Food and Bioprocess Technology

, Volume 9, Issue 8, pp 1367–1379 | Cite as

Role of Whey Components in the Kinetics and Thermodynamics of β-Lactoglobulin Unfolding and Aggregation

  • Jeremy Petit
  • Anne Moreau
  • Gilles Ronse
  • Pascal Debreyne
  • Laurent Bouvier
  • Pascal Blanpain-Avet
  • Romain Jeantet
  • Guillaume Delaplace
Original Paper


The optimisation of dairy unit operations involving heat transfer requires the control of fouling and aggregation phenomena following the denaturation of thermosensitive proteins, in particular β-lactoglobulin (β-lg). This study intends to give a better view of the influence of whey components (whey proteins, lactose, and minerals such as calcium) on β-lg denaturation through a combined kinetic and thermodynamic approach. β-lg denaturation in model solutions of increasing complexity (pure β-lg solution, whey protein solution, and two model wheys differing in mineral content) was characterised at temperatures ranging from 64.5 to 98 °C by following the evolution of soluble β-lg concentration with HPLC. It was demonstrated that whatever the model solution composition, a two-step mechanism (unfolding followed by aggregation) of 1.5-order kinetics could be adopted to describe β-lg denaturation reaction, as the temperature dependence of the denaturation reaction rate was properly fitted by Arrhenius equation. The dependency of kinetic and thermodynamic parameters on solution composition indicated that the presence of whey proteins enhanced β-lg aggregation, whereas lactose showed a small protective effect against β-lg unfolding. Additionally, minerals, especially calcium, tended to stabilise β-lg native state while increasing β-lg aggregation rates. However, at high mineral content, calcium influence could be hindered or even reversed, presumably owing to a lower bioavailability due to complexation with anions such as inorganic phosphates.


β-Lactoglobulin Whey components Heat treatment Denaturation Kinetics Thermodynamics 



This work, constituting a part of the PERE (Procédé Electromagnétique de Réduction d’Encrassement, i.e. Eletromagnetic device for the reduction of milk fouling) project, has been funded by INRA Transfert within the framework of the 2010 patent valorisation campaign.


  1. Allen, E., & Smith, P. (2001). A review of particle agglomeration. AEAT/R/PSEG, Vol. 398. Accessed 7 Apr 2016.
  2. Ames, J. M. (1998). Applications of the Maillard reaction in the food industry. Food Chemistry, 62(4), 431–439.CrossRefGoogle Scholar
  3. Anandharamakrishnan, C., Rielly, C. D., & Stapley, A. G. F. (2008). Loss of solubility of alpha-lactalbumin and beta-lactoglobulin during the spray drying of whey proteins. LWT - Foos Science and Technology, 41(2), 270–277.CrossRefGoogle Scholar
  4. Anema, S. G., & McKenna, A. B. (1996). Reactions kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. Journal of Agricultural and Food Chemistry, 44, 422–428.CrossRefGoogle Scholar
  5. Anema, S. G., Lee, S. K., & Klostermeyer, H. (2006). Effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible thermal denaturation of β-lactoglobulin and α-lactalbumin in skim milk. Journal of Agricultural and Food Chemistry, 54(19), 7339–7348.CrossRefGoogle Scholar
  6. Belmar-Beiny, M. T., Gotham, S. M., Paterson, W. R., & Fryer, P. J. (1993). The effect of Reynolds number and fluid temperature in whey protein fouling. Journal of Food Engineering, 19, 119–139.CrossRefGoogle Scholar
