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Department of Food Science, University of Wisconsin, Madison, WI 53706
3To whom correspondence should be addressed. E-mail: etzel{at}engr.wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT CONCLUSIONSLITERATURE CITED |
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3.0 were clear after thermal processing (88°C, 120 s). Beverages at higher pH were made clear after heating by addition of food-grade lauryl sulfate, which prevented aggregation of the denatured proteins formed during thermal processing. Alternatively, thermally processed clear beverages at pH 3–7 were possible using the whey protein glycomacropeptide. Because of the balance between sweetness and acidity, beverages with a pH greater than carbonated soft drinks and juices (pH 2.5–3) might remain palatable using less sugar. Development of high-protein low-carbohydrate beverages might provide health benefits for individuals suffering from diabetes, obesity, and hypercholesterolemia, especially when these beverages contain dairy protein fractions known to be high in essential amino acids and branched-chain amino acids.
KEY WORDS: • turbidity • leucine • ß-lactoglobulin • 
-lactalbumin • recovery
Nutritional attributes of dairy proteins
Milk contains a mixture of proteins, each having unique attributes for nutritional, biological, and food ingredient applications (1). The major proteins present in milk include -lactalbumin, ß-lactoglobulin, immunoglobulin, bovine serum albumin, and the caseins: 
-casein, ß-casein, and the -caseins. Minor but commercially important proteins are lactoferrin and lactoperoxidase. In addition, rennet whey contains glycomacropeptide that is cleaved from -casein by chymosin to initiate precipitation of the caseins forming curd.
Dairy proteins commonly available today are typically concentrates of the caseins or whey proteins, but not fractionated into individual proteins. For example, cheese is essentially a precipitate of all the caseins in milk. The caseins are much more concentrated in cheese than in milk, but the relative amounts of -casein, ß-casein, and the -caseins are not altered significantly. Similarly, the proteins in cheese whey are concentrated by membrane filtration, vacuum evaporation, and spray drying to form whey protein concentrate powder. The proteins are much more concentrated in whey protein concentrate powder than in whey, but the relative amounts of -lactalbumin, ß-lactoglobulin, immunoglobulin, bovine serum albumin, glycomacropeptide, lactoferrin, and lactoperoxidase are not significantly different.
Fractionation of the mixture of proteins found in milk and whey to form pure, individual dairy protein fractions might allow individuals with special nutritional needs to tailor their diet to improve health. As more is learned about the link between the human genome, diet, and health the need for specific proteins for individuals with different health concerns is expected to increase. This new knowledge must be developed hand-in-hand with new inexpensive separation process technologies to manufacture these protein fractions. Furthermore, palatable foods such as high-protein beverages must be developed to provide attractive routes for consumption.
Much has been learned recently about the role of proteins in improving health. Individuals interested in losing weight, controlling diabetes, losing fat, and building muscle protein might benefit from reducing their consumption of carbohydrates and increasing their consumption of protein (2). The Recommended Daily Allowance (RDA)4 of protein is set at the minimum needed to prevent deficiency. Protein consumption in the United States is at levels above the RDA. Nevertheless, most Americans satisfy much of their daily energy intake by carbohydrate consumption in the form of refined carbohydrates from breads, cereals, starchy snacks, and pasta, and from refined sugars in sodas and fruit juice drinks.
Energy restricted diets containing more than the RDA of protein increase weight loss and body fat loss, while sparing loss of muscle protein, compared to diets of the same calorie intake that are high in carbohydrates (2). Conversely, high carbohydrate diets reduce oxidation of body fat, increase blood triglycerides, and increase appetite. Fasting blood glucose titers are lower and less stable for individuals on equal-calorie high-carbohydrate diets compared to diets with reduced carbohydrates and increased protein, insulin titers are higher, and appetite is higher. High carbohydrate diets increase insulin titers by 40% compared to high protein diets.
Furthermore, proteins are not all equal in impact. There is increasing evidence that proteins high in BCAAs, and leucine (Leu) in particular, may play a unique role in human metabolism beyond the role of simply providing amino acids as substrates for protein synthesis (2). BCAAs are the only amino acids not degraded in the liver. Metabolism of the BCAAs occurs primarily in the skeletal muscle. In muscle, BCAAs are directed to protein synthesis and energy production. Muscle protein synthesis is triggered by Leu, which is sensed by the insulin-signaling pathway (2) and initiates the protein synthesis process. For energy production, the first step is removal of the amino group via transamination with the remaining carbon chain available for oxidation. The amino group is transferred to alanine or glutamine, which are released from muscle and ultimately converted into glucose in the liver by the process of gluconeogenesis. This pathway provides an alternative to dietary carbohydrates for providing essential levels of glucose. Thus, dietary protein has the potential to substitute for dietary carbohydrates in maintaining blood glucose levels, but only the BCAA Leu has the unique regulatory ability to stimulate muscle protein synthesis.
