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 (WPC) contains significant amounts of lipid, minerals and lactose. Whey lipids include phospholipids and milk fat globule membrane material. The fat level rises with the protein content of WPC i.e. 2.1% fat in 35% WPC, 3.7% fat in 50% WPC, and 7.2% fat in 80% WPC. The fat content reduces functionality such as foaming potential. That lipid composition is very similar to bulk milk.
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.
Raw liquid whey needs to be concentrated usually by evaporation 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.
As well as ion-exchange for removal of lipids, microfiltration can also be employed. The pores of a microfilter are large enough to remove large lipid-containing material. The cost however of allowing fat to be present in whey protein mixes is more cost-effective however than using a microfiltration method to remove fat. As methods improve the option for fat removal may well become economically viable (Zadow, 2003). Another alternative method for fat removal is the addition of calcium which aggregates lipoproteins in whey. It is technically effective but poses issues for scaling-up (Zadow, 2003).
Some processes for removal of fat produce a preferential transfer of lipids into one particular fraction. The Australian process which uses thermal aggregation preferentially concentrates lipid in the alpha fraction (Zadow, 2003).
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.
Whey Powder
Liquid whey can be left as is in a frozen format but it is best to just stabilize it by drying to whey powder. The liquid is usually evaporated by thermal methods. When the degree of concentration is correct it can be crystallized which usually means precipitated into a general whey protein mass. The alternative is to dry using spray drying or drum drying.
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.
Demineralised whey powder
Liquid whey is demineralised which means removing minerals using electrodialysis and/or nanofiltration. Evaporation is then performed to raise the solids content. It is then dried to a powder using spray drying to give DMWP. See the article on demineralized whey.
Whey Protein Concentrate (WPC)
Liquid whey is usually ultrafiltered to produce two streams. The retentate is converted to whey protein concentrate (WPC 34 – 80) by evaporation to increase solids and then dried.
The ultrafiltration membranes have a molecular weight cut-off of between 10 and 20kDa. The permeate contains lactose and minerals. These are evaporated too to increase solids content to well over 60%w/w. The lactose is crystallized out by slow cooling over 12 hours and collected. Further drying produces a dried powder. Further work-up produces a purified lactose sugar of over 99.9% purity.
All WPCs contain lipid. Given that to produce a retentate which contains protein using ultrafiltration, the presence of lipid reduces flux across the membrane. It is the same issue with diafiltration. Hence the desire to remove lipids before ultrafiltration or any other processing.
The retentate is further processed using evaporation and then spray drying. The protein content of the final product is affected by the degree of concentration. The 35 to 60% WPCs are obtained when the volume concentration ratio ranges from 4.5 to 20 respectively (Zydney, 1998)
A WPC of 8 to 10% w/w is obtained using PVDF membranes of 6 to 8 kDa molecular weight cut-off (Atra et al., 2005). Such concentrates are reused for cheese making.
The permeates from UF contain 0.1 to 0.5% protein and 5% lactose. These are further treated using nanofiltration to produce concentrated lactose.
Modified Whey Protein Concentrate
In most cases for WPC production, liquid whey is skimmed to remove fat and demineralised. This has to take place to obtains a protein content greater than 90%. Unlike WPC 34-80, a WPC 50-80 is generated using microfiltration or a centrifugal separator to remove large lipid molecules rather than just going straight to evaporation. The two streams generated are split. The retentate contains fat, large proteins, minerals and lactose. This is ultrafiltered further to increase protein content as required. The retentate is then spray dried to generate modified WPC 50-80.
The permeate from the microfiltration earlier contains some fats and lipids, small proteins, minerals and lactose which can be found in the retentate. This permeate is also ultrafiltered an second time but to remove lactose as well as increase protein content. The retentate from ultrafiltration can be diafiltered and concentrated so that the protein content rises to above 90% (only when the fat has been removed). This is dried to become Whey Protein Isolate (WPI 90). It is also possible use an ion-exchange tower that separates materials using ionic charge rather than molecular size. Some producers use this method is conjunction with microfiltration.
The permeate from the 2nd ultrafiltration is actually added to the permeate from the 1st ultrafiltration step. The permeate contains lactose and minerals too. The pH is adjusted and by applying heat, a mineral complex forms. The mineral complex can then be removed by decanting or clarification. The mineral complex can be concentrated further and then dried to form either an ingredient called milk minerals or whey mineral complex. The demineralised lactose can be treated as before.
