Journal of the American Oil Chemists' Society

, Volume 74, Issue 11, pp 1441–1446 | Cite as

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

  • Yuji Shimada
  • Kazuaki Maruyama
  • Akio Sugihara
  • Shigeru Moriyama
  • Yoshio Tominaga


Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.

Key words

Docosahexaenoic acid hydrolysis lipase Pseudomonas sp Rhizopus delemar selective esterification tuna oil 


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  1. 1.
    Fischer, S., Dietary Polyunsaturated Fatty Acids and Eicosanoid Formation in Humans, in Advances in Lipid Research, edited by R. Paoletti and D. Kritchevsky, Academic Press, New York, 1989, Vol. 23, pp. 169–198.Google Scholar
  2. 2.
    Harris, W.S., Fish Oils and Plasma Lipid and Lipoprotein Metabolism in Humans: A Critical Review. J. Lipid Res. 30:785–809 (1989).PubMedGoogle Scholar
  3. 3.
    Yamaguchi, M., I. Tanaka, and Y. Ohtsu, New Method for Separation and Purification of Polyunsaturated Fatty Acids Using a Silver Ion-Exchanged Spherical Clay Mineral, Yukagaku (in Japanese) 40:959–964 (1991).Google Scholar
  4. 4.
    Shimada, Y., A. Sugihara, K. Maruyama, T. Nagao, S. Nakayama, H. Nakano, and Y. Tominaga, Enrichment of Arachidonic Acid: Selective Hydrolysis of a Single-Cell Oil from Mortierella with Candida cylindracea Lipase, J. Am. Oil Chem. Soc. 72:1323–1327 (1995).CrossRefGoogle Scholar
  5. 5.
    Shimada, Y., A. Sugihara, H. Nakano, T. Nagao, M. Suenaga, S. Nakai, and Y. Tominaga, Fatty Acid Specificity of Rhizopus delemar Lipase in Acidolysis. J. Ferment. Bioeng. 83:321–327 (1997).CrossRefGoogle Scholar
  6. 6.
    Yang, L.Y., A. Kuksis, and J.J. Myher, Lipolysis of Menhaden Oil Triacylglycerols and the Corresponding Fatty Acid Alkyl Esters by Pancreatic Lipase in vitro: A Reexamination, J. Lipid Res. 31:137–148 (1990).PubMedGoogle Scholar
  7. 7.
    Shimada, Y., K. Maruyama, S. Okazaki, M. Nakayama, A. Sugihara, and Y. Tominaga, Enrichment of Polyunsaturated Fatty Acids with Geotrichum candidum Lipase, J. Am. Oil Chem. Soc. 71:951–954 (1994).CrossRefGoogle Scholar
  8. 8.
    Shimada, Y., K. Maruyama, M. Nakamura, S. Nakayama, A. Sugihara, and Y. Tominaga, Selective Hydrolysis of Polyunsaturated Fatty Acid-Containing Oil with Geotrichum candidum Lipase, Ibid.:1577–1581 (1995).CrossRefGoogle Scholar
  9. 9.
    Hoshino, T., T. Yamane, and S. Shimizu, Selective Hydrolysis of Fish Oil by Lipase to Concentrate n-3 Polyunsaturated Fatty Acids, Agric. Biol. Chem. 54:1459–1467 (1990).Google Scholar
  10. 10.
    Syed Rahmatullah, M.S.K., V.K.S. Shukla, and K.D. Mukherjee, Enrichment of γ-Linolenic Acid from Evening Primrose Oil and Borage Oil via Lipase-Catalyzed Hydrolysis, J. Am. Oil Chem. Soc. 71:569–573 (1994).CrossRefGoogle Scholar
  11. 11.
    Hills, M.J., I. Kiewitt, and K.D. Mukherjee, Enzyme Fractionation of Fatty Acids: Enrichment of γ-Linolenic Acid and Docosahexaenoic Acid by Selective Esterification Catalyzed by Lipases, Ibid.:561–564 (1990).CrossRefGoogle Scholar
  12. 12.
    Syed Rahmatullah, M.S.K., V.K.S. Shukla, and K.D. Mukherjee, γ-Linolenic Acid Concentrates from Borage and Evening Primrose Oil Fatty Acids via Lipase-Catalyzed Esterification, Ibid.:563–567 (1994).CrossRefGoogle Scholar
  13. 13.
    Foglia, T.A., and P.E. Sonnet, Fatty Acid Selectivity of Lipases: γ-Linolenic Acid from Borage Oil, Ibid.:417–420 (1995).CrossRefGoogle Scholar
  14. 14.
    Shimada, Y., A. Sugihara, H. Nakano, T. Kuramoto, T. Nagao, M. Gemba, and Y. Tominaga, Purification of Docosahexaenoic Acid by Selective Esterification of Fatty Acids from Tuna Oil with Rhizopus delemar Lipase, Ibid.:97–101 (1997).CrossRefGoogle Scholar
  15. 15.
    Sugihara, A., Y. Shimada, and Y. Tominaga, Separation and Characterization of Two Molecular Forms of Geotrichum candidum Lipase, J. Biochem. 107:426–430 (1990).PubMedGoogle Scholar
  16. 16.
    Maruyama, K., and M. Nishikawa, Function of Fish Oil and Its Application to Foods, Food Chemicals (in Japanese) 1995- 4:31–37 (1995).Google Scholar

Copyright information

© AOCS Press 1997

Authors and Affiliations

  • Yuji Shimada
    • 2
  • Kazuaki Maruyama
    • 1
  • Akio Sugihara
    • 2
  • Shigeru Moriyama
    • 1
  • Yoshio Tominaga
    • 2
  1. 1.Maruha Corp.IbarakiJapan
  2. 2.Osaka Municipal Technical Research InstituteOsakaJapan

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