Purification of lactoperoxidase from bovine milk by integrating the technique of salting-out extraction with cation exchange chromatographic separation

  • Tianqi Li
  • Ling Ma
  • Dongxue Sun
  • Lili Liu
  • Abdul Qayum
  • Zhanmei JiangEmail author
  • Juncai HouEmail author
Original Paper


In the current experiment, the integrated techniques of salting-out extraction followed by cation exchange chromatographic separation were used to purify lactoperoxidase (LP) from milk whey. In order to optimize the purification condition of LP, the different conditions of salting out extraction as well as chromatographic separation were tried and the efficiency of these conditions were evaluated in terms of activity recovery and purification fold. LP extracted with ammonium sulphate for 8 h at pH 6.5, followed by centrifugation at 7000 rpm was revealed as optimum salting condition due to maximum value activity recovery (85.74%) and purification fold (3.13%). LP from this optimum salting out extraction process were furthermore separated in 100 GigaCap S-650M strong cation chromatographic column (20 cm × 1.6 cm, 50–100 µm) by using phosphate buffer (0.03M, pH 7.2) with flow rate of 2 mL/min which was shown highest activity recovery (82.62%) and purification fold (126.65 times). It was also verified that there was a single band presence and the molecular weight of LP was approximately 85 kDa by SDS-PAGE analysis. Therefore, it is suggested that combination of salting-out and GigaCap S-650M strong cation exchange chromatography could be an efficient and practical method to purify bovine LP.


Lactoperoxidase Salting-out Cation exchange chromatography 



This study was supported by project for the Research and Development of Harbin Application Technology (No. 2016RAQXJ046), The National Key Research and Development Program of China (No. 2016YFD0400605) and The National “Twelfth Five-Year” Plan for Science and Technology Support Program of China (No. 2013BAD18B06).


