European Food Research and Technology

, Volume 237, Issue 3, pp 377–384 | Cite as

Bleaching of colored whey and milk by a multiple-enzyme system

  • Renata T. Szweda
  • Katharina Schmidt
  • Holger Zorn
Original Paper


Carotenoids are broadly used to enhance the color of cheese types like Cheddar and Gouda. While ∼80 % of the colorants are transferred into the cheese, the rest remains in the whey and impedes its commercial utilization. Therefore, cheese whey is currently bleached chemically by addition of either hydrogen peroxide or benzoyl peroxide in the industrial practice. To avoid heat- and peroxide-induced protein denaturation and the formation of off-flavors, an enzymatic bleaching process was developed. The ability of the fungal peroxidase MsP1 to degrade carotenoids was successfully employed for the bleaching of colored whey and milk in two- and three-enzyme systems, respectively. The systems were composed of MsP1, glucose oxidase, and when necessary, acid lactase. The initial step of the three-enzyme system was the lactase-catalyzed hydrolysis of lactose to galactose and glucose. The latter served as a substrate for the enzyme glucose oxidase in the second step which yielded gluconic acid and hydrogen peroxide. Finally, MsP1 oxidatively degraded the carotenoids. The activities of the involved enzymes were fine-tuned to optimize the bleaching process.


Peroxidase Basidiomycete Bleaching Annatto β-Carotene 



This work was supported by the excellence initiative of the Hessian Ministry of Science and Art which encompasses a generous grant for the LOEWE research focus ‘Insect Biotechnology.’

Conflict of interest


Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.