Whey Protein Hydrolysate
One final fraction of note is whey protein hydrolysate (also known as hydrolysed whey protein) which is another important protein fraction found in adult nutrition products. The protein beta-lactoglobulin is the main protein in whey but it is also the main allergen especially in baby foods. The protein is difficult to hydrolyse using pepsin because the cleavage sites are all hidden inside the core of the protein. Other proteases having pH optima at higher values are used.
The hydrolysate is composed of di- and tripeptides. Being hydrolysed protein already, it is much more easily and readily absorbed than the other protein products. It is also ideal for those with a dairy allergy because beta-lactoglobulin is broken down.
The product is manufactured in a stirred tank usually as a batch process using proteases. It may require buffering to a specific pH using either acid or alkali.
We will discuss a bit on evaporation and then cover off the purification of proteins in whey using ion-exchange and filtration methods in more detail.
The Main Process Operations
Evaporation
Evaporation of whey liquid and permeates is still the most convenient method for dewatering a liquid to produce a concentrate. In 1933, the long-tube multiple-effect evaporator was introduced. The multiple-effect evaporator can boil water in a sequence of tanks at successively lover pressure. The boiling point of water decreases when pressure drops. The vapour that is boiled off is used to heat the next so an external heat source is used on the first vessel only. On that basis, evaporation in the first effect takes place at 77C and in the second at 45C.
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 90ºC 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).
Different forms of fouling do occur especially with membrane filtration Ultrafiltration accounts for 75% of all processing operations involving whey proteins. The issues relate to pore blockage and to the formation of protein cake layers on the membrane surface. This not only means loss of protein but also loss of processing time. The main protein that causes membrane fouling is β-lactoglobulin in whey. Even after diafiltration, there is still lactose and ash in whey protein concentrates (Wen-Qoing et al., 2019).
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.
A final method worth considering is selective precipitation which is sometimes found as part of another type of process.
Precipitation
All the why proteins have different isoelectric points (pIs) which means they have different solubilities depending on the pH. Such a difference can be exploited by selective precipitation. Proteins generally are least soluble at their pI and in low ionic strength solutions. They tend to aggregate and form precipitates rather than crystallise. When the pH pf whey is adjusted to 4.2 and then heated to about 65ºC, the alpha-lactalbumin denatures, aggregates and precipitates out. The supernatant then contains mostly the remaining protein which will largely be beta-lactoglobulin (Pearce, 1983).
When whey is demineralised at pH 4.65 using electrodialysis, it forms a precipitate of beta-lactoglobulin. No heat is needed. The approach has some value in producing protein reduced supernatants.
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 (RO) which has 99% protein with salt retention and produces a whey concentrate because only water and some salt is removed. The whey needs to be heated to 50 -55ºC and then pumped at high pressure between 2.7 and 10 MPa. This removes more minerals. Membrane-fouling is an issue because the molecular weight cut-off is 150 Da. RO is very helpful because removing 66% of the water is possible which means liquid retentate can be shipped about.
A looser membrane process is nanofiltration which leaves a retentate of protein and lactose but allows water with more salt through. The permeates from UF following treatment of whey to obtain whey protein concentrates contain 0.1 to 0.5% protein and 5% lactose (Adegoke et al., 2021).
These are further treated using nanofiltration to produce concentrated lactose for use in confectionary. 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; Adegoke et al., 2021).
The higher protein content of WPCs is generated using diafiltration after ultrafiltration, The retentate which is rich in proteins is diluted with water so that membrane permeable molecules are further removed. This increases the amount of protein in the retentate in terms of solids content. The retentate is cooled to 4C and stored until enough is available for evaporation before spray drying. At this point there may be a pasteurisation or UHT type treatment again to reduce spores and bacteria which get concentrated during membrane processing.
Treatment of Whey Prior To Microfiltration
We’ve mentioned elsewhere that lipids are a processing issue when producing whey protein fractions. Lipids reduce flux rates in ultrafiltration so any pretreatment is welcome. Not only do they prevent fouling of membranes they produce low-fat products (O’Regan et al., 2009).