  1. 1.
    K.D. Kussendrager, A.C.M.V. Hooijdonk, Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications. Br. J. Nutr. 84, 19–25 (2000)CrossRefGoogle Scholar
  2. 2.
    F.A. Fonteh, A.S. Grandison, M.J. Lewis, Variations of lactoperoxidase activity and thiocyanate content in cows’ and goats’ milk throughout lactation. J. Dairy Res. 69, 401 (2002)CrossRefGoogle Scholar
  3. 3.
    M.M. Cals, P. Mailliart, G. Brignon, P. Anglade, B.R. Dumas, Primary structure of bovine lactoperoxidase, a fourth member of a mammalian heme peroxidase family. Eur. J. Biochem. 198, 733–739 (1991)CrossRefGoogle Scholar
  4. 4.
    De Wit JN, Van Hooydonk ACM Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Neth. Milk Dairy J. 50, 227–244 (1996)Google Scholar
  5. 5.
    E. Seifu, E.M. Buys, E.F. Donkin, Significance of the lactoperoxidase system in the dairy industry and its potential applications: a review. Trends Food Sci. Technol. 16, 137–154 (2005)CrossRefGoogle Scholar
  6. 6.
    R. Uceda, A.M. Guillen, P. Gaya, M. Medina, M. Nunez, Chemical and rheological characteristics of Manchego cheese manufactured from ewe raw milk preserved by activation of the lactoperoxidase system. Milchwiss. Milk Sci. Int. 49, 494–499 (1994)Google Scholar
  7. 7.
    M. Nakada, S. Dosako, R. Hirano, M. Oooka, I. Nakajima, Lactoperoxidase suppresses acid production in yoghurt during storage under refrigeration. Int. Dairy J. 6, 33–42 (1996)CrossRefGoogle Scholar
  8. 8.
    R. Montiel, I. Martín-Cabrejas, M. Medina, Reuterin, lactoperoxidase, lactoferrin and high hydrostatic pressure onthe inactivation of food-borne pathogens in cooked ham. Food Control. 51, 122–128 (2015)CrossRefGoogle Scholar
  9. 9.
    M. Yousefi, M. Farshidi, A. Ehsani, Effects of lactoperoxidase system-alginate coating on chemical, microbial, and sensory properties of chicken breast fillets during cold storage. J. Food Saf. 1, 24–49 (2018)Google Scholar
  10. 10.
    N. Urtasun, M.F. Baieli, D.B. Hirsch, M.C. Martínez-Ceron, O. Cascone, F.J. Wolman, Lactoperoxidase purification from whey by using dye affinity chromatography. Food Bioprod. Process. 103, 58–65 (2017)CrossRefGoogle Scholar
  11. 11.
    A.C.M.V. Hooijdonk, K.D. Kussendrager, J.M. Steijns, In vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence. Br. J. Nutr. 84, 127–134 (2000)Google Scholar
  12. 12.
    A. Atasever, H. Ozdemir, I. Gulcin, O. Irfan, Kufrevioglu, One-step purification of lactoperoxidase from bovine milk by affinity chromatography. Food Chem. 136,864 – 70(2013)Google Scholar
  13. 13.
    K.E. Nandini, N.K. Rastogi, Reverse micellar extraction for downstream processing of lipase: effect of various parameters on extraction. Process Biochem. 44, 1172–1178 (2009)CrossRefGoogle Scholar
  14. 14.
    D. Barba, F. Beolchini, F. Veglió, Minimizing water use in diafiltration of whey protein concentrates. Sep. Sci. Technol. 35, 951–965 (2000)CrossRefGoogle Scholar
  15. 15.
    S. Yoshida, Isolation of lactoperoxidase and lactoferrins from bovine milk acid whey by carboxymethyl cation exchange chromatography. J. Dairy Sci. 74, 1439–1444 (1991)CrossRefGoogle Scholar
  16. 16.
    K.E. Nandini, N.K. Rastogi, Integrated downstream processing of lactoperoxidase from milk whey involving aqueous two-phase extraction and ultrasound-assisted ultrafiltration. Appl. Biochem. Biotechnol. 163, 173–185 (2011)CrossRefGoogle Scholar
  17. 17.
    F. Beolchini, Whey protein concentrate production in a pilot scale two-stage diafiltration process. Sep. Sci. Technol. 36, 587–603 (2001)CrossRefGoogle Scholar
  18. 18.
    Y. Liang, X. Wang, M. Wu, W. Zhu, Simultaneous isolation of lactoferrin and lactoperoxidase from bovine colostrum by SPEC 70 SLS cation exchange resin. Int. J. Environ. Res. Public Health. 8, 3764 (2011)CrossRefGoogle Scholar
  19. 19.
    A. Atasever, H. Ozdemir, I. Gulcin, OI Kufrevioglu, One-step purification of lactoperoxidase from bovine milk by affinity;chromatography. Food Chem. 136, 864–870 (2013)Google Scholar
  20. 20.
    Y. Morita, A. Ono, A. Serizawa, K. Yogo, N. Ishida-Kitagawa, T. Takeya, T. Ogawa, Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis. J. Dairy Sci. 94, 2270 (2011)CrossRefGoogle Scholar
  21. 21.
    L. Voswinkel, U. Kulozik, Fractionation of all major and minor whey proteins with radial flow membrane adsorption chromatography at lab and pilot scale. Int. Dairy J. 39, 209–214 (2014)CrossRefGoogle Scholar
  22. 22.
    E.I. Elagamy, R. Ruppanner, A. Ismail, C.P. Champagne, R. Assaf, Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel’s milk. Int. Dairy J. 6, 129–145 (1996)CrossRefGoogle Scholar
  23. 23.
    C.K. Chiu, M.R. Etzel, Fractionation of lactoperoxidase and lactoferrin from bovine whey using a cation exchange membrane. J. Food Sci. 62, 996–1000 (1997)CrossRefGoogle Scholar