Other researchers have found that essential amino acids (EAAs) are more important than nonessential amino acids in muscle protein synthesis (3,4). The nonessential amino acids were not necessary for muscle protein synthesis and increases in the amount of EAA fed increased muscle protein synthesis. It is well established that consumption of significantly more protein than the RDA, especially protein high in EAA content, leads to increased muscle protein synthesis.
Based on the evidence above, diets high in protein, and particularly proteins having elevated contents of EAA, BCAA, and Leu, may have nutritional benefits not seen in diets containing an equal amount of an average protein. The amino acid compositions of pure, fractionated dairy proteins are closer to this ideal than a theoretical "average" protein (Table 1). The amino acid composition of a theoretical "average" protein was calculated from frequency of occurrence of each amino acid in 207 unrelated proteins of known sequence (5). As seen in Table 1, the EAA and BCAA contents are greater for the major whey proteins compared to an average for most proteins. For example, ß-lactoglobulin contains 17% more EAA, 33.5% more BCAA, and 74% more Leu than an average for proteins. These differences may have important nutritional consequences. Development of separation processes to manufacture specific dairy protein fractions such as ß-lactoglobulin may allow formulation of new high-protein foods designed to address emerging health crises such as obesity and diabetes.
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Dairy proteins have different molecular masses, concentrations, and isoelectric points (Table 2). Separation processes for proteins are designed to exploit these differences to the maximum extent. Processes proposed for commercial-scale production of whey protein fractions fall into 4 main categories: (1) selective precipitation (6–8), (2) membrane filtration (9), (3) selective adsorption (10–12), and (4) selective elution (13–17).
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Selective precipitation.
This separation process involves adjusting the solution physical properties to promote insolubility. Proteins are typically least soluble at a pH near the isoelectric point (pI) and in low ionic strength solutions, and most likely to aggregate under these conditions. For example, Pearce (7) adjusted whey to pH 4.2 and heated the solution to 65°C to denature, aggregate, and precipitate -lactalbumin. The supernatant was depleted in -lactalbumin and enriched in the remaining proteins such as ß-lactoglobulin. Similarly, Amundson et al. (8) demineralized whey at pH 4.65 by electrodialysis forming a precipitate of ß-lactoglobulin without using heat. Selective precipitation fractionates the feed solution producing a more dilute supernatant solution and a more concentrated precipitate solution.
Membrane filtration.
These separation processes traditionally have been based solely on differences in molecular mass. Concentration, not fractionation, is the most common method of use. For example, membranes with a 5,000 to 10,000 g/mol molecular mass rating are commonly used to make whey protein concentrate, a mixture of all the proteins in whey. These membranes retain essentially all the proteins listed in Table 2 with the exception of glycomacropeptide. Using membranes for fractionation of proteins is possible in 2 cases: (1) proteins where differences in molecular mass are great (e.g., -lactalbumin vs. immunoglobulin G), and (2) proteins where the combined differences in charge and size are great. Until recently, membrane processes were thought to only achieve separation between proteins differing in molecular mass by at least a factor of 10. However, careful adjustment of the solution pH and ionic strength has been used to separate proteins having little or no difference in molecular mass (9). Membranes commonly have a slight residual charge. By adjusting the solution pH, electrostatic rejection by the membrane can be enhanced or reduced. Furthermore, the effective diameter of a protein increases with decreasing ionic strength. Separation is enhanced by operating near the pI of the smaller protein and far from the pI of the larger protein to maximize the difference in effective hydrodynamic size. Low salt concentrations (1–20 mmol/L) increase electrostatic and steric rejection by the membrane. Multi-step adjustment of the pH and ionic strength of the whey may allow fractionation of proteins using a sequence of membrane separation processes.
Selective adsorption.
There are many examples of selective adsorption processes for whey proteins. For -lactalbumin adsorption, Gurgel et al. (10) used the immobilized hexapeptide WHWRKR, and Noppe et al. (11) used immobilized phenyl groups. For ß-lactoglobulin adsorption, Wang et al. (12) used immobilized retinal. In all of these selective adsorption processes, a single purified protein is produced in conjunction with a treated whey solution depleted in that protein. The cost of manufacture must be borne by revenue generated from that single purified protein product and the depleted whey solution.
