Whey protein is an important ingredient. Whey is used for a range of food applications including gel formation, bolstering the nutritional status of foods and beverages, clinical and therapeutic foods, and especially sports nutrition products. We know that 70% of all whey is now turned into various food products with 30% used to feed pigs. Some is used as fertilizer whilst more unscrupulous producers dump it in river systems. The environmental impact of wasting whey is high. So, the purification of whey proteins has taken on special nutritional, pharmaceutical and commercial significance (Buchanan et al., 2023).
In many applications the whey protein is concentrated to whey protein concentrate (WPC) or to whey protein isolate (WPI) which makes it a more cost-effective ingredient. WPC is a mixture of all the whey proteins. Whey itself is usually derived from skimmed milk from which casein has been removed for cheese making or for casein powder as an ingredient. In some cases small amounts of casein type protein and peptides are retained which are removed by further processing so that as much casein as possible is returned to a casein processing line. The subject of whey processing is well reviewed by Pearce (1992).
The global demand for whey was 10.3 billion US dollars in 2021. According to Statista in 2022, that demand will increase to 18.1 billion US dollars in 2029.
Composition
Whey is a waste stream generated when curds are collected for cheese production or for casein production. It is estimated that the global annual production of whey is 160 million tonnes, That 94% comes from cheese production and the remainder for casein manufacture (Božanić et al., 2014).
The whey from a mozzarella cheese process has a water content of at least 94% w/w. The remainder on a total solids basis is 5.9% of which proteins are 0.72%, fat is 0.79%, lactose is 3.9% and salt is 0.46% (Rektor & Vatai, 2004).
Whey proteins correspond to about 18–20% of the total milk proteins and its major components are β-Lactoglobulin (β-Lg) [50 to 55% w/w], α-Lactalbumin (α-La) [20-25% w/w], bovine serum albumin (BSA) [5-10% w/w] and immunoglobulin (Ig) [10-15%]. There is a general catch all protein group called glycomacropeptides [10-15%]. As well, whey protein contains numerous minor proteins, such as lactoferrin (LF) [1 -2 %w/w], lactoperoxidase (LPO), proteose peptone (PP), osteopontin (OPN), lysozyme (LZ), amongst others (Hahn et al., 1998; Jovanovic et al., 2007; Guo, 2019).
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.
Processing Whey
Whey which contains protein, lactose and water is the byproduct of the cheese production process. It is primarily separated from casein in milk. Given cheese was the main product of milk processing, whey has oft been seen as a by-product, a waste to be fed only to animals. It is only since the end of the second world war that whey protein began to attract greater commercial interest. However, to obtain the ‘gold’ which is whey protein takes quite a bit of processing.
Liquid raw whey needs to be concentrated to make subsequent unit operations viable and effective. Before concentration, whey liquid is clarified to remove any particulates and remnants of the cheese making process. Processors either use centrifugation which is preferred or microfiltration. There is then a subsequent separation process to produce a fully clarified whey followed by pasteurization to stabilize it.
Separation means removal of fat (whey cream) and other fine particulates and is sometimes called skimming. The skimmer can also be a centrifuge or membrane filter. The fat can then processed for addition to other products.
The cheese fines can be separated further from residual whey using a decanter. The whey can then returned to the raw whey for further processing.
Pasteurization has a profound effect on whey protein because it denatures it. It is also unusable if casein micelles are to be produced. Some key thermal facts: the minor whey proteins such as immunoglobulins and serum albumin denature at about 65ºC, whilst the major ones such as β-lactoglobulin (β-LG) and α-lactalbumin (α-LA)) begin to denature significantly above 70 to 75ºC (Oldfield et al., 1998). A popular non-thermal option is to use centrifugation which involves double bacterial removal prior to centrifugation. It is not feasible to use a centrifuge as an alternative to a pasteuriser but there are some whey producers who use aseptic microfiltration.
In some processes, an ion-exchange process is used to remove fat and reduce lactose content. The whey stream can then be membrane filtered so that what is still a watery stream of protein is split up further into a permeate of low molecular weight molecules and a retentate containing concentrated protein.
