Food and Bioprocess Technology

, Volume 6, Issue 12, pp 3456–3461 | Cite as

Aqueous Two-Phase Micellar System for Nisin Extraction in the Presence of Electrolytes

  • Angela Faustino Jozala
  • André Moreni LopesEmail author
  • Letícia Celia de Lencastre Novaes
  • Priscila Gava Mazzola
  • Thereza Christina Vessoni Penna
  • Adalberto Pessoa Júnior
Original Paper


Liquid–liquid extraction for aqueous two-phase micellar systems (ATPMS) is a promising technique that can either replace or be used as a complementary process to more typical chromatographic operations in order to reduce the costs of downstream processing of many biological products. This method offers attractive conditions when exploited in the extraction/purification of a target protein directly on the culture medium. Nisin, an extracellular antimicrobial peptide, is produced by Lactococcus lactis and is effective at controlling a wide range of Gram-positive bacteria, including multidrug-resistant pathogens. This study evaluates ATPMS composed by a nonionic surfactant, Triton X-114, in the presence or absence of electrolytes, to improve the extraction of nisin. The partitioning behavior of nisin showed that it can be directly extracted from the fermentation media. The partitioning coefficient (K nis) of the commercial nisin in the presence of electrolytes showed K nis values of 5.6 and 5.4 for MgSO4 and (NH4)2SO4, respectively. Similar behavior was observed for the produced nisin where K nis values were 4.1 and 5.1 for MgSO4 and (NH4)2SO4. After partition, the commercial nisin activity in the micelle-rich phase, in the absence of electrolytes, was 3.3 logAU/mL, and in the presence of MgSO4 and (NH4)2SO4, the activity was 5.0 logAU/mL. The produced nisin activity with and without MgSO4 was around 3.5 logAU/mL; however, in the presence of (NH4)2SO4, nisin activity was 4.5 logAU/mL. The increase in nisin activity after partitioning with salts encourages further researches for the optimization of nisin extraction.


Liquid–liquid extraction Electrolytes Lactococcus lactis Peptide purification Nisin Triton X-114 



This research was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil), and Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil).


