Protein Expression and Purication 21, 165169 (2001) doi:10.1006/prep.2000.1350, available online at http://www.idealibrary.

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One-Step Separation from Lactose: Recovery and Purification of Major Cheese-Whey Proteins by HydroxyapatiteA Flexible Procedure Suitable for Small- and Medium-Scale Preparations 1
Rocco Rossano, Assunta DElia, and Paolo Riccio
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, 85100 Potenza, Italy

Received July 26, 2000, and in revised form September 18, 2000

The recovery of cheese-whey proteins and lactose represents an important task both in environmental and in food sciences. Optimization of whey processing requires the quantitative separation of whey proteins from lactose, lower costs, harmless environmental impact, exibility in protein recovery, and adaptability of the process to type and amount of available whey. Here we present a method based on the use of self-made, lowprice, and nontoxic hydroxyapatite for one-step separation of lactose (non adsorbed) from bovine whey proteins (adsorbed). Recovery of proteins can be performed with high exibility. Total protein fraction can be eluted with 0.4 M phosphate at pH 7.0. In alternative, proteins can be recovered in pairs with 0.4 M phosphate but at different pHs. About 56% of the proteins, primarily -lactalbulmin and IgG, were eluted at pH 5.0. The other major proteins, -lactoglobulin and BSA, were eluted at pH 6.0. Fractions eluted with the two rst eluants at pH 5.0 and pH 6.0 were applied to a Superdex 75 column for nal purication by gel ltration. This method provides exibility in whey protein recovery and quantitative separation of proteins from lactose before ultraltration and nanoltration. 2001 Academic Press

industry, as functional food ingredients with added value, and in the cosmetic, pharmaceutical, and medical industries. The development of market strategies and the advent of new technologies have made it now possible to set up whey processing systems for the concentration and separation of its components. These processes are mainly based on ion exchange, electrodialysis, and membrane ltration. The aim of this study was the search for alternative/ additional methods that can be applied on both small and medium scales, are t to different sources of whey, and, when used before other processes, can improve their effectiveness while reducing their costs.
MATERIALS AND METHODS

Whey Samples Bovine cheese whey was collected from the draining curd in a local factory producing provolone cheese. Whey pH was 5.0. Samples were defatted and claried by centrifugation at 12,000g, 4C for 10 min, and stored at 20C until use. Protein content was determined using the Bradford (1) reagent from Bio-Rad with microassay procedure and BSA 2 as a standard. Protein concentration was 4 mg/ml and lactose content, determined using the enzymatic kit (lactose/D-galactose UV test) of Boehringer Mannheim, was 37 mg/ml. Hydroxyapatite Chromatography Hydroxyapatite, a form of calcium phosphate, Ca 3 (PO 4 ) 2 Ca(OH) 2 was prepared in our laboratory as described by Tiselius et al. (2). Briey, 500 ml of each of 0.5 M solutions of CaCl 2 and Na 2 HPO 4 was added under stirring (210 rpm) and at an equal rate of ow
2 Abbreviations used: BSA, bovine serum albumin; IgG, immunoglobulin.

Whey, the by-product of cheesemaking, is a diluted mixture containing proteins, lactose, minerals, and vitamins. For a long time, because of the very low concentration of its components and the technical problems to be faced for a low-cost recovery process, whey has been considered as a waste or only as useful, in part, for animal feeding. However, whey components, if isolated, are highly valuable for their nutritional and functional properties and can be used both in the food
1

Italian patent pending, BA99000028.

1046-5928/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1.

Flow sheet of cheese-whey protein fractionation and purication.

(250 ml/h) to 50 ml 1 M NaCl in a stainless-steel vessel. The precipitate was decanted and washed two times with deionized water. After the addition of 40% (w/v) NaOH, the mixture was boiled under stirring (100 rpm) for 1 h. The precipitate was decanted and washed two times with deionized water. The hydroxyapatite thus obtained (about 75 80 ml, corresponding to about 25 g of dry adsorbent material), was stored in 10 mM sodium phosphate in the cold. The cost of chemical reagents for the production of 25 g hydroxyapatite, as described here, was of only 1.1 USD. Chromatography was performed at room temperature (about 20C). Sodium phosphate (NaPi) was used as eluant at different pHs. The maximum load was 4 mg protein per milliliter bed volume (or about 13 mg protein/g hydroxyapatite). The adsorbent material was regenerated ve times without changes of the chromatographic behavior of the proteins applied to the column.

Gel Electrophoresis SDSpolyacrylamide gel electrophoresis was performed according to Scha gger and von Jagow (3), using a 4% spacer gel and 10% running gel. Proteins were solubilized with a medium containing 4% SDS, 2% -mercaptoethanol, 12% glycerol, 0.01% bromphenol blue, 50 mM TrisHCl at pH 6.8. Following electrophoresis, proteins were stained for 2 h in 0.2% Coomassie blue R-250 and 0.05% Coomassie blue G-250 in methanol:acetic acid:water (4:1:4, v/v/v), and excess dye was removed with acetic acid:methanol:water (1:4:5, v/v/v). Densitometric analysis of the gels was carried out using an Ultroscan XL enhanced laser spectrodensitometer with Gel Scan XL software (Pharmacia Biotech). Native polyacrylamide gel electrophoresis was performed according to Schagger and von Jagow (4), using a 4% spacer gel and 8% running gel. Proteins were solubilized with a medium containing 12% glycerol,

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FIG. 2.