  1. 1.
    National Agricultural Statistics Service (2011) Dairy products 2010 summary April 2011. Accessed 6 February 2013
  2. 2.
    Barnicoat CR (1937) 151. The reactions and properties of annatto as a cheese colour, with particular reference to the chemistry of cheese discoloration. J Dairy Res 8:61–73CrossRefGoogle Scholar
  3. 3.
    Kosikowski FV (1979) Whey utilization and whey products. J Dairy Sci 62:1149–1160CrossRefGoogle Scholar
  4. 4.
    Childs JL, Yates MD, Drake MA (2007) Sensory properties of meal replacement bars and beverages made from whey and soy proteins. J Food Sci 72:S425–S434CrossRefGoogle Scholar
  5. 5.
    Alfaifi MS, Stathopoulos CE (2009) Effect of egg yolk substitution by sweet whey protein concentrate on some Gelato ice cream physical properties during storage. J Food Nutr Res 48:183–188Google Scholar
  6. 6.
    Foegeding EA, Davis JP, Doucet D, McGuffey MK (2002) Advances in modifying and understanding whey protein functionality. Trends Food Sci Technol 13:151–159CrossRefGoogle Scholar
  7. 7.
    US Food and Drug Administration (2012) Code of federal regulations title 21. FDA, Silver Spring. Accessed 5 Feb 2013
  8. 8.
    McDonough FE, Hargrove RE, Tittsler RP (1968) Decolorization of annatto in Cheddar cheese whey. J Dairy Sci 51:471–472CrossRefGoogle Scholar
  9. 9.
    Carrie MS (1938) 179. Annatto as a cheese colour. J Dairy Res 9:72–79CrossRefGoogle Scholar
  10. 10.
    Kang E, Campbell R, Bastian E, Drake M (2010) Invited review: annatto usage and bleaching in dairy foods. J Dairy Sci 93:3891–3901CrossRefGoogle Scholar
  11. 11.
    Croissant AE, Kang EJ, Campbell RE, Bastian E, Drake MA (2009) The effect of bleaching agent on the flavor of liquid whey and whey protein concentrate. J Dairy Sci 92:5917–5927CrossRefGoogle Scholar
  12. 12.
    Listiyani MAD, Campbell RE, Miracle RE, Barbano DM, Gerard PD, Drake MA (2012) Effect of temperature and bleaching agent on bleaching of liquid Cheddar whey. J Dairy Sci 95:36–49CrossRefGoogle Scholar
  13. 13.
    Damodaran S (2011) Straightforward process for removal of milk fat globule membranes and production of fat-free whey protein concentrate from cheese whey. J Agric Food Chem 59:10271–10276CrossRefGoogle Scholar
  14. 14.
    Zhu D, Damodaran S (2012) Short communication: annatto in Cheddar cheese-derived whey protein concentrate is primarily associated with milk fat globule membrane. J Dairy Sci 95:614–617CrossRefGoogle Scholar
  15. 15.
    Zorn H, Langhoff S, Scheibner M, Nimtz M, Berger RG (2003) A peroxidase from Lipista irina cleaves β, β-carotene to flavor compounds. Biol Chem 384:1049–1056CrossRefGoogle Scholar
  16. 16.
    Pühse M, Szweda RT, Ma Y, Jeworrek C, Winter R, Zorn H (2009) Marasmius scorodonius extracellular dimeric peroxidase—Exploring its temperature and pressure stability. Biochim Biophys Acta 1794:1091–1098CrossRefGoogle Scholar
  17. 17.
    Scheibner M, Hülsdau B, Zelena K, Nimtz M, de Boer L, Berger RG, Zorn H (2008) Novel peroxidases of Marasmius scorodonius degrade β-carotene. Appl Microbiol Biotechnol 77:1241–1250CrossRefGoogle Scholar
  18. 18.
    Faraco V, Piscitelli A, Sannia G, Giardina P (2007) Identification of a new member of the dye-decolorizing peroxidase family from Pleurotus ostreatus. World J Microbiol Biotechnol 23:889–893CrossRefGoogle Scholar
  19. 19.
    Commission internationale de L’Eclairage (1986) Colorimetry. Central Bureau of the CIE, ViennaGoogle Scholar
  20. 20.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  21. 21.
    Theorell H, Maehly AC (1950) Untersuchungen an künstlichen peroxydasen. Acta Chem Scand 4:422–434CrossRefGoogle Scholar
  22. 22.
    Eggert C, Temp U, Eriksson KEL (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158Google Scholar
  23. 23.
    US Food and Drug Administration (2012) Code of federal regulations title 21. FDA, Silver Spring. Accessed 5 Feb 2013
  24. 24.
    US Food and Drug Administration (2012) Code of federal regulations title 21. FDA, Silver Spring. Accessed 5 Feb 2013
  25. 25.
    Listiyani MAD, Campbell RE, Miracle RE, Dean LO, Drake MA (2011) Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey proteins. J Dairy Sci 94:4347–4359CrossRefGoogle Scholar
  26. 26.
    Law AJR, Leaver J (2000) Effect of pH on the thermal denaturation of whey proteins in milk. J Agric Food Chem 48:672–679CrossRefGoogle Scholar
  27. 27.
    Bernal V, Jelen P (1985) Thermal stability of whey proteins—a calorimetric study. J Dairy Sci 68:2847–2852CrossRefGoogle Scholar
  28. 28.
    Cooney CM, Morr CV (1972) Hydrogen peroxide alteration of whey proteins in whey and concentrated whey systems. J Dairy Sci 55:567–573CrossRefGoogle Scholar
  29. 29.
    Zelena K, Hardebusch B, Hülsdau B, Berger RG, Zorn H (2009) Generation of norisoprenoid flavors from carotenoids by fungal peroxidases. J Agric Food Chem 57:9951–9955CrossRefGoogle Scholar
  30. 30.
    Tirimanna ASL (1981) Study of the carotenoid pigments of Bixa orellana L. seeds by thin layer chromatography. Microchim Acta 2:11–16Google Scholar
  31. 31.
    Kussendrager KD, van Hooijdonk ACM (2000) Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications. Br J Nutr 84:19–25CrossRefGoogle Scholar
  32. 32.
    Otte J, Qvist KB (1998) Crosslinking of whey proteins by enzymatic oxidation. J Agric Food Chem 46:1326–1333CrossRefGoogle Scholar
  33. 33.
    Arnao MB, Acosta M, Delrio JA, Varon R, Garciacanovas F (1990) A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide. Biochim Biophys Acta 1041:43–47CrossRefGoogle Scholar
  34. 34.
    Baynton K, Bewtra J, Biswas N, Taylor K (1994) Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: a kinetic investigation. Biochim Biophys Acta 1206:272–278CrossRefGoogle Scholar
  35. 35.
    Khosraneh M, Mahmoudi A, Rahimi H, Nazari K, Moosavi-Movahedi AA (2007) Suicide-peroxide inactivation of microperoxidase-11: a kinetic study. J Enzyme Inhib Med Chem 22:677–684CrossRefGoogle Scholar
  36. 36.
    Gil-Rodriguez P, Ferreira-Batista C, Vazquez-Duhalt R, Valderrama B (2008) A novel heme peroxidase from Raphanus sativus intrinsically resistant to hydrogen peroxide. Eng Life Sci 8:286–296CrossRefGoogle Scholar
  37. 37.
    Ogola HJ, Hashimoto N, Miyabe S, Ashida H, Ishikawa T, Shibata H, Sawa Y (2010) Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues. Appl Microbiol Biotechnol 87:1727–1736CrossRefGoogle Scholar
  38. 38.
    Ogola HJ, Kamiike T, Hashimoto N, Ashida H, Ishikawa T, Shibata H, Sawa Y (2009) Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120. Appl Environ Microbiol 75:7509–7518CrossRefGoogle Scholar
  39. 39.
    Keilin D, Hartree EF (1948) Properties of glucose oxidase (notatin). Biochem J 42:221–229Google Scholar
  40. 40.
    Liers C, Bobeth C, Pecyna M, Ullrich R, Hofrichter M (2010) DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes. Appl Microbiol Biotechnol 85:1869–1879CrossRefGoogle Scholar
  41. 41.
    Çankaya M, Şişecioğlu M, Barış Ö, Güllüce M, Özdemir H (2010) Effects of bovine milk lactoperoxidase system on some bacteria. Appl Biochem Microbiol 46:57–60CrossRefGoogle Scholar
  42. 42.
    Zhou Y, Lim L (2009) Activation of lactoperoxidase system in milk by glucose oxidase immobilized in electrospun polylactide microfibers. J Food Sci 74:C170–C176CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Renata T. Szweda
    • 1
  • Katharina Schmidt
    • 1
  • Holger Zorn
    • 1
  1. 1.Institute of Food Chemistry and Food BiotechnologyJustus Liebig University GiessenGiessenGermany

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