  7. Bouvier, L., Moreau, A., Ronse, G., Six, T., Petit, J., & Delaplace, G. (2014). A CFD model as a tool to simulate β-lactoglobulin heat-induced denaturation and aggregation in a plate heat exchanger. Journal of Food Engineering, 136, 56–63.CrossRefGoogle Scholar
  8. Boxler, C., Augustin, W., & Scholl, S. (2013). Fouling of milk components on DLC coated surfaces at pasteurization and UHT temperatures. Food and Bioproducts Processing, 91(4), 336–347.CrossRefGoogle Scholar
  9. Burton, H. (1968). Deposits of whole milk in treatment plants: a review and discussion. Journal of Dairy Reasearch, 35, 317–330.CrossRefGoogle Scholar
  10. Changani, S. D., Belmar-Beiny, M. T., & Fryer, P. J. (1997). Engineering and chemical factors associated with fouling and cleaning in milk processing. Experimental Thermal and Fluid Science, 14, 392–406.CrossRefGoogle Scholar
  11. Chobert, J.-M. (2012). Milk protein tailoring to improve functional and biological properties. Journal of Bioscience and Biotechnology, 1(3), 171–197.Google Scholar
  12. Croguennec, T., Leng, N., Hamon, P., Rousseau, F., Jeantet, R., & Bouhallab, S. (2014). Caseinomacropeptide modifies the heat-induced denaturation-aggregation process of β-Lactoglobulin. International Dairy Journal, 36, 55–64.CrossRefGoogle Scholar
  13. Dannenberg, F., & Kessler, H.G. (1986). In Le Maguer, M. & Jelen, P. (Ed.), Food engineering and process applications (pp. 335–346). Amsterdam (Netherlands): Elsevier Applied Science.Google Scholar
  14. De Jong, P., Bouman, S., & Van der Linden, H. J. L. J. (1992). Fouling of heat treatment equipment in relation to the denaturation of ß-lactoglobulin. Journal of the Society of Dairy Technology, 45, 3–8.CrossRefGoogle Scholar
  15. De Wit, J. N. (1990). Thermal stability and functionality of whey proteins. Journal of Dairy Science, 73, 3602–3612.CrossRefGoogle Scholar
  16. Donato, L., Schmitt, C., Bovetto, L., & Rouvet, M. (2009). Mechanism of formation of stable heat-induced ß-lactoglobulin microgels. International Dairy Journal, 19, 295–306.CrossRefGoogle Scholar
  17. Doultani, S., Tuhran, K. N., & Etzel, M. R. (2004). Fractionation of proteins from whey using cation exchange chromatography. Process Biochemistry, 39, 1737–1743.CrossRefGoogle Scholar
  18. Erabit, N., Flick, D., & Alvarez, G. (2013). Effect of calcium chloride and moderate shear on β-lactoglobulin aggregation in processing-like conditions. Journal of Food Engineering, 115, 63–72.CrossRefGoogle Scholar
  19. Erabit, N., Flick, D., & Alvarez, G. (2014). Formation of β-lactoglobulin aggregates during thermomechanical treatments under controlled shear and temperature conditions. Journal of Food Engineering, 120, 57–68.CrossRefGoogle Scholar
  20. Erabit, N., Ndoye, F. T., Flick, D., & Alvarez, G. (2015). A Population Balance Model integrating some specificities of the β-lactoglobulin thermally-induced aggregation. Journal of Food Engineering, 144, 66–76.CrossRefGoogle Scholar
  21. Ferreira, I. M. P. L. V. O., Mendes, E., & Ferreira, M. A. (2001). HPLC/UV analysis of proteins in dairy products using a hydrophobic interaction chromatographic column. Analytical Sciences, 17, 499–501.CrossRefGoogle Scholar
  22. Fryer, P. J., Christian, G. K., & Liu, W. (2006). How hygiene happens: physics and chemistry of cleaning. International Journal of Dairy Technology, 59, 76–84.CrossRefGoogle Scholar
  23. Gaiani, C., Mullet, M., Arab-Tehrany, E., Jacquot, M., Perroud, C., Renard, A., & Scher, J. (2011). Milk proteins differentiation and competitive adsorption during spray-drying. Food Hydrocolloids, 25, 983–990.CrossRefGoogle Scholar