Selective elution. Selective elution is an attractive alternative to selective adsorption (13). In selective elution, all the proteins in a mixture are trapped simultaneously onto the adsorbent, rinsed free of contaminants, and then eluted one-by-one to manufacture many different purified proteins. Ideally, the process uses an adsorbent and buffers that are inexpensive and food-grade, and it is operated at a high flow rate. Using such a process, the cost of manufacture is spread among many purified protein products, and the processor has the day-to-day flexibility to manufacture different products simply by using different elution buffers.
Precipitation and membrane separation processes are volume-dependent separation methods, wherein the equipment capacity and cost of manufacture is proportional to the volume of solution processed and not the mass of product produced. For dilute protein solutions such as whey, large volumes of liquid must be processed to recover a fixed mass of protein. Selective adsorption and selective elution processes are less volume dependent because adsorbent capacity depends mostly on the mass of protein recovered, not the volume of liquid processed (18).
For example, recovery of 1 kg of lactoferrin from whey by membrane filtration or process chromatography requires processing 10,000 L of whey, because the concentration of lactoferrin in whey is about 0.1 g/L (Table 2). Membrane filtration would require passing 10,000 L of permeate through a membrane that rejects the lactoferrin, but not the other whey proteins. Although such a membrane does not exist, processing this volume of whey in 1 day would require a membrane area of at least 6 m2, because the permeate flux for ultrafiltration membranes is at most 20 mL/s per m2 (19). Flux rates for lactoferrin recovery using process chromatography are about 5,800 mL/s per m2 (290X greater), which translates to a column cross-sectional area of about 0.02 m2 and a column volume of about 2 L (20). Flux does not set the capacity limit for process chromatography and lactoferrin recovery. Instead, the adsorbent capacity for lactoferrin is reached after processing about 1500 L of whey (20), requiring regeneration of the adsorbent by desorption of the bound lactoferrin before starting another cycle. Completion of about 7 adsorption-desorption cycles per day using a 2 L column would be required to recover the lactoferrin from 10,000 L of whey. Therefore, the capacity of membrane filtration is mostly set by the volume of whey processed, whereas the capacity of process chromatography is set mostly by the mass of lactoferrin recovered.
Process chromatography to fractionate whey proteins
The objective of our work was to develop process scale separation processes to fractionate proteins from milk and cheese whey. Selective adsorption and selective elution processes were considered for the reasons mentioned above. Selective elution was preferred over selective adsorption for 2 reasons. First, selective elution both concentrates and fractionates the protein. The protein is concentrated during the adsorption step, because all the proteins in the feed stream that can possibly bind are captured onto the adsorbent. The volume of the adsorbent is much less than the volume of the whey. That makes the concentration of the proteins bound to the adsorbent much greater than the concentration in the feed stream. The proteins are fractionated during the elution step, because the bound proteins are released separately into several different elution solutions. In comparison, selective adsorption concentrates and fractionates the target protein only, leaving unconcentrated and unfractionated all the remaining proteins in the feed solution.
The second reason to choose selective elution over selective adsorption is that only with selective elution can a single piece of equipment be used to make different protein products day-to-day. With selective elution, once the proteins are bound to the adsorbent, simply changing the elution buffers produces different protein fractions. There is no need to change the process equipment. For example, the system could be used to bind the proteins in the feed solution and then elute 1 protein using a first elution buffer, and then a second protein using a second elution buffer and so on. On another day, the system could be used to elute the first and second proteins together as a mixture followed by separate elution of the remaining bound proteins. On yet another day, the system could be used to elute all the proteins at once as a mixture, concentrating but not fractionating the proteins. The ability to switch between different protein fractions day-to-day and to manufacture different protein fractions all-at-once from a single feed stream offers the flexibility to respond quickly to changing market and customer demands. In addition, the cost of manufacture can be spread among several protein products, lowering costs per protein fraction.
Column chromatography was chosen over batch adsorption for several reasons. In batch adsorption, whey is stirred in a tank with the ion-exchange beads and bound protein recovered from the beads by adjusting the solution to alkaline pH (21–23). Limitations of the stirred tank batch process are: (1) recovery is low because of equilibrium considerations (24); (2) throughput is low because of long processing times; (3) the equipment is large because the tanks must hold all the whey (about 95% of the total volume), not just the beads; and (4) fractionation is difficult because changing the buffer requires emptying the large tank. In column chromatography, the beads are packed into a column and the solutions pass through the packed bed, which overcomes the limitations of batch adsorption processes.
