Journal of the American Oil Chemists' Society

, Volume 83, Issue 2, pp 93–99 | Cite as

Enrichment of CLA isomers by selective esterification with l-menthol using Candida rugosa lipase

  • Takashi Kobayashi
  • Toshihiro Nagao
  • Yomi Watanabe
  • Yoshie Yamauchi-Sato
  • Satoshi Negishi
  • Vuji Shimada


Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.

Key Words

Candida rugosa lipase CLA l-menthol purification selective esterification 


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  1. 1.
    Pariza, M.W., CLA, a New Cancer Inhibitor in Dairy Products, Bull. Int. Dairy Fed. 257:29–30 (1991).Google Scholar
  2. 2.
    Ip, C., S.F. Chin, J.A. Scimeca, and M.W. Pariza, Mammary Cancer Prevention by Conjugated Dienoic Derivatives of Linoleic Acid, Cancer Res. 51:6118–6124 (1991).Google Scholar
  3. 3.
    Ha, Y.L., N.K. Grimm, and M.W. Pariza, Anticarcinogens from Field Ground Beef: Heat-Altered Derivatives of Linoleic Acid, Carcinogenesis 8:1881–1887 (1987).Google Scholar
  4. 4.
    Lee, K.N., D. Kritchevsky, and M.W. Pariza, Conjugated Linoleic Acid and Atherosclerosis in Rabbits, Atherosclerosis 108:19–25 (1994).CrossRefGoogle Scholar
  5. 5.
    Nicolosi, R.J., E.J. Rogers, D. Kritchevsky, J.A. Scimeca, and P.J. Huth, Dietary Conjugated Linoleic Acid Reduces Plasma Lipoproteins and Early Aortic Atherosclerosis in Hypercholesterolemic Hamsters, Artery 22:266–277 (1997).Google Scholar
  6. 6.
    Park, Y., K.J. Albright, W. Liu, J.M. Storkson, M.E. Cook, and M.W. Pariza, Effect of Conjugated Linoleic Acid on Body Compisition in Mice, Lipids 32:853–858 (1997).CrossRefGoogle Scholar
  7. 7.
    Ostrowska, E., M. Muralitharan, R.F. Cross, D.E. Bauman, and F.R. Dunshea, Dietary Conjugated Linoleic Acids Increase Lean Tissue and Decrease Fat Deposition in Growing Pigs, J. Nutr. 129:2037–2042 (1999).