Adsorption chromatography of whey proteins on hydroxyapatite.

0.01% bromphenol blue, 50 mM TrisHCl, pH 6.8. Whey proteins purchased from Sigma were used as a standard both in SDS and in native gel electrophoresis. Gel Filtration Gel permeation chromatography was carrier out at room temperature on a column (16 400 mm) of Superdex 75 prep grade (Pharmacia Biotech), connected to a FPLC system using as buffer 40 mM TrisHCl, 100 mM NaCl, pH 7.0, at a ow rate of 1.0 ml/min.
RESULTS

An example of the purication procedure is shown in Fig. 1. The cheese-whey fraction (1 ml, 32 mg protein) was ltered through a 0.45- m cellulose nitrate membrane lter (Whatman) and applied at a ow rate of 30 ml/h to a column (16 65 mm) of hydroxyapatite connected to a Pharmacia FPLC system, previously equilibrated with 10 mM phosphate buffer pH 5.0. The column was washed with 2 vol of the same buffer and the nonadsorbed material (passthrough fraction) was collected. More than 96% of the lactose and only about 0.51% of the proteins were found in this passthrough fraction. All other applied proteins remained bound to the column. Bound proteins were eluted in pairs with 2 column vol of 400 mM phosphate at different pHs. The rst elution of the proteins bound to hydroxyapatite was carried with 2 vol of 400 mM phosphate buffer at pH 5.0. About 56 58% of the proteins applied, primarily -lactalbumin and IgG, were eluted with this buffer. Then the column was washed with 2 vol of 10 mM phosphate at pH 6.0 and the proteins, still bound to the hydroxyapatite column, were eluted with two

volumes of 400 mM phosphate at pH 6.0. The second eluate contained about 3738% of the proteins applied to hydroxyapatite, primarily -lactoglobulin and BSA. Finally, the column was washed with 3 vol of 400 mM phosphate at pH 7.0 yielding about 57% of the applied proteins. This fraction contained the residual -lactoglobulin. The chromatographic prole is shown in Fig. 2. In the alternative, total recovery of whey proteins was possible at one time by eluting with 400 mM phosphate at pH 7.0. Analysis of HTP eluate fractions, carried out both with SDS gel electrophoresis (Fig. 3A) and with native gel electrophoresis (Fig. 3B), showed the presence of -lactalbumin and IgG in the eluate obtained at pH 5.0 and the presence of BSA and -lactoglobulin in the eluate obtained at pH 6.0. Fractions eluted at pH 5.0 and 6.0 were pooled and concentrated by ultraltration on YM 10 Amicon membrane (cutoff 10 kDa). The concentrate fractions were applied for nal purication on gel permeation chromatography on a column of Superdex 75 prep grade (Pharmacia Biotech) (not shown).
DISCUSSION

Cheese-whey proteins are molecules with high nutritional value and several interesting functional properties useful for gel formation, emulsication, whipping and foaming, water binding, avor, and solubility. On these grounds, their recovery in puried form is convenient for many industrial applications. Different laboratory methods have been used for the separation of whey proteins. They are based on elec-

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FIG. 3. (A) SDS gel electrophoresis. Lane a, whey protein standards; lane b, cheese whey; lane c, hydroxyapatite eluate at pH 5.0; lane d, puried IgG; lane e, puried -lactalbumin; lane f, hydroxyapatite eluate at pH 6.0; lane g, puried -lactoglobulin; lane h, puried BSA. (B) Native gel electrophoresis. Lane a, whey protein standards; lane b, cheese whey; lane c, hydroxyapatite eluate at pH 5.0; lane d, puried -lactalbumin; lane e, puried IgG; lane f, hydroxyapatite eluate at pH 6.0; lane g, puried -lactoglobulins: A (top band), B (bottom band); lane h, puried BSA.

