Food and Bioprocess Technology

, Volume 5, Issue 3, pp 1010–1018 | Cite as

Separation and Purification of Bromelain by Reverse Micellar Extraction Coupled Ultrafiltration and Comparative Studies with Other Methods

  • Umesh H. Hebbar
  • B. Sumana
  • A. B. Hemavathi
  • K. S. M. S. RaghavaraoEmail author
Original Paper


Reverse micellar extraction (RME) is a promising liquid-liquid extraction technique for downstream processing of biomolecules from dilute solutions. An integrated approach of coupling RME with ultrafiltration is attempted to improve the overall efficiency of extraction and purification of bromelain from aqueous extract of pineapple core. The performance of RME is compared with aqueous two-phase extraction (ATPE), another potential liquid-liquid extraction technique and conventional ammonium sulphate precipitation technique. The reverse micellar system of cationic surfactant cetyltrimethylammoniumbromide/isooctane/hexanol/butanol used for RME resulted in an activity recovery of 95.8% and purification of 5.9-fold. The purification of bromelain increased to 8.9-fold after ultrafiltration. Alteration of aqueous phase pH during RME facilitated the differential partitioning of bromelain and polyphenoloxidase. Comparison of RME results with ATPE (activity recovery of 93.1% and purification of 3.2-fold) and the conventional ammonium sulphate precipitation (activity recovery of 82.1% and purification of 2.5-fold) indicated the improved performance of RME.


Activity recovery ATPE Bromelain Partition coefficient Reverse micelles Ultrafiltration 



The authors thank Dr. V Prakash, Director, CFTRI, for the encouragement and keen interest in the area of downstream processing. Authors wish to thank Department of Biotechnology, New Delhi for funding (No.BT/PR-6418/PID/20/259/2005) the project.