All sorts of approaches have been tried from pH adjustment to changes in temperature and combinations of these. Gesan et al., 2009 studied the pretreatment of clarification of whey by ultrafiltration. In this process, they assessed a control pretreatment by increasing ionic calcium and pH accompanied with heat (50ºC, 15 minutes). This causes the complex lipid-calcium phosphate to precipitate. Calcium complexing agents, quiescent standing, centrifugation, microfiltration to dissolve colloidal calcium phosphate and/or remove insoluble cheese curd or casein fines, milkfat and producing calcium lipophosphoprotein complexes have all been tried.
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). In their study which is a good example of the application of the method, they used carboxymethyl- (CM) and sulphopropyl- (SP) cation-exchange derivatives from a chemically modified regenerated cellulose. The feed was whey protein. The CM derivative has a much narrower pH range for best performance compared to SP-ion exchange. If the whey source was demineralised beforehand the performance of both ion exchange systems was better.
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) which is now used routinely for whey protein isolation.
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.
Immunoglobulins of up to 90% purity are obtained from a two-step purification process. This includes includes a macroscopic MF pretreatment to remove fat globules and casein micelles prior to UF treatment with a membrane of 100 kDa (Piot et al., 2004).
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.
Protein Losses
The losses of protein in any process nowadays amount to less than 10% in terms of solids content. It is the same for casein production. What is more concerning is the loss of protein functionality which is a better measure of loss.
Heating causes denaturation of proteins. Some are more resistant to loss than others. Heat-induced denaturation of whey proteins has been studied in fresh milk in oil and water baths to make such studies as simple as possible (Hillier et al., 1979). In a simple heating system, the more concentrated the total solids in solution the slower the denaturation of beta-lactoglobulin but the more rapid the denaturation of lactalbumin. If lactose was present however, the denaturation rates were slowed down. It is thought that might be by preventing the formation of what would normally be heat-induced complexes.
In a study by Anema and McKenna (1996) α-Lactalbumin denaturation was first order, whereas both β-lactoglobulin variants had a reaction order of 1.5. In more cent times, Wijayanti et al., 2014 .
Heat processing which involves pasteurisation and evaporation, even spray drying usually causes a loss of about 20% of the general protein functionality (Mulihill & Donovan, 1987; Anema, 2020). Spray drying is one of the most likely causes of functional loss because of the level of heat required to drive off water. It could be the case that sizeable amounts of protein is denatured without physical loss. Protein denaturation is much more serious for those products which rely on functionality such as baby and infant formula.
The rates of denaturation of both lactalbumin and lactoglobulin increases significantly over the temperature range of 80 to 120C when milk is preheated prior to any processing let alone spray drying (Oldfield et al., 2005).
Loss of protein functionality whatever the protein is mainly due to denaturation. This means that the protein structure is disrupted. A variety of conditions including temperature, pH, ionic strength, chemical disruption etc. causes the three-dimensional structure of the protein to change by unfolding to such an extent that a new set of linkages are formed. The different types of cross-linkages in whey proteins include electrostatic, hydrogen-bonding etc. Denaturation can be so extensive that as well as aggregation and coagulation, the protein finally precipitates out of solution.
The process of spray drying of any sort causes some unavoidable losses in protein functionality. This is due to difficulties with reconstitution as well as the dehydration process that occurs during such an operation which causes aggregation and denaturation (Augustin and Udabage, 2007).
Some whey proteins are more susceptible than others. We know that beta-lactoglobulin loses a greater degree of functional solubility than alpha-lactalbumin (Ferreira et al., 2001). It also means that alpha-lactalbumin is more heat stable and this may be due to the binding of calcium ions. Only lactalbumin as a whey protein is capable of this. It is also the case that the loss of calcium ions leads to denaturation of this protein. The losses of functionality in beta-lactoglobulin can amount to 40%.
Whey proteins and casein are easily denatured and then aggregated. Aggregation reactions are highly influential (Gotham et al., 1992).
The concentration of each protein in solution prior to spray drying is directly related to the loss of solubility. .
During processing it is thought that the general amount losses are expected to be less than 10% across the process:-
- Spray Drying – less than 1% because there can be some coating of nozzles and retention in the spray chamber.
- Ultrafiltration – roughly 1% physical losses.
Economics
Economies of scale apply for whey manufacture as they do for any other process. Plant size is by far the biggest factor which affects the costs of each unit operation. The differences can be up to 40% according to unpublished analysis (FoodWrite,2019).
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