  1. Acuna, L., Dionisio-Morero, R., & Bellomio, A. (2011). Development of wide-spectrum hybrid bacteriocins for food biopreservation. Food and Bioprocess Technology, 4(6), 1029–1049.CrossRefGoogle Scholar
  2. Anacker, E. W., & Ghose, H. M. (1963). Counterions and micelle size. 1. Light scattering by solutions of dodecyltrimethylammonium salts. The Journal of Physical Chemistry, 67(8), 1713–1716.CrossRefGoogle Scholar
  3. Arauz, L. J., Jozala, A. F., Baruque-Ramos, J., Mazzola, P. G., Pessoa-Jr, A., & Penna, T. C. V. (2011). Culture medium of diluted skimmed milk for the production of nisin in batch cultivations. Annals of Microbiology, 62(1), 419–426.CrossRefGoogle Scholar
  4. Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science and Technology, 20(3–4), 146–154.CrossRefGoogle Scholar
  5. Balzer, D., & Lüders, H. (Eds.). (2000). Nonionic surfactants: alkyl polyglucosides, New York: Marcel Dekker.Google Scholar
  6. Brochsztain, S., Berci, P., Toscano, V. G., Chaimovich, H., & Politi, M. J. (1990). Ion binding and selectivity in zwitterionic micelles. Journal of Physical Chemistry, 94(17), 6781–6785.CrossRefGoogle Scholar
  7. Carale, M. T. R. (1993). Salt effects on micellization, micellar growth, and phase behavior of aqueous solutions of nonionic surfactants. Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, USA.Google Scholar
  8. Cheigh, C. I., Kook, M. C., Kim, S. B., Hong, Y. H., & Pyun, Y. R. (2004). Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. Lactis A164 using expanded bed ion exchange chromatography. Biotechnology Letters, 26(17), 1341–1345.CrossRefGoogle Scholar
  9. Chol, H. J., Cheigh, C. I., Kim, S. B., & Pyun, Y. R. (2000). Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi. Journal of Applied Microbiology, 88(4), 563–571.CrossRefGoogle Scholar
  10. Delves-Broughton, J., Blackburn, P., Evans, R. J., & Hugenholtz, J. (1996). Applications of the bacteriocin, nisin. Antonie Van Leeuwenhoek, 69(2), 193–202.CrossRefGoogle Scholar
  11. Dreyer, S.E. (2008) Aqueous two-phase extraction of proteins and enzymes using tetraalkylammonium-based ionic liquids. Ph.D. thesis, Mathematisch-Naturwissenschaftlichen Fakultät, Universität Rostock, Rostock, GER.Google Scholar
  12. Gagnon, P. (2012). Technology trends in antibody purification. Journal of Chromatography A, 1221, 57–70.CrossRefGoogle Scholar
  13. Garcia-Parra, M. D., Garcia-Ahmendarez, B. E., Guevara-Olvera, L., Guevara-Gonzalez, R. G., Rodriguez, A., Martinez, B., Dominguez-Dominguez, J., & Regalado, C. (2011). Effect of sub-inhibitory amounts of nisin and mineral salts on nisin production by Lactococcus lactis UQ2 in skim milk. Food and Bioprocess Technology, 4(4), 646–654.CrossRefGoogle Scholar
  14. Gujarathi, S. S., Bankar, S. B., & Ananthanarayan, L. A. (2008). Fermentative production, purification and characterization of nisin. International Journal of Food Engineering, 5(4).Google Scholar
  15. Hao, L. S., Hu, P., & Nan, Y. Q. (2010). Salt effect on the rheological properties of the aqueous mixed cationic and anionic surfactant systems. Colloids and Surfaces A: Physicochemical and Engineering Aspects—Colloid Surface A, 36(1), 187–195.CrossRefGoogle Scholar
  16. Hebbar, U. H., Sumana, B., Hemavathi, A. B., & Raghavarao, K. S. M. S. (2012). Separation and purification of bromelain by reverse micellar extraction coupled ultrafiltration and comparative studies with other methods. Food and Bioprocess Technology, 5(3), 1010–1018.CrossRefGoogle Scholar
  17. Imran, M., Revol-Junelles, A. M., René, N., Jamshidian, M., Akhtar, M. J., Arab-Tehrany, E., Jacquot, M., & Desobry, S. (2012). Microstructure and physico- chemical evaluation of nano-emulsion-based antimicrobial peptides embedded in bioactive packaging films. Food Hydrocolloids, 29(2), 407–419.CrossRefGoogle Scholar
  18. Jozala, A. F., Lopes, A. M., Mazzola, P. G., Magalhães, P. O., Penna, T. C. V., & Pesso-Jr, A. (2008). Liquid–liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems. Enzyme and Microbial Technology, 42(2), 107–112.CrossRefGoogle Scholar
  19. Jozala, A. F., Silva, D. P., Vicente, A. A., Teixeira, J. A., Pessoa-Jr, A., & Penna, T. C. V. (2011). Processing of byproducts to improve nisin production by Lactococcus lactis. African Journal of Biotechnology, 10(66), 14920–14925.CrossRefGoogle Scholar
  20. Lam, H., Kavoosi, M., Haynes, C. A., Wang, D. I. C., & Blankschtein, D. (2004). Affinity-enhanced protein partitioning in decyl beta-d-glucopyranoside two-phase aqueous micellar systems. Biotechnology and Bioengineering, 89(4), 381–392.CrossRefGoogle Scholar
  21. Liu, C. L., Kamei, D. T., King, J. A., Wang, D. I. C., & Blankschtein, D. (1998). Separation of proteins and viruses using two-aqueous micellar systems. Journal of Chromatography B: Biomedical Sciences and Applications, 711(1–2), 127–138.CrossRefGoogle Scholar
  22. Liu, C. L., Nikas, Y. J., & Blankschtein, D. (1996). Novel bioseparations using two-phase aqueous micellar systems. Biotechnology and Bioengineering, 52(2), 185–192.CrossRefGoogle Scholar
  23. Lopes, A. M., Magalhães, P. O., Mazzola, P. G., Rangel-Yagui, C. O., Carvalho, J. C. M., Penna, T. C. V., & Pessoa-Júnior, A. (2011). Green fluorescent protein extraction and LPS removal from E. coli fermentation medium using aqueous two-phase micellar system. Separation and Purification Technology, 81, 339–346.CrossRefGoogle Scholar