  1. Andersson, E., Johansson, A., & Hagerdal, B. (1985). α-amylase production in aqueous two-phase system with Bacillus subtilis. Enzyme and Microbial Technology, 7(7), 333–338.CrossRefGoogle Scholar
  2. Asenjo, J. A., Mistry, S. L., Andrews, B. A., & Merchuk, J. C. (2002). Phase separation rates of aqueous two-phase systems: correlation with system properties. Biotechnology and Bioengineering, 79(2), 217–223.CrossRefGoogle Scholar
  3. Babu, B. R., Rastogi, N. K., & Raghavarao, K. S. M. S. (2008). Liquid-liquid extraction of bromelain and polyphenoloxidase using aqueous two-phase system. Chemical Engineering and Processing, 47(1), 83–89.CrossRefGoogle Scholar
  4. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.CrossRefGoogle Scholar
  5. Chen, Y. L., Su, C. K., & Chiang, B. H. (2006). Optimization of reversed micellar extraction of chitosanases produced by Bacillus cereus. Process Biochemistry, 41(4), 752–758.CrossRefGoogle Scholar
  6. Das, J. R., Bhat, S. G., & Gowda, L. R. (1997). Purification and characterization of polyphenol oxidase from the kew cultivar of Indian pineapple fruit. Journal of Agricultural and Food Chemistry, 45(6), 2031–2035.CrossRefGoogle Scholar
  7. Dekker, M., Riet, K. V., Bijsterbosch, B. H., Wolbert, R. B. G., & Hilhorst, R. (1989). Modeling and optimization of the reversed micellar extraction of α–amylase. AIChE Journal, 35(2), 321–324.CrossRefGoogle Scholar
  8. Devakate, R. V., Patil, V. V., Waje, S. S., & Thorat, B. N. (2009). Purification and drying of bromelain. Separation and Purification Technology, 64(3), 259–264.CrossRefGoogle Scholar
  9. Gaikaiwari, R., Shendye, A., & Kulkarni, M. N. (1996). Two phase separation of xylanase from alcoholic thermophilic bacillus using a poly (ethyleneglycol)-KH2PO4 System. Biotechnology and Applied Biochemistry, 23(3), 237–241.Google Scholar
  10. Grossman, P. D., & Gainer, J. L. (1988). Correlation of aqueous two-phase partitioning of proteins with change in free volume. Biotechnology Progress, 4(1), 6–11.CrossRefGoogle Scholar
  11. Gunduz, U., & Korkmaz, K. (2000). Bovine serum albumin partitioning in an aqueous two-phase system: effect of pH and sodium chloride concentration. Journal of Chromatography. B, 743, 225–258.CrossRefGoogle Scholar
  12. Harikrishna, S., Srinivas, N. D., Raghavarao, K. S. M. S., & Karanth, N. G. (2002). Reverse micellar extraction for downstream processing of proteins/enzymes. Advances in Biochemical Engineering/Biotechnology, 75, 119–183.CrossRefGoogle Scholar
  13. Hasmann, F. A., Cortez, D. V., Gurpilhares, D. B., Santos, V. C., Roberto, I. C., & Pessoa, A., Jr. (2007). Continuous counter-current purification of glucose-6-phosphate dehydrogenase using liquid-liquid extraction by reverse micelles. Biochemical Engineering Journal, 34(3), 236–241.CrossRefGoogle Scholar
  14. Hebbar, U. H., & Raghavarao, K. S. M. S. (2007). Extraction of bovine serum albumin using nanoparticulate reverse micelles. Process Biochemistry, 42(12), 1602–1608.CrossRefGoogle Scholar
  15. Hebbar, U. H., Sumana, B., & Raghavarao, K. S. M. S. (2008). Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes. Bioresource Technology, 99(11), 4896–4902.CrossRefGoogle Scholar
  16. Hemavathi, A. B., Hebbar, U. H., & Raghavarao, K. S. M. S. (2007). Reverse micellar extraction of bromelain from Ananas comosus L Merryl. Journal of Chemical Technology and Biotechnology, 82(11), 985–992.CrossRefGoogle Scholar
  17. Hemavathi, A. B., Hebbar, U. H., & Raghavarao, K. S. M. S. (2008). Reverse micellar extraction of β-Galactosidase from Barley (Hordeum vulgare). Applied Biochemistry and Biotechnology, 151(2–3), 522–531.CrossRefGoogle Scholar
  18. Ichikawa, S., Imai, M., & Shimizu, M. (1992). Solubilizing water involved in protein extraction using reversed micelles. Biotechnology and Bioengineering, 39(1), 20–26.CrossRefGoogle Scholar
  19. Lakshmi, M. C., Madhusudan, M. C., & Raghavarao, K. S. M. S. (2009). Extraction and purification of lipoxygenase from soybean using aqueous two phase system. Food and Bioprocess Technology. doi: 10.1007/s11947-009-0278-8.
  20. Lammeli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.CrossRefGoogle Scholar
  21. Lee, H. Y., & Dungan, S. R. (1998). Selective solubilization of α-lactalbumin and β-lactoglobulin into reversed micelles from their mixtures. Journal of Food Science, 63(4), 601–605.CrossRefGoogle Scholar
  22. Liu, J. G., Xing, J. M., Shen, R., Yang, C. L., & Liu, H. Z. (2004). Reverse micelles extraction of nattokinase from fermentation broth. Biochemical Engineering Journal, 21(3), 273–278.CrossRefGoogle Scholar
  23. Lopes, F. L. G., Junior, J. B. S., de Souza, R. R., Ehrhardt, D. D., Santana, J. C. C., & Tambourgi, E. B. (2009). Concentration by membrane separation processes of a medicinal product obtained from pineapple pulp. Brazilian Archives of Biology and Technology, 52(2), 457–464.CrossRefGoogle Scholar
  24. Murachi, T. (1976). Bromelain enzymes. In L. Lorand (Ed.), Methods in enzymology (Vol. 45, pp. 475–485). New York: Academic Press.Google Scholar
  25. Nagaraj, N., Srinivas, N. D., & Raghavarao, K. S. M. S. (2002). Acoustic filed assisted demixing of aqueous two-phase systems. Journal of Chromatography. A, 977(2), 163–172.CrossRefGoogle Scholar
  26. Nandini, K. E., & Rastogi, N. K. (2008). Liquid-liquid extraction of lipase using aqueous two-phase system. Food and Bioprocess Technology. doi: 10.1007/s11947-008-0160-0.
  27. Rabelo, A. P. B., Tambourgi, E. B., & Pessoa, A., Jr. (2004). Bromelain partitioning in two phase aqueous systems containing PEO-PPO-PEO block copolymers. Journal of Chromatography. B, 807(1), 61–68.CrossRefGoogle Scholar
  28. 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
  29. Rairkar, M. E., Hayes, D. G., & Harris, J. M. (2007). Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant. Biotechnological Letters, 29(5), 767–771.CrossRefGoogle Scholar
  30. Regalado, C., Asenjo, J. A., & Pyle, D. L. (1996). Studies on the purification of peroxidase from horseradish roots using reverse micelles. Enzyme and Microbial Technology, 18(5), 332–339.CrossRefGoogle Scholar
  31. Rowan, A. D., Buttle, D. J., & Barrett, A. J. (1990). The cysteine proteinases of the pineapple plant. The Biochemical Journal, 266(3), 869–875.Google Scholar
  32. Silva, M. E., & Franco, T. T. (2000). Liquid-liquid extraction of biomolecules in downstream processing–a review paper. Brazilian Journal of Chemical Engineering, 17(1), 1–17.CrossRefGoogle Scholar
  33. Srinivas, N. D., Rashmi, K. R., & Raghavarao, K. S. M. S. (1999). Extraction and purification of a plant peroxidase by aqueous two-phase extraction coupled with gel filtration. Process Biochemistry, 35(1–2), 43–48.CrossRefGoogle Scholar
  34. Sriwatanapongse, A., Balaban, M., & Teixeira, A. (2000). Thermal inactivation kinetics of bromelain in pineapple juice. Transactions of the ASAE, 43(6), 1703–1708.Google Scholar
  35. Tanuja, S., Srinivas, N. D., Gowthaman, M. K., & Raghavarao, K. S. M. S. (2000). Aqueous two-phase extraction coupled with ultrafiltration for purification of amyloglucosidase. Bioprocess and Biosystems Engineering, 23(1), 63–68.Google Scholar
  36. Vaidya, B. K., Suthar, H. K., Kasture, S., & Nene, S. (2006). Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system. Biochemical Engineering Journal, 28(2), 161–166.CrossRefGoogle Scholar
  37. Wu, Y. T., Zhu, Z. Q., & Mei, L. H. (1996). Interfacial tension of poly (ethylene glycol) + salt + water systems. Journal of Chemical & Engineering Data, 41(5), 1032–1035.CrossRefGoogle Scholar
  38. Zaslavsky, B. Y. (1995). Aqueous two phase partitioning: physical chemistry and bioanalytical applications. New York: Marcel Dekker.Google Scholar
  39. Zhang, W., Liu, H., & Chen, J. (2002). Forward and backward extraction of BSA using mixed reverse micellar system of CTAB and alkyl halides. Biochemical Engineering Journal, 12(1), 1–5.CrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2010

Authors and Affiliations

  • Umesh H. Hebbar
    • 1
  • B. Sumana
    • 1
  • A. B. Hemavathi
    • 1
  • K. S. M. S. Raghavarao
    • 1
    Email author
  1. 1.Department of Food EngineeringCentral Food Technological Research Institute, Council of Scientific and Industrial ResearchMysoreIndia

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