Chromatographic fractionation of proteins from milk and cheese whey at commercial scale requires conditions quite different from analytical scale chromatography: (1) all buffers and materials must be food grade and inexpensive; (2) relative flow rates in column volumes per hour must be 10–20 times greater for economic viability; (3) columns must be 20,000 to 30,000 times larger in volume; (4) recovery must be >90%; and (5) the column must be operated for many cycles prior to cleaning without loss in capacity.
An example flow sheet for a process chromatography system is shown in Figure 1. Two pumps are used to mix buffer concentrates with purified water to reduce storage tank volumes. Solutions pass through detectors for conductivity, temperature, and pH, before passing through an air trap to remove bubbles, and a column prefilter to remove particles that may plug the column. For a bed of perfect spheres, the voids between the beads have a size representing 15.5% of the bead diameter. Because filtration to 25% of that size is needed to prevent plugging of the packed bed by bridging of particles, filtration to about 4% of the bead diameter is required. For process chromatography systems, using beads that are 200 µm in diameter, filtration to 8 µm is sufficient to avoid plugging of the column.
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Figure 2 contains the UV absorbance trace (panel a) and SDS-PAGE analysis (panel b) of the protein fractions from a laboratory scale chromatography column packed with SP Sepharose Big Beads (13). Mozzarella cheese whey was adjusted to pH 4.0, filtered to 3.0 µm, and passed through a column packed with 80 mL of beads to a bed depth of 15 cm. The procedure involved sequentially pumping different solutions into the column: (1) whey, (2) equilibration buffer consisting of 10 mmol/L sodium lactate, pH 4.0 to rinse unbound material from the column, (3) a first elution buffer consisting of 100 mmol/L sodium acetate, pH 4.9 to selectively recover -lactalbumin bound to the beads, and (4) a second elution buffer consisting of 10 mmol/L sodium hydroxide to recover the remaining whey proteins (enriched in ß-lactoglobulin). These are all inexpensive food grade buffers.
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-lactalbumin, 3% for ß-lactoglobulin, and essentially zero for immunoglobulin G, lactoperoxidase, and lactoferrin (Fig. 2b, lane 2). The column bound 95% of the combined total mass of these 5 proteins from the whey feed solution. The first elution peak contained 92% of the -lactalbumin loaded in the feed solution and was not contaminated with ß-lactoglobulin, immunoglobulin G, or bovine serum albumin (Fig. 2b, lane 3). The second elution peak contained no -lactalbumin, and most of the ß-lactoglobulin, immunoglobulin G, and bovine serum albumin loaded in the feed solution (Fig. 2b, lane 4). In other words, this process produced 2 products: -lactalbumin, and whey protein depleted in -lactalbumin. Based on these results, a 2 m diameter column with 20 cm bed depth, holding 630 L of beads, could produce 500 kg per day of -lactalbumin and 1800 kg per day of whey protein depleted in -lactalbumin from 500,000 L per day of whey. Beverage applications
The United States beverage marketplace is very large and growing. Major nonalcoholic products include: carbonated soft drinks ($63 billion/y), juice drinks and 100% juice ($20 billion/y), functional-food beverages ($11 billion/y), and bottled water ($8 billion/y). Essentially none of these products contain protein. All are of low nutrient density, consisting mainly of sugar water or simply just water. Fluid milk sales are $12 billion/y and stagnant, while other beverage sales are increasing. Yet, milk is less expensive than any of these beverages, including bottled water, and is nutritionally balanced. Increasing consumption of high-carbohydrate drinks such as soda and juice and declining consumption of higher protein drinks such as milk can be linked to increases in body fat, blood lipid titers, insulin secretion, and appetite as mentioned above.
One alternative to low-protein high-sugar beverages is to develop a soft drink-like high-protein low-sugar beverage having a slightly higher pH than current soft drinks. There is a balance between pH and sugar content. Fruits with a higher pH taste sweeter (Fig. 3). Balancing sweetness and acidity plays an important role in beverage formulation. Acidic beverages (cola is pH 2.5) are balanced using sugar: some soft drinks contain 28 g sugar per 240 mL. To be considered "high protein" requires adding 10 g of protein per 240 mL of this beverage (FDA reference amount for beverages = 240 mL). Calories that are added from protein can be compensated for by reducing sugar by 10 g and increasing the pH. Thus, a beverage containing 18 g sugar and 10 g protein per 240 mL, and having a pH between 3.0 and 4.6 is a suitable target. This beverage would qualify for a "high protein" and "reduced sugar" label.