  24. Galani, D., & Apenten, R. K. O. (1999). Heat-induced denaturation and aggregation of ß-lactoglobulin: kinetics of formation of hydrophobic and disulphide-linked aggregates. International Journal of Food Science and Technology, 34, 467–476.CrossRefGoogle Scholar
  25. Gernigon, G., Schuck, P., & Jeantet, R. (2010). Processing of Mozzarella cheese wheys and stretchwaters: A preliminary review. Dairy Science and Technology, 90, 27–46.Google Scholar
  26. Gotham, S. M., Fryer, P. J., & Pritchard, A. M. (1989). Model studies of food fouling. In H. G. Kessler & D. B. Lund (Eds.), Fouling and cleaning in food processing (pp. 1–13). Prien (Germany): University of Munich.Google Scholar
  27. Gotham, S. M., Fryer, P. J., & Pritchard, A. M. (1992). ß-lactoglobulin denaturation and aggregation reactions and fouling deposit formation: a DSC study. International Journal of Food Science and Technology, 27, 313–327.CrossRefGoogle Scholar
  28. Grijspeerdt, K., Mortier, L., De Block, J., & Van Renterghem, R. (2004). Applications of modelling to optimise ultra-high temperature milk heat exchangers with respect to fouling. Food Control, 15, 117–130.CrossRefGoogle Scholar
  29. Havea, P., Singh, H., & Creamer, L. K. (2001). Characterization of heat-induced aggregates of ß-lactoglobulin, α-lactalbumin and bovine serum albumin in a whey protein concentrate environment. Journal of Dairy Research, 68, 483–497.CrossRefGoogle Scholar
  30. Jeyarajah, S., & Allen, J. C. (1994). Calcium binding and salt-induced structural changes of native and preheated beta-lactoglobulin. Journal of Agricultural and Food Chemistry, 42, 80–85.CrossRefGoogle Scholar
  31. Khaldi, M., Ronse, G., André, C., Blanpain-Avet, P., Bouvier, L., Six, T., Bornaz, S., Croguennec, T., Jeantet, R., & Delaplace, G. (2015). Denaturation kinetics of whey protein isolate solutions and fouling mass distribution in a plate heat exchanger. International Journal of Chemical Engineering, 2015(139638), 10. doi: 10.1155/2015/139638.Google Scholar
  32. Khaldi, M., Ronse, G., André, C., Blanpain-Avet, P., Bouvier, L., Six, T., Bornaz, S., Croguennec, T., Jeantet, R., & Delaplace, G. (2016). Denaturation kinetics of whey protein isolate solutions and fouling mass distribution in a plate heat exchanger. International Journal of Chemical Engineering, 2016(4924250), 2. doi: 10.1155/2016/4924250.Google Scholar
  33. Kilara, A. (1994). Chapter 11—whey protein functionality. In N. S. Hettiarachchy & G. R. Ziegler (Eds.), Protein functionality in food systems (pp. 335–355). New York (USA): CRC Press.Google Scholar
  34. Labouré, H., Cases, E., & Cayot, P. (2004). Heat induced ß-lactoglobulin polymerization: role of change in medium permittivity. Food Chemistry, 85, 399–406.CrossRefGoogle Scholar
  35. Labuza, T. P. (1980). Enthalpy/entropy compensation in food reactions. Food Technology, 67, 67–77.Google Scholar
  36. Loveday, S. M. (2016). β-lactoglobulin heat denaturation: a critical assessment of kinetic modelling. International Dairy Journal, 52, 92–100.CrossRefGoogle Scholar
  37. Lowe, E. K., Anema, S. G., Bienvenue, A., Boland, M. J., Creamer, L. K., & Jiménez-Flores, R. (2004). Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine β-lactoglobulin and ĸ-casein. Journal of Agricultural and Food Chemistry, 52(25), 7669–7680.CrossRefGoogle Scholar
  38. Mahdi, Y., Mouheb, A., & Oufer, L. (2009). A dynamic model for milk fouling in a plate heat exchanger. Applied Mathematical Modelling, 33, 648–662.CrossRefGoogle Scholar
