Whey protein fractionation

Whey protein concentrate

Following its introduction in the early 1970s, ultrafiltration of whey has grown to become commonplace throughout the dairy industry today. Ultrafiltration membranes with a molecular weight cut-off in the range of 10 000-30 000 Da are used to concentrate the whey proteins, while the lactose and minerals readily pass through the membrane into the ultrafiltration permeate. The ultrafilter concentrates the whey protein concentrate (WPC) to 15-20% total solids. The spray-dried WPC contains approximately 34% protein, dry weight, the remainder consists of lactose and minerals. WPC 34 has a similar gross composition to skim milk powder and can be used in foods as a skim milk replacement.

Diafiltration can be employed to remove lactose and minerals, purifying the whey protein up to 90% protein dry weight. In this process, water is added to the whey retentate during ultrafiltration to wash out lactose and minerals. There are a range of WPCs on the market, with enhanced protein levels such as WPC 50, WPC 75 and WPC 90.

WPCs possess a range of functional properties: gelation, water binding, foaming and emulsification. The 'quality' or functionality of the protein concentrate depends on the processing history of the whey, including the time/temperature history, pH, fat content, pump shear experienced and the whey pretreatments.

Whey contains many high-value biologically active proteins and peptides, with health and nutritional benefits that are attractive to an increasingly sophisticated market. In response, dairy processors continue to develop technologies to recover these fractions, with separations based on size, density, charge or hydrophobicity of the different components (see Table 14.18). Techniques include salting out, selective precipitation, heating at low pH, affinity chromatography, anion exchange chromatography, cation exchange chromatography using conventional resins or ion exchange membranes, size exclusion chromatography, hydrophobic chromatography, or combinations of enzymatic hydrolysis and membrane filtration. Different techniques are often combined to develop elaborate processes of sequential fractionation and purification resulting in an array of high-value by-products.

Whey protein isolate

Whey protein isolates (WPI) containing over 90% protein dry weight, are prepared using ion exchange. Whey protein purification by ion exchange adsorption utilises the amphoteric nature of whey proteins. Whey proteins have a net positive or negative charge depending on the pH of the medium. The isoelectric point of whey proteins is less than pH 5.5 and sweet whey is pH 6.1. Whey proteins therefore have a net negative charge at the pH of

Table 14.18 Types of proteins found in whey. Source: Marshall (1982)

Concentration (g/L)




Whey protein

of protein





(Da x 103)











Proteose peptones





Bovine serum albumins























sweet whey and can adsorb on to the positively charged anion exchange resins.

The ion exchange process is carried out in a stirred tank, a batch column or on a continuous separator such as a simulated moving bed or using annular chromatography. Typically the whey is eluted through an anion exchange resin to adsorb the whey protein. The lactose and minerals are rinsed from the resin, then the protein is desorbed with acid or brine solutions. Variations on this process may employ cation exchange resins with whey adjusted to less than pH 4.5 so the proteins carry a positive charge, the purified whey proteins can then be eluted from the resin by raising the pH above 5.5. For example, a high-flow process described by Doultani et al. (2004) uses Sepharose big beads to adsorb the whey proteins, which are eluted with a single buffer for WPI, or with combinations of buffers to separate a-lactalbumin, lactoperoxidase and lactoferrin fractions.

There are some difficulties associated with ion exchange protein separation. Large volumes of dilute rinse and regenerant solutions are required, particularly for batch separations. The protein fraction needs to be ultrafil-tered to separate the protein from the regenerant. The highly concentrated brine, acid or alkali used for more efficient protein desorption can affect protein functionality and solubility. Contaminants in whey can also foul the resin, necessitating regular cleaning cycles (Morr 1989).

Separation of a-lactalbumin and b-lactoglobulin a-Lactalbumin is the major protein in breast milk and has a large market in the manufacture of infant formula. The P-lactoglobulin fraction is a highly functional protein that is soluble in acidic beverages, suitable for egg-white replacement in meringues and for heat-set gels in manufactured meat products.