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4.6. However, normal thermal processing of hot-filled shelf-stable beverages includes exposure to 88°C for 120 s to prevent yeast and mold growth. Turbidity of the WPI solution (Fig. 4, sample A) increased dramatically after thermal processing at the conditions required for shelf stable beverages (Fig. 5) Beverages with a pH 3.0 remained clear, and beverages at pH 4.0 were turbid without sediment formation, but beverages at pH 4.6 were heavily precipitated forming sediment layers on standing. Thus, clear high-protein beverages using WPI could be developed that have a pH 2.5 to 3.0, similar to current carbonated soft drinks. However, the sugar content would probably have to be just as high as soft drinks to balance the high acidity.
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The health advantages of a soft-drink-like beverage containing glycomacropeptide would be numerous. First, it would help shift the diet of the consumer from a high-carbohydrate high-calorie to a high-protein reduced-calorie diet. This would have the health benefits mentioned above regarding obesity and diabetes. Second, glycomacropeptide is higher in BCAAs and EAAs than a theoretical "average" protein (Table 1), which increases the health benefits as mentioned earlier. It might be desirable to supplement the beverage with Leu or all 3 BCAAs because of the special metabolic role of Leu mentioned above, and because glycomacropeptide is low in Leu compared to an average protein. Note that although supplementation of beverages with BCAAs maybe useful, it remains to be proven effective or safe. Third, glycomacropeptide has been shown to prevent viral and bacterial infection, neutralize endotoxin, promote growth of bifidobacteria, and modulate immunity (26). In 1 study (27), a control group of rats fed pathogenic E. coli all died from diarrhea, but another group of rats fed 5 mg/kg glycomacropeptide first followed by E. coli all lived. Nesser et al. (28) have shown that glycomacropeptide prevents dental plaque and caries. Consumers would have many health benefits from such a beverage and would not have to stop drinking the type of beverages they prefer.
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CONCLUSIONS |
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TOPABSTRACTCONCLUSIONSLITERATURE CITED |
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Our aim is to develop separation processes to manufacture these dairy protein fractions inexpensively at commercial scale. Process chromatography using ion exchange was chosen for the separation process. Selective elution was the preferred mode of operation because several protein fractions can be made from a single feed stream and single piece of equipment simply by changing buffers. The column bound 95% of the 5 major proteins in whey. The first elution peak contained -lactalbumin that was free of ß-lactoglobulin, immunoglobulin G, and bovine serum albumin. The second elution peak contained the remaining proteins (mostly ß-lactoglobulin) and no -lactalbmin. In another mode of operation, all the proteins were desorbed all at once, instead of separately, to produce WPI.
Whey protein fractions and WPI were tested for clarity in a prototype high-protein beverage. Beverages containing WPI at pH 2–7 were clear before thermal processing, but only beverages at pH 3.0 remained clear after heating to 88°C for 120 s. Thus, WPI could be added to thermally processed juices and carbonated soft drinks, which have a pH 2.5–3.0. Clear beverages containing WPI at pH > 3.0 were possible after addition of 16 g/L lauryl sulfate which prevented aggregation of the denatured proteins formed during thermal processing. Alternatively, thermally processed clear beverages were made at pH 3–7 using the whey protein fraction glycomacropeptide. Because of the balance between sweetness and acidity, it may be possible to develop beverages containing glycomacropeptide that have a higher pH than carbonated soft drinks and juices and that contain less sugar. Development of high-protein low-carbohydrate beverages might provide health benefits for individuals suffering from diabetes, obesity, and hypercholesterolemia, especially if these beverages contain dairy protein fractions known to be high in EAAs and BCAAs.
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FOOTNOTES |
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2 This research was supported in part by the College of Agricultural and Life Sciences and the Wisconsin Center for Dairy Research through funding from Dairy Management Inc.
4 Abbreviations used: EAA, essential amino acid; Leu, leucine; NTU, Nephelos turbidity units; pI, isoelectric point; RDA, Recommended Daily Allowance; WPI, whey protein isolate.
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LITERATURE CITED |
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TOPABSTRACTCONCLUSIONSLITERATURE CITED |
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1 column volume of effluent liquid) for recovery of 