  39. Mekmene, O. (2010). Comportement de la fraction minérale laitière en fonction des conditions physicochimiques : de la précipitation des sels de phosphate de calcium à la prédiction des équilibres minéraux du lait. PhD Thesis, AgroCampus Ouest, Rennes, France.Google Scholar
  40. Mulvihill, D. M., & Donovan, M. (1987). Whey proteins and their thermal denaturation—a review. Irish Journal of Food Science and Technology, 11, 43–75.Google Scholar
  41. Mulvihill, D. M., & Kinsella, J. E. (1987). Gelation characteristics of whey proteins and ß-lactoglobulin. Food Technology, 41(9), 102–111.Google Scholar
  42. Nicolai, T., Britten, M., & Schmitt, C. (2011). β-lactoglobulin and WPI aggregates: formation, structure and applications. Food Hydrocolloids, 25(8), 1945–1962.CrossRefGoogle Scholar
  43. Nielsen, B. T., Singh, H., & Latham, J. M. (1995). Aggregation of bovine ß-lactoglobulin A and B on heating at 75 °C. International Dairy Journal, 6, 519–527.CrossRefGoogle Scholar
  44. Oldfield, D. J., Singh, H., & Taylor, M. W. (2005). Kinetics of heat induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration. Journal of Dairy Research, 72, 369–378.CrossRefGoogle Scholar
  45. Palazolo, G., Rodriguez, F., Farrugia, B., Pico, G., & Delorenzi, N. (2000). Heat treatment of ß-lactoglobulin: structural changes studied by partioning and fluorescence. Journal of Agricultural and Food Chemistry, 48, 3817–3822.CrossRefGoogle Scholar
  46. Parris, N., & Baginski, M. A. (1991). A rapid method for the determination of whey protein denaturation. Journal of Dairy Science, 74, 58–64.CrossRefGoogle Scholar
  47. Patel, H. A., Anema, S. G., Holroyd, S. E., Singh, H., & Creamer, L. K. (2007). Methods to determine denaturation and aggregation of proteins in low-, medium- and high-heat skim milk powders. Dairy Science and Technology, 87, 251–268.CrossRefGoogle Scholar
  48. Pereira, R. N., Teixeira, J. A., & Vicente, A. A. (2011). Exploring the denaturation of whey proteins upon application of moderate electric fields: a kinetic and thermodynamic study. Journal of Agricultural and Food Chemistry, 59, 11589–11597.CrossRefGoogle Scholar
  49. Petit, J., Herbig, A.-L., Moreau, A., & Delaplace, G. (2011). Calcium influence on beta-lactoglobulin denaturation kinetic rates: implications in unfolding/aggregation mechanisms. Journal of Dairy Science, 94, 5794–5810.CrossRefGoogle Scholar
  50. Petit, J., Herbig, A.-L., Moreau, A., Le Page, J.-F., Six, T., & Delaplace, G. (2012). Granulomorphometry: a suitable tool for identifying hydrophobic and disulfide bonds in beta-lactoglobulin aggregates. Application to the study of beta-lactoglobulin aggregation mechanism between 70 and 95 °C. Journal of Dairy Science, 95, 4188–4202.CrossRefGoogle Scholar
  51. Petit, J., Six, T., Moreau, A., Ronse, G., & Delaplace, G. (2013). β-lactoglobulin denaturation, aggregation, and fouling in a plate heat-exchanger: pilot-scale experiments and dimensional analysis. Chemical Engineering Science, 101, 432–450.CrossRefGoogle Scholar
  52. Prabakaran, S., & Damodaran, S. (1997). Thermal unfolding of beta-lactoglobulin: characterization of initial unfolding events responsible for heat-induced aggregation. Journal of Agricultural and Food Chemistry, 45, 4303–4308.CrossRefGoogle Scholar
  53. Relkin, P. (1996). Thermal unfolding of ß-lactoglobulin, α-lactalbumin, and bovine serum albumin. A thermodynamic approach. Critical Reviews in Food Science and Nutrition, 36, 565–601.CrossRefGoogle Scholar
