The Purification Of Whey Proteins

cheese whey
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Whey protein is an important ingredient. It is a byproduct of cheese manufacture. Whey is used for a range of food applications including gel formation, bolstering the nutritional status of foods and beverages, especially sports nutrition products. So it is that the purification of whey proteins has takes on special significance.

In many applications the whey protein is concentrated to whey protein concentrate (WPC) which makes it a more cost-effective ingredient. WPC is a mixture of all the whey proteins.

Whey proteins correspond to about 18–20% of the total milk proteins and its major components are β-Lactoglobulin (β-Lg), α-Lactalbumin (α-La), bovine serum albumin (BSA) and immunoglobulin (Ig), representing, 50%, 20%, 10% and 10% of the whey
fraction, respectively. As well, whey protein contains numerous minor proteins, such as lactoferrin (LF), lactoperoxidase (LPO), proteose peptone (PP), osteopontin (OPN), lizozyme (LZ), amongst others (Hahn et al., 1998; Jovanovic et al., 2007).

Whey protein concentrate contains significant amounts of lipid, minerals and lactose. Whey protein isolate (WPI) is a higher value mixture of whey proteins. It is usually produced from sweet whey.

Fractionation Of Whey Proteins

There are two distinct methods of separation which either uses ultrafiltration or ion-exchange for the manufacture of whey protein concentrates. These concentrates can then be processed further either through selective fractionation to yield individual proteins or for the creation of spray dried agglomerates.

Considering ion-exchange methods; a number of studies have examined a variety of ion-exchange matrices for the very purpose of recovering whey protein (Ayers & Petersen, 1985).

One that can be used successfully for large-scale purification is the Spherosil ion exchange matrix. Another suitable and similar matrix is Indion S (Ion Exchange (India) Ltd) (Ayers et al., 1986).

Spherosil-S is a tradename for microporous silica beads containing a high concentration of strongly acidic sulphonic acid ligands (Spherosil-S). This operates as a cation-exchange resin where the pH of the whey is below 4.2.

As a matrix it has a high ion exchange capacity, a large surface area, a large pore diameter but low degree of compressability. That latter aspect makes it suitable for large-scale chromatography.

The following method was developed by Rhone-Poulenc in the late 60s. The ion-exchange medium is prepared and acid whey solution pumped through the column. The matrix is ideal for acidic solutions as the acidic whey has a pH of between pH4.5 and 4.6 but has its pH reduced to less than 3 for optimal binding. Rinsing of the column now containing the bound whey is achieved with a dilute acid solution to remove any lactose and minerals.

All the protein is eluted using dilute alkali solution based on dilute ammonia (actually ammonium hydroxide). The eluted proteins are then concentrated by ultrafiltration and then freeze dried (Nichols & Morr, 1985).

The ammonia is removed by volatilization which produces a high protein concentration and lower ash content. Ammonia needs to be handled with care in an industrial context because it is a severe irritant. If sodium hydroxide is used the health & safety issues are reduced but the ash content of the WPC is slightly higher.

In such a process the WPC contains 85% of material by dry weight. The WPC contains 80.9% protein, 7.0% milkfat, 10.3% moisture, 7.6% ash and less than 0.3% lactose.  

Other porous silica media have found similar benefits (Skudder, 1985; Schutyser et al., 1987; Ye et al., 2000). Schutyser (1987) examined a strongly acidic ion-exchange matrix composed of microporous silica coated with a thin layer of hydrophilic copolymer using sulphonic acid groups. To improve reliability and strength, the coating was cross-linked and covalently linked to the silica surface via its diol groups. These researchers compared Spherosil-S with this new material in both batch and column operation. The new silica product performed better than Spherosil-S in terms of breakthrough capacities.

Cellulosic and agarose matrices have been tried and found to have specifically better properties than silica-based variants (Doultani et al., 2003). 

Agarose beads were used to successfully recover 96% of the lactoferrin (LF) and 94% of the lactoperoxidase from sweet whey at flow rates 10 to 20 times greater than cellulose or silica beads (Kussendrager et al., 1997). The ability to recover proteins at higher flowrates is a major cost benefit and illustrates the resilience of the agarose matrix.

Fouling Of Ion Exchange Matrices

The whey needs to be defatted because residual milkfat upsets the functionality of WPC (Burgess & Kelly, 1979). Ideally it should be removed before ion-exchange chromatography.

Columns will foul more so when whey is applied. The column is flushed with a 1:4 mixture of diethyl ether and ethanol (v/v), then distilled water although the benefit is stated to be only partial. Fouling is due to residual binding of polar lipids hence the desire to remove these fats before ion-exchange.

As the column is used, the exchange capacity of the Spherosil-S media declines from 0.9 to 0.38 meq/g with continuous use. Recovery is often not feasible because the ion-exchange groups are either removed or lost from wear and tear.

Purification Of Individual Proteins

Further fractionation of whey proteins is possible with ion-exchange because the technology can exploit the differences in the pI of the individual whey proteins. The pI for a protein is good way to predict the protein’s behaviour during separation. The implication is that no protein can be retained at its isoelectric point because it has no residual charge.

A protein is retained by anion exchange resins when it is above its pI because the protein has a residual positive charge and binds to negatively charged groups on the resin. Likewise, cation exchange resins are used when proteins are below their pI.

Adjusting pH of the whey media helps in the selective fractionation of individual proteins. The pIs of beta-lactoglobulin and alpha-lactalbumin which are the two major fractions are 4.9 to 5.4 and 4.8 respectively. These are in the acid range. On this basis, anion exchange matrices are preferred because the pH of the feedstock is neutral. On this basis weak (DEAE) and strong (Mono Q) anion-exchange materials are used in the purification of these two proteins (Santos et al., 2012; Stojadinovic et al., 2012).

Using DEAE-C anion-exchange chromatography, Neyestani et al., (2003) fractionated β-Lg, α-La and BSA. In this study α-La and BSA were found to co-elute. Furthermore, to separate β-Lg into its two variants the authors reported the need to perform a second
chromatographic step, which could be achieved in a single step in the current work

References

Ayers, J. S., & Petersen, M. J. (1985). Whey protein recovery using a range of novel ion-exchangers. New Zealand Journal of Dairy Science and Technology. 20 pp. 129

Ayers, J. S., Elgar, D.F. & Petersen, M. J. (1985). Whey protein recovery using Indion S, an industrial ion exchanger for proteins. New Zealand Journal of Dairy Science and Technology. 21 pp. 21

Bottomley, R.C., Evans, M.T.A., Parkinson, C.J. (1990). Whey proteins. In: Harris P, editor. Food Gels. New York: Elsevier Applied Science pp. 435–66

Burgess. K.J. and Kelly. J. (1979). Selected functional properties of whey protein isolate. J. Food Technol. 14: pp. 325

Doultani, S., Turhan, K.N., Etzel, M.R. (2003). Whey protein isolate and glycomacropeptide recovery from whey using ion-exchange chromatography. J Food Sci
68 pp. 1389–95

Doultani, S., Turhan, K.N., Etzel, M.R. (2004). Fractionation of proteins from whey using cation-exchange chromatography. Process Biochem.

Etzel, M.R. (1995). Whey protein isolation and fractionation using ion exchangers. In: Singh RK, Rizvi SSH, editors. Bioseparation Processes in Foods. New York: Marcel Dekker, Inc. p 389–416.

Etzel, M.R., Dermawan, S., Budiman, M.N., Hendriadi, V.V., Rosalina, I. (1998). Protein separation by ion exchange in columns. Int. Dairy Fed. (Spec. Iss.) 9804 pp. 66–72

Gerberding, S.J., Byers, C.H. (1998). Preparative ion-exchange chromatography of proteins from dairy whey. J. Chromat. A 808 pp. 141–51

R. Hahn, P.M. Schulz, C. Schaupp, A. Jungbauer, (1998) Bovine whey fractionation based on cation-exchange chromatography. J. Chromatogr. A 795  pp. 277–287

S. Jovanovic, M. Barac, O. Macej, T. Vucic, C. Lacnjevac, (2007) SDS–PAGE analysis ofsoluble proteins in reconstituted milk exposed to different heat treatments,
Sensors 7 pp. 371–383.

Kussendrager, K.D., Kivits, M.G.C., Verver, A.B. (1997) Jan 21. Process for isolating lactoferrin and lactoperoxidase from milk and milk products and products obtained
by such process. Campina Mclkunie BV, Netherlands, assignee. U.S. Patent 5,596,082

Nichols, J.A., Morr, C.V. (1985) Spherosil-S Ion Exxchange Process for preparing Whey Protein Concentrate. J. Food Sci. 50 pp. 610-614

Schutyser, J.A.J., Buser, T.J.W., van Olden, D., Overeem, T. (1987). The isolation of proteins from whey with a new strongly acidic silica-based ion exchanger. J. Liq.
Chromat. 10 pp. 2151–75

Skudder, P. J. (1985). Evaluation of a porous silica-based ion-exchange medium for the production of protein fractions from rennet-and acid-whey. Journal of Dairy Research52(1), pp. 167-181.

Ye, X., Yoshida, S., Ng, T.B. (2000). Isolation of lactoperoxidase, lactoferrin, alphalactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography. Int. J. Biochem. Cell Biol. 32 pp. 1143–1150

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