  24. Lopes, A. M., Magalhães, P. O., Mazzola, P. G., Rangel-Yagui, C. O., Carvalho, J. C. M., Penna, T. C. V., & Pessoa-Júnior, A. (2010). LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system. Biotechnology Progress, 26(6), 1644–1653.CrossRefGoogle Scholar
  25. Lopes, A. M., Rangel-Yagui, C. O., & Pessoa-Jr, A. (2008). Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems? Química Nova, 31, 998–1003.CrossRefGoogle Scholar
  26. Mageste, A. B., Lemos, L. R., Ferreira, G. M. D., Silva, M. C. H., Silva, L. H. M., Bonomo, L. H. M., & Minim, L. A. (2009). Aqueous two-phase systems: an efficient, environmentally safe and economically viable method for purification of natural dye carmine. Journal of Chromatography A, 1216(45), 7623–7629.CrossRefGoogle Scholar
  27. Mazzola, P. G., Lam, H., Kavoosi, M., Haynes, C. A., Pessoa-Júnior, A., Vessoni-Penna, T. C., & Blankschtein, D. (2006). Affinity-tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two-phase aqueous micellar system. Biotechnology and Bioengineering, 93(5), 998–1004.CrossRefGoogle Scholar
  28. Mazzola, P. G., Lopes, A. M., Hasmann, F. A., Jozala, A. F., Penna, T. C. V., Magalhaes, P. O., Rangel-Yagui, C. O., & Pessoa-Júnior, A. (2008). Liquid–liquid extraction of biomolecules: an overview and update of the main techniques. Journal of Chemical Technology and Biotechnology, 83(2), 143–157.CrossRefGoogle Scholar
  29. Norhana, M. N. W., Poole, S. E., Deeth, H. C., & Dykes, G. A. (2012). Effects of nisin, EDTA and salts of organic acids on Listeria monocytogenes, Salmonella and native microflora on fresh vacuum packaged shrimps stored at 4 °C. Food Microbiology, 31(1), 43–50.CrossRefGoogle Scholar
  30. O’Sullivan, L., Ross, R. P., & Hill, C. (2002). Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality. Biochimie, 84(5–6), 593–604.CrossRefGoogle Scholar
  31. Parada, J. L., Caron, C. R., Medeiros, A. B. P., & Soccol, C. R. (2007). Purification, properties and use as biopreservatives. Brazilian Archives of Biology and Technology, 50(3), 521–542.CrossRefGoogle Scholar
  32. Quina, F. H., & Chaimovich, H. (1979). Ion-exchange in micellar solutions. 1. Conceptual-framework for ion-exchange in micellar solutions. Journal of Physical Chemistry, 83(14), 1844–1850.CrossRefGoogle Scholar
  33. Raghavarao, K. S. M. S., Rastogi, N. K., Gowthaman, M. K., & Karanth, N. G. (1995). Aqueous two-phase extraction for downstream processing of enzymes/proteins. Advances in Applied Microbiology, 41, 97–171.CrossRefGoogle Scholar
  34. Sanjurjo, K., Flores, S., Gerschenson, L., & Jagus, R. (2006). Study of the performance of nisin supported in edible films. Food Research International, 39(6), 749–754.CrossRefGoogle Scholar
  35. Santos, V. S., Hasmann, F. A., Converti, A., & Pessoa-Jr, A. (2011). Liquid–liquid extraction by mixed micellar systems: a new approach for clavulanic acid recovery from fermented broth. Biochemical Engineering Journal, 56(1–2), 75–83.CrossRefGoogle Scholar
  36. Sarubbo, L. A., Oliveira, L. A., Porto, A. L. F., Duarte, H. S., Carneiro-Leão, A. M. A., Lima-Filho, J. L., Campos-Takaki, G. M., & Tambourgi, E. B. (2000). New aqueous two-phase system based on cashew-nut tree gum and poly(ethylene glycol). Journal of Chromatography B: Biomedical Sciences and Applications, 743(1–2), 79–84.CrossRefGoogle Scholar
  37. Tonova, K., & Lazarova, Z. (2005). Influence of enzyme aqueous source on RME-based purification of α-amylase. Separation and Purification Technology, 47(1–2), 43–51.CrossRefGoogle Scholar
  38. Ulloa, G., Coutens, C., Sánchez, M., Sineiro, J., Rodríguez, A., Deive, F. J., & Núñez, M. J. (2012). Sodium salt effect on aqueous solutions containing Tween 20 and Triton X-102G. The Journal of Chemical Thermodynamics, 47, 62–67.CrossRefGoogle Scholar
  39. Wang, T., Qin, Y., He, H., Lv, J., & Fan, Y. (2011). An extraction technique for analytical sample preparation in aqueous solution based on controlling dispersion of ionic surfactant assemblies in isotachophoretic migration. Journal of Chromatography A, 1218(1), 185–189.CrossRefGoogle Scholar
  40. Wan Norhana, M. N., Poole, S. E., Deeth, H. C., & Dykes, G. A. (2012). Effects of nisin, EDTA and salts of organic acids on Listeria monocytogenes, Salmonella and native microflora on fresh vacuum packaged shrimps stored at 4oC. Food Microbiology, 31, 43–50.CrossRefGoogle Scholar
  41. Yanjie, Z., & Paul, S. C. (2006). Interactions between macromolecules and ions: the Hofmeister series. Current Opinion in Chemical Biology, 10, 658–663.CrossRefGoogle Scholar
  42. Zhang, F., Wu, Z., Wu, Z., & Wang, H. (2011). Effect of ionic strength on the foam separation of nisin from the fermentation broth. Separation and Purification Technology, 78(1), 42–48.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Angela Faustino Jozala
    • 1
  • André Moreni Lopes
    • 1
    • 3
    Email author
  • Letícia Celia de Lencastre Novaes
    • 1
  • Priscila Gava Mazzola
    • 2
  • Thereza Christina Vessoni Penna
    • 1
  • Adalberto Pessoa Júnior
    • 1
  1. 1.Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil
  2. 2.Department of Clinical Pathology, Faculty of Medical SciencesUniversity of CampinasCampinasBrazil
  3. 3.Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas—FCFUSPSão PauloBrazil

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