  54. Rouyer, B. (2012). World trade of dried dairy products (Proceedings of the 5th International Symposium on Spray-Dried Dairy Products, p. 25).Google Scholar
  55. Santos, O., Nylander, T., Paulsson, M., & Tragardh, A. C. (2006a). Whey protein adsorption onto steel-surfaces—effect of temperature, flow rate, residence time and aggregation. Journal of Food Engineering, 74, 468–483.CrossRefGoogle Scholar
  56. Santos, O., Nylander, T., Schillén, K., Paulsson, M., & Tragardh, A. C. (2006b). Effect of surface and bulk solution properties on the adsorption of whey protein onto steel surfaces at high temperature. Journal of Food Engineering, 73, 174–189.CrossRefGoogle Scholar
  57. Sava, N., Van der Plancken, I., Claeys, W., & Hendrickx, M. (2005). The kinetics of heat-induced structural changes of ß-lactoglobulin. Journal of Dairy Science, 88, 1646–1653.CrossRefGoogle Scholar
  58. Schmitt, C., Bovay, C., Rouvet, M., Shojaei-Rami, S., & Kolodziejczyk, E. (2007). Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial and foaming properties. Langmuir, 23, 4155–4166.CrossRefGoogle Scholar
  59. Schokker, E. P., Singh, H., & Creamer, L. K. (2000). Heat-induced aggregation of beta-lactoglobulin A and B with alpha-lactalbumin. International Dairy Journal, 10(12), 843–853.Google Scholar
  60. Simmons, M. J. H., Jayaraman, P., & Fryer, P. J. (2007). The effect of temperature and shear rate upon the aggregation of whey protein and its implication for milk fouling. Journal of Food Engineering, 79, 517–528.CrossRefGoogle Scholar
  61. Simons, J.-W. F. A., Kosters, H. A., Visschers, R. W., & De Jongh, H. H. J. (2002). Role of calcium as trigger in thermal beta-lactoglobulin aggregation. Archives of Biochemistry and Biophysics, 406, 143–152.CrossRefGoogle Scholar
  62. Tolkach, A., & Kulozik, U. (2007). Reaction kinetic pathway of reversible and irreversible thermal denaturation of ß-lactoglobulin. Dairy Science and Technology, 87, 301–315.CrossRefGoogle Scholar
  63. Verheul, M., Roefs, S. P. F. M., & De Kruif, K. G. (1998). Kinetics of heat-induced aggregation of beta-lactoglobulin. Journal of Agricultural and Food Chemistry, 46, 896–903.CrossRefGoogle Scholar
  64. Vetri, V., & Militello, V. (2004). Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin. Biophysical Chemistry, 113, 83–91.CrossRefGoogle Scholar
  65. Visser, J., & Jeurnink, T. J. M. (1997). Fouling of heat exchangers in the dairy industry. Experimental Thermal and Fluid Science, 14, 407–424.CrossRefGoogle Scholar
  66. Xiong, Y. L. (1992). Influence of pH and ionic environment on thermal aggregation of whey proteins. Journal of Agricultural and Food Chemistry, 40, 380–384.CrossRefGoogle Scholar
  67. Zúñiga, R. N., Tolkach, A., Kulozik, U., & Aguilera, J. M. (2010). Kinetics of formation and physicochemical characterization of thermally-induced β-lactoglobulin aggregates. Journal of Food Science, 75(5), 261–268.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jeremy Petit
    • 1
    • 2
  • Anne Moreau
    • 2
  • Gilles Ronse
    • 2
  • Pascal Debreyne
    • 2
  • Laurent Bouvier
    • 2
  • Pascal Blanpain-Avet
    • 2
  • Romain Jeantet
    • 3
  • Guillaume Delaplace
    • 2
  1. 1.Université de Lorraine - LIBio, Laboratoire d’Ingénierie des BiomoléculesVandœuvre-lès-NancyFrance
  2. 2.INRA, UR638 Processus aux Interfaces et Hygiène des MatériauxVilleneuve-d’AscqFrance
  3. 3.Agrocampus Ouest, INRA, UMR1253, Science et Technologie du Lait et de l’ŒufRennesFrance

Personalised recommendations