  24. 24.
    B. Langbakk, T. Flatmark, Demonstration and partial purification of lactoperoxidase from human colostrum. Febs Lett. 174, 300–303 (1984)CrossRefGoogle Scholar
  25. 25.
    W. Leicht, S. Pundak, Large-scale purification of halophilic enzymes by salting-out mediated chromatography. Anal. Biochem. 114, 186–192 (1981)CrossRefGoogle Scholar
  26. 26.
    X. Xin, R.R. Ambati, Z. Cai, B. Lei, Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. Biotech. 8, 90 (2018)Google Scholar
  27. 27.
    E.S. Girgis, A.A. Ismail, S.M. El-Dieb, W.M. Zaky, Isolation, purification and characterisation of buffaloes and cows milk Lactoperoxidase. Egypt. J. Food Sci. 49, 158–162 (2002)Google Scholar
  28. 28.
    K.C. Duongly, S.B. Gabelli, Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol. 541, 85–94 (2014)CrossRefGoogle Scholar
  29. 29.
    X. Ye, S. Yoshida, T.B. Ng Isolation of lactoperoxidase, lactoferrin, a-lactalbumin, b-lactoglobulin B and b-lactoglobulin A from bovine rennet whey using ion exchange chromatography. Int. J. Biochem. Cell Biol. 32,1143–1150(2000).CrossRefGoogle Scholar
  30. 30.
    S. Ainsworth, Steady-state enzyme kinetics. Palgrave, Place (1977), pp. 29–42Google Scholar
  31. 31.
    U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685 (1970)CrossRefGoogle Scholar
  32. 32.
    P.R. Foster, P. Dunnill, M.D. Lilly, The kinetics of protein salting-out: precipitation of yeast enzymes by ammonium sulfate. Biotechnol. Bioeng. 18, 1496–1496 (2010)CrossRefGoogle Scholar
  33. 33.
    H. Fu, X. Wang, Y. Sun, L. Yan, J. Shen, J. Wang, S.T. Yang, Z. Xiu, Effects of salting-out and salting-out extraction on the separation of butyric acid. Sep. Purif. Technol. 180, 44–50 (2017)CrossRefGoogle Scholar
  34. 34.
    M.O. Ji-Xian, X.J. Gao, Study on the technological conditions of extracting IGF-1 from bovine colostrum by salting-out. Sci. Technol. Food Ind. 32, 309–312 (2011)Google Scholar
  35. 35.
    C.R. Lowe, Protein purification: principles and practice. Cell Biochem. Funct. 2, 63–63 (2010)CrossRefGoogle Scholar
  36. 36.
    L. Bian, X. Ji, W. Hu, Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out. Biomed. Chromatogr. 28, 957 (2014)CrossRefGoogle Scholar
  37. 37.
    M.S. Izydorczyk, C.G. Biliaderis, Gradient ammonium sulphate fractionation of galactomannans. Food Hydrocoll. 10, 295–300 (1996)CrossRefGoogle Scholar
  38. 38.
    B. Purschke, Centrifugal fractionation of mealworm larvae (Tenebrio molitor, L.) for protein recovery and concentration. 89,224–228(2018)Google Scholar
  39. 39.
    K.C. Duong-Ly, S.B. Gabelli, Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol. 541, 85 (2014)CrossRefGoogle Scholar
  40. 40.
    J.H. Zhou, C.S. Zhou, A study of pectin salting-out from shaddock peel. J. Central South Univ. For. Technol. 29, 95–99 (2009)Google Scholar
  41. 41.
    S.Q. Li, Pectin extraction from potato residues by different methods of salting out and process optimization. Adv. Mater. Res. 926–930, 174–177 (2014)Google Scholar
  42. 42.
    X.D. Sun, S.D. Arntfield, Dynamic oscillatory rheological measurement and thermal properties of pea protein extracted by salt method: effect of pH and NaCl. J. Food Eng. 105, 577–582 (2011)CrossRefGoogle Scholar
  43. 43.
    M. Amid, M. Shuhaimi, M.Z.I. Sarker, M.Y.A. Manapa, Purification of serine protease from mango (Mangifera Indica Cv. Chokanan) peel using an alcohol/salt aqueous two phase system. Food Chem. 132, 1382–1386 (2012)CrossRefGoogle Scholar
  44. 44.
    T. Ozawa, Separation of the components in black tea infusion by chromatography on Toyopearl®. Agric. Biol. Chem.. 46, 1079–1081 (2006)Google Scholar
  45. 45.
    C. Mecitoğlu,A. Yemenicioğlu, Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films. Food Chem. 104, 726–733 (2007)CrossRefGoogle Scholar
  46. 46.
    J. Pilbrow, B. Ael-D, A. Carne, Fractionation of sheep cheese whey by a scalable method to sequentially isolate bioactive proteins. Food Chem. 203, 165–174 (2016)CrossRefGoogle Scholar
  47. 47.
    L.U. Rong-Rong, X.U. Shi-Ying, Z. Wang, Isolation and purification of lactoperoxidase and its enzymatic properties. Food Sci. 27, 100–104 (2006)Google Scholar
  48. 48.
    T. Nagai, W. Worawattanamateekul, N. Suzuki, T. Nakamura, T. Ito, K. Fujiki, M. Nakao, T. Yano, Isolation and characterization of collagen from rhizostomous jellyfish (Rhopilema asamushi). Food Chem. 70, 205–208 (2000)CrossRefGoogle Scholar
  49. 49.
    R. Hahn, P.M. Schulz, C. Schaupp, A. Jungbauer, Bovine whey fractionation based on cation-exchange chromatography. J. Chromatogr A. 795,277–287(1998)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Dairy ScienceNortheast Agricultural University, Ministry of EducationHarbinPeople’s Republic of China

Personalised recommendations