The whey cream can be standarised using a Standomat (GEA, Germany)which is an automated unit. Arla Foods currently have one in their German plant.
The whey in liquid format is now prepared for concentration.
Most operations use evaporation such as thin-film. The alternative is the use of reverse osmosis (RO) which is a membrane technique that removes water and may be a small amount of salt depending on the pore-size. Evaporation is a high energy thermal process whilst RO is more benign but limited by fouling. It is at this specific point that whey protein concentrate is manufactured.
Whey can be left as is. When the degree of concentration is correct it can be crystallized which usually means precipitated into a general whey protein mass – whey powder. That is the principal ingredient of many formulations. Whey and its permeate can however be dried using conventional methods such as spray-drying. A couple of forms are available – hygroscopic and non-hygroscopic. This refers to the degree of potential for picking up water and remaining dry on storage.
Spray drying takes many forms but there are a number of options available (i) a straight-through Instantizing process, (ii) drying with post-crystallization and (iii) drying in an integrated fluid bed.
Evaporation
Evaporation of whey liquid and permeates is still the most convenient method for dewatering a liquid to produce a concentrate.
When methods of water removal are compared, each unit operation can be quoted in terms of the separation cost per unit volume of water removed.
| Method of water removal | Separation costs per unit volume of water removed (equivalent units) |
| Spray drying | 17-50 |
| Drum Drying | 10-25 |
| Centrifugation | 0.1-10 |
| Ultrafiltration and Reverse Osmosis | 0.2-7 |
| Evaporation | 0.2-5 |
Fouling and Cleaning
One of the major issues of processing is fouling especially of heated surfaces such as pasteurisation which use heat exchangers or concentration by evaporation. Milk deposit in a heat exchanger when heated to between 70 and 90C will contain between 50 and 60% w/w protein, 30-35% minerals and 4-8% fat. This is termed a milk type A deposit (Jeunink & Brinkman, 1994).
Fractionation Of Whey Proteins
To isolate and purify whey proteins is challenging because all of these proteins occur in relatively low concentrations in a medium which has a complex chemistry. Separation has been described in two exceptionally good reviews: El-Sayed and Chase (2011) and by Zydney (1998).
There are two important and distinct methods of separation which either use ultrafiltration or ion-exchange for the manufacture of general whey protein products such as the concentrates and isolates. These will often be processed further as a composite for spray-dried agglomeration. Combinations of both techniques are possible too. When it comes to isolating the specific components then a technique such as affinity chromatography shows better promise because it offers greater selectivity.
Using Membrane Filtration Technology
The whey processing industry was one of the first to adopt large-scale membrane processing as an alternative to evaporation because of a desire to reduce thermal damage of proteins (Zadow, 1987; Maubois, 1991). It is also the case that milk itself can be treated using various forms of cross-flow filtration to separate off or to concentrate casein micelles for cheese-making (Carvalho & Maubois, 2009).
One specific approach is the use of microfiltration (MF) to take out lactic acid bacteria (LAB) alongside Listeria and other food safety and spoilage microorganisms. As well as serving as a sterilization method it is also possible to defat whey as well. It is also the first unit operation in a sequence of steps when microfiltration. The permeate is then processed further because it contains all the proteins of interest.
The permeate can be sent to three types of membrane process after microfiltration depending on whey is required of the permeate. The tightest is reverse osmosis which has 99% protein with salt retention and produces a whey concentrate because only water and some salt is removed. A looser membrane process is nanofiltration which leaves a retentate of protein and lactose but allows water with more salt through. The retentate is ideal as a raw diary product for ice-cream production. The investment costs are appropriate for small- and medium-sized businesses.
A looser membrane process again is ultrafiltration where water and milk fat forms the permeate so a protein concentrate is still generated. Very often, a second filtration process following ultrafiltration is used because of the interest in a lactose rich concentrate which is the retentate. That type of process will be nanofiltration. The lactose in the retentate can be crystallised out (Kümmel & Robert, 2000).
Using Ion-Exchange Technology
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 (isoelectric point) 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.
