achieved for example by microfluidization with or without heating. The protein based microparticulated fat replacers such as Simplesse® (NutraSweet Co., Deerfield, IL) and Dairy-Lo®

(Pfizer Inc., New York, NY) are also produced with the use of heat and high shear (Akoh, 1998). The microparticulated WP behave like fats, for example, they considerably improve texture and taste in low-fat food products. Therefore, this technique is used to improve most cheese types, milk desserts and yoghurts, dressings and sauces and also other fat containing food products. If simultaneous heat and shear are applied, the characteristics of the resulting aggregates depend on the shear controlled aggregate growth and shear induced aggregate break-up as well (Spiegel, 1999). Extending the value of this technique, the microfluidization

has been used to improve the solubility of denatured whey protein isolates as described by Iordache and Jelen (2003). This study focused on sedimentation behaviour of heat denatured microfluidized WP. It revealed that microfluidization disrupted the heat-induced aggregates and produced non-sedimenting WP particles. This effect was obvious when the heat denaturation was carried out at pH 3.8 and thus produced micro-particles showed complete resistance to sedimentation. Also, as Paquin et al., (1993) previously reported, microparticulation of denatured WP at pH ~ 6.7 has reduced > 80% of the particle size of aggregates below 10 µm.

2.7.2 Microfluidization

The microfluidizer (MicrofluidicsTM, Newton, Massachusetts, USA) is a special fluid processor for ultra high pressure mixing, homogenizing, uniform particle/droplet sized reduction, cell disruption and creation of nanoparticles (Iordache and Jelen, 2003). This innovative technique, the microfluidization, can be used in many applications of food/nutraceutical, pharmaceutical, biotechnology, chemical, cosmetic and energy to research, develop and improve products efficiently. The principle of operation of the microfludizer is based on pumping a liquid product at constant high pressure of up to 275 MPa with subsequent division of the main flow into 2 smaller streams, which are then forced to collide against each other within an interaction chamber. When the product travels along the walls of micro channels of the microfluidizer, shear forces are applied to it at high velocity with an impact against the walls of the interaction chamber and the two streams colliding. In addition, the cavitations in the streams can occur by bubble forming and collapsing when they pass the different pressure zones within the interaction chamber (Barnadas-Rodriguez and Sabes, 2001).

Figure 2.4 Principle of operation of a microfluidizer (Adapted from: www.equilabo.com/MICROFLUIDICS_Presentation.html and modified).

Inlet reservoir Intensifier pump Outlet Pressure gauge Pressurized material Interaction chamber

The efficiency of process primarily depends on the extent of pressure and the number of microfluidizing passes (Iordache and Jelen, 2003). The generation of heat during the processing can be minimized by efficient cooling with an iced water jacket.

Microfluidization has become known as a very effective alternative method of reducing size of fat globules compared to conventional homogenization. As an conventional homogenization method, approximately 15 MPa are used in dynamic high pressure treatments in the production of stable emulsions in dairy and other food industries (Iordache and Jelen, 2003). The nanostructure of Mozzarella cheese could be altered using microfluidization and the temperature and pressure affected the extent of modifications (McCrae, 1994). Also, compared to sonication, the microfluidization was used to produce nano-emulsions with narrower size distributions (Jafari, et al., 2006). In addition, microfluidization creates fine emulsions, which would be advantageous in ice cream like dairy desserts. As reported by Olson et al., (2003), microfluidization produced non-fat and low-fat ice-cream that had a slower meltdown without affecting sensory properties.

2.7.3 Effect of dynamic high pressure shearing on whey proteins

The application of high pressure shear on globular WP has shown particularly different physico-chemical properties from those of native proteins. During this process, the collision, compression, shearing and flowing may take place with the contraction and more importantly stretching and elongation of protein molecules leading to the conformational rearrangements. In addition, the cavitation, turbulence and temperature rise in the medium are also possible as a result of forced induced occurrences. Also, the impact time of dynamic high pressure is very short when compared to that of static high pressure (Bouaouina, et al., 2006). As a result of mechanical forces, the quaternary and tertiary structures of proteins

are more susceptible to be disrupted compared to their secondary conformation owing to the perturbation of comparatively weaker inter- and intra-molecular hydrophobic and electrostatic interactions, however, depending on the extent of applied pressure and number of microfluidizing passes, β-sheets and α-helixes are also prone to be affected via the disruption of intra-molecular hydrogen bonds (Bouaouina, et al., 2006). Although, it is impossible to rupture the already existing covalent disulfide bonds in high pressure shearing, it may enhance the formation of new covalent bonds via exposed and newly formed reactive sites. Eventually, all of these high pressure induced conformational changes may be reflected as the altered or modified functionality of the globular WP.