There are many methods developed for the separation of the a-/p-fractions of whey protein with a few commercially practicable, such as that developed by Pearce (1995). This process involves heating a membrane-concentrated whey to >55 °C at pH 4-4.5 resulting in reversible aggregation of the a-lactalbumin, which is then separated by centrifugation. The solu-bilised a-fraction can then be purified by microfiltration to remove phos-pholipids, while the supernatant is recovered as P-fraction. Another method for the separation of a-/p-fractions is based on precipitation using acidified sodium citrate to precipitate a-lactalbumin; this is separated, re-solubilised with calcium chloride and then dialysed to give a 70% pure a-lactalbumin fraction; the liquid P-lactoglobulin is dialysed to give a 90% pure P-fraction (Alomirah & Alli 2004). There are many more processes described in the literature, for example: a-/p-fraction separation based on size exclusion chromatography (Garcia Rojas et al. 2004); two-stage ultrafiltration with 30 000 and 100 000 Da membranes in series (Beelin & Zydeny 2004); continuous annular chromatography (Giovannini & Freitag 2002); colloidal gas aphrons from cationic detergent (i.e. surfactant stabilised microbubbles)

(Fuda et al. 2005); ceramic hydroxyapatite chromatography and size exclusion chromatography to further purify and recover lactoferrin and immunoglobulins (Schlatterer et al. 2004).

Bioactive proteins

Whey contains a range of bioactive proteins. Lactoperoxidase is a prominent enzyme in whey that has a natural antimicrobial action. It is used to catalyse the inactivation of micro-organisms, yet is harmless to mammalian cells (Kussendrager & Hooijdonk 2000). Lactoferrin is an iron-binding protein with health benefits related to intestinal health, anti-inflammatory and anti-tumour activity (Naidu 2005). GMP is a K-casein-derived protein naturally free of phenylalanine. It has physiological functions such as promotion of bifidobacteria, and immunomodulatory and bacterial toxin-binding effects (Thoma & Kulozik 2004).

Ion exchange chromatography is normally employed for the separation of lactoferrin and lactoperoxidase from whey. These proteins have isoelec-tric points above pH 9 and so are positively charged at the pH of whey and can be separated on cation exchange resins, then purified using ultrafiltration (Heeboll-Neilsen et al. 2004).

Novel ion exchange techniques are also being developed to recover lactoferrin and lactoperoxidase. Noh et al. (2005) describe the use of reverse micelles formed by cationic detergent to separate lactoferrin, based on solubilization behaviours of the proteins manipulated by pH, salt and surfactant concentrations. Fuda et al. (2004) describe the use of colloidal gas aphrons from anionic detergent, i.e. surfactant-stabilised microbubbles that act as ion exchangers for separation of lactoferrin and lactoperoxidase from whey.

Membrane adsorbers are also suited for separation of large volumes containing small amounts of the desired compound - such as whey growth factors, lactoferrin and lactoperoxidase (Regester et al. 1996). Ion exchange microporous membranes bind the desired component, yet allow passage of the remainder of the whey through the membrane, the desired component is then eluted from the membrane using dilute brine, acid or alkali and then concentrated by ultrafiltration (Splitt et al. 1996).

Uniform transmembrane pressure (UTP) filtration is opening up scope for more control over membrane separations for whey protein fractiona-tion. In the past, concentration polarisation at the membrane surface limited selectivity of the membrane. In this mode the permeate is recycled to maintain a uniform pressure differential across the membrane (Kulozik & Kersten 2004). So far the application has been limited to microfiltration separation of native caseins, but there is scope for fractionation of many of the proteins in whey.

GMP is normally separated using ion exchange chromatography, whereby GMP carries a negative charge in whey at acidic pH, thus the GMP flows through the ion exchanger while the other positively charged proteins bind to it. The GMP is then recovered from this final liquid (Tek et al. 2005). An alternative method, which reduces the volume of ion exchange resin and regeneration brine required, involves separating the GMP by ultrafiltration with a 100 000 Da membrane, then recovering the GMP by anion exchange chromatography (Xu et al. 2000).

Whey protein hydrolysates

Milk and whey proteins have been found to have different physiological functions due to the numerous bioactive peptides that are encrypted within the intact protein. Whey peptides contain 3-20 amino acid residues with the sequence of amino acids defining the function. Whey peptides have been found to exhibit various properties such as antioxidative, anti-hypertensive, antimicrobial, cytomodulatory, immunoregulatory, opioid, angiotensin-converting enzyme inhibition and mineral carrying capacity (Korhonan & Pihlanto 2003).

Bioactive peptides are commonly produced by enzymic hydrolysis of the whole protein, batch or continuously with immobilised enzymic membrane reactors. Hydrolysis is coupled with various separation procedures depending on the target peptide. Stepwise ultrafiltration (with diafiltration) can be used to fractionate the peptides based on size, while ion exchange/size exclusion chromatography is used for separations based on size and charge (Korhonan & Pihlanto 2003). New techniques have been reported by Groleau et al. (2004) who describe peptide fractionation by electro-nanofiltration which involves inserting a cathode into the permeate compartment of nanofiltration to increase separation of neutral and basic peptides.

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