hydroxyapatite can be considered as a mixed bed exchanger capable of recognizing with high afnity the different distribution and density of multiple charges on the surface of proteins (23), without binding single charged molecules. On these grounds, hydroxyapatite offers the possibility of ne tuning and exibility of protein binding/eluting with great advantage when compared to ion exchange. The method has general applicability, since we have not found great differences when it is applied to goat cheese-whey proteins. In this case, about 70% of the applied proteins, -lactalbulmin and IgG, are eluted at pH 5.0, whereas the remaining major proteins, -lactoglobulin and BSA, are eluted at pH 7.0 and not at pH 6.0. In conclusion, the major advantages offered by this method are: (i) relative low cost and lack of toxicity of the material used, as well as possibility to regenerate the material; (ii) one-step quantitative separation between lactose and proteins; (iii) high exibility in protein recovery, ranging from specic proteins to total protein fractions; and (iv) purication of whey proteins in the native form.
REFERENCES
1. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248 254. 2. Tiselius, A., Hjrtien, S., and Levin, O. (1956) Protein chromatog raphy on calcium phosphate columns. Arch. Biochem. Biophys. 65, 132155. 3. Schagger, H., and von Jagow, G. (1987) Tricinesodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 379 386. 4. Schagger, H., and von Jagow, G. (1991) Blue native electrophore sis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223231. 5. Kaiser, K. P., and Krause, I. (1985) Analysis of proteins in foods by means of electrophoretic and chromatographic methods. Z. Lebensm. Unters. Forsch. 180, 181. 6. Amigo, L., Santamaria, G., Gonzales De Llano, D., and Ramos, M. (1986) Polyacrylamide gel electrophoresis of whey proteins in cheeses made from milk of different species in XXII International Dairy Congress, The Hague, Vol. 202, p. 175. 7. Addeo, F., Moio, L., Chianese, L., and Di Luccia, A. (1989) Detection of bovine milk in ovine milk or cheese by isoelectro focusing of -lactoglobulin: application and limitations. Ital. J. Food Sci. 1, 45. 8. Vogt, S., and Freitag, R. (1997) Comparison of anion-exchange and hydroxyapatite displacement chromatography for the isolation of whey proteins. J. Chromatogr. 760(1), 125137. 9. Felipe, X., and Law, A. J. (1997) Preparative-scale fractionation of bovine, caprine and ovine whey proteins by gel permeation chromatography. J. Dairy Res. 64(3), 459 464. 10. Mucchetti, G., Taglietti, P., Gatti, M., and Neviani, E. (1993) Whey ultraltration: Evaluation of protein retention with different membranes. Ital. J. Food Sci. 2, 99 106.

trophoretic (57) and chromatographic techniques (8 22). The commonest chromatographic approach is ion exchange. To our knowledge, there is only one report on the use of hydroxyapatite beside the present study. It compares adsorption chromatography on hydroxyapatite and ion-exchange chromatography (8). The authors conclude that ion exchange is the best choice. However, ion-exchange material is too expensive for use on a large scale, although it can be necessary when the purication of specic proteins and/or the complete separation from lactose are required. In fact, membrane ltration, which is the most convenient method presently used on industrial basis for the recovery of whey proteins and their separation from salts and lactose, is neither sufcient for the complete removal of lactose, nor for the isolation of single proteins. Moreover, the poor exibility of membrane ltration complicates its use when whey characteristics are highly variable. Hydroxyapatite appears to be an ideal tool for whey protein recovery both in the laboratory and in mediumsize processing systems, since it can be prepared in laboratory at very low cost with a simple procedure that does not require special apparatus. Furthermore,

PURIFICATION OF WHEY PROTEINS AND LACTOSE 11. Morais, F., and San Jose, C. (1991) Suero de queseria: Procesos industriales de transformacion clasicos y nuevas tecnologias. ` ` Rev. Esp. Lecheria 26, 27. 12. De Wit, I. N., Klarembeek, G., and Adamse, E. (1986) Evaluation of functional properties of whey protein concentrates and whey protein isolates. Netherland Milk Dairy J. 40 41. 13. Evans, M. T. A., and Gordon, J. F. (1980) Whey proteins in Applied Protein Chemistry (Grant, R. A., Ed.), pp. 31 67, Applied Science Publishers, London. 14. Coton, S. G. (1980) Whey technology: The utilization of permeates from the ultraltration of whey and skim milk. J. Soc. Dairy Technol. 33, 89 94. 15. Kosikowski, F. V. (1979) Whey utilization and whey products. J. Dairy Sci. 62, 1149 1160. 16. Bramaud, C., Aimar, P., and Daun, G. (1997) Optimisation of a whey protein fractionation process based on the selective precipitation of -lactalbumin. Lait 77, 411 423. 17. Maubois, J. L., Pierre, A., Fauquant, J., Piot, M. (1987) Industrial fractionation of main whey proteins. Bull. Int. Dairy Fed. 212, 154 159

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18. Pearce, R. J. (1988) Fractionation of whey proteins. Bull. Int. Dairy Fed. 212, 150 153. 19. Pierre, A., and Fauquant, J. (1986) Principes pour un procede industriel de fractionement des proteins du lactoserum lait. Lait 66, 405 419. 20. Zydney, A. L. (1998) Protein separation using membrane ltration: new opportunities for whey fractionation. Int. Dairy J. 8, 243250. 21. Pearce, R. J. (1992) Whey protein recovery and whey protein fractionation in Whey and Lactose Processing (Zadow, J. G., Ed.), pp 271316, Elsevier, New York. 22. Kristiansen, K. R., Otte, J., Ipsen, R., and Qvist, K. B. (1998) Large-scale preparation of -lactoglobulin A and B by ultraltration and ion-exchange chromatography. Int. Dairy J. 8, 113 118. 23. Riccio, P. (1989) Hydroxyapatite chromatography as a tool for the isolation of anion carriers and other membrane proteins in Anion Carrier of Mitochondrial Membranes (Azzi, A., et al., Eds.), pp. 35 44, Springer-Verlag, Berlin/Heidelberg.