The Use of Affinity Chromatography
Affinity chromatography offers better selectivity than ion-exchange processing methods. Heparin affinity chromatography has been tried for separating out minor protein components as they call them from whey protein isolates (WPI) (Ounis et al., 2008). The WPI was produced by ion-exchange chromatography (IEC-WPI) and the second by microfiltration/ ultrafiltration (MF/UF-WPI).
Using heparin affinity chromatography meant only between 1 and 13% by weight of the main whey proteins were bound. The minor components however such as insulin-like growth factor-I (IGF-I), transforming growth factor-beta2 (TGF-β2) and lactoferrin were concentrated by factors of 24–38, 10–30 and 32–93, respectively. These were found in a fraction eluted with 0.5M NaCl from either source of WPI.
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
Božanić, R., Barukčić, I. and Lisak, K., 2014. Possibilities of whey utilisation. Austin Journal
Nutr. Food Science., 2(7), pp. 1036
Buchanan, D., Martindale, W., Romeih, E., & Hebishy, E. (2023). Recent advances in whey processing and valorisation: Technological and environmental perspectives. International Journal of Dairy Technology, 76(2), pp. 291-312.
Burgess. K.J. and Kelly. J. (1979). Selected functional properties of whey protein isolate. J. Food Technol. 14: pp. 325
Carvalho, A. and Maubois, J. L. (2009) Applications of Membrane Technologies in the Dairy Industry. USA: CRC Press. pp. 33–56.
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.
El‐Sayed, M. M., & Chase, H. A. (2010). Purification of the two major proteins from whey concentrate using a cation‐exchange selective adsorption process. Biotechnology Progress, 26(1), pp. 192-199.
El-Sayed, M. M., & Chase, H. A. (2011). Trends in whey protein fractionation. Biotechnology Letters, 33, pp. 1501-1511.
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
(2019). Whey protein production, chemistry, functionality, and applications ( 1st ed.). John Wiley & Sons.
Hahn, R., P.M. Schulz, C. Schaupp, A. Jungbauer, (1998) Bovine whey fractionation based on cation-exchange chromatography. J. Chromatogr. A 795 pp. 277–287
Jeurnink, T. J., & Brinkman, D. W. (1994). The cleaning of heat exchangers and evaporators after processing milk or whey. International Dairy Journal, 4(4), pp. 347-368.
Jovanovic, S., M. Barac, O. Macej, T. Vucic, C. Lacnjevac, (2007) SDS–PAGE analysis of soluble proteins in reconstituted milk exposed to different heat treatments. Sensors 7 pp. 371–383.
Kümmel, R.; J. Robert, J. (2000) Application of membrane processes in food technologies, in: K. Bélafi-Bakó, L. Gubicza and M. Mulder, eds., Integration of Membrane Processes into Bioconversions, Kluwer
Academic/Plenum, New York.
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
Maubois, J.L. (1991) New applications of membrane technology in the dairy industry. Aust. J. Dairy Technol., 46(11) pp. 91–95
Nichols, J.A., Morr, C.V. (1985) Spherosil-S Ion Exchange Process for preparing Whey Protein Concentrate. J. Food Sci. 50 pp. 610-614
Oldfield, D.J., Singh, H., Taylor, M.W. and Pearce, K.N., 1998. Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot plant. International Dairy Journal, 8(4), pp. 311-318
Ounis, W. B., Gauthier, S. F., Turgeon, S. L., Roufik, S., & Pouliot, Y. (2008). Separation of minor protein components from whey protein isolates by heparin affinity chromatography. International Dairy Journal, 18(10-11), pp. 1043-1050 (Article).
Pearce, R. J. (1992). Whey processing. Whey and Lactose Processing, pp. 73-89
Rektor, A., & Vatai, G. (2004). Membrane filtration of Mozzarella whey. Desalination, 162, pp. 279-286 (Article)
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 Research, 52(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
Zadow, J.G. (1987) Whey production and utilization in Oceania in trends in whey uitilization. IDF Bull., 212 pp. 6
Zydney, A. L. (1998). Protein separations using membrane filtration: new opportunities for whey fractionation. International Dairy Journal 8(3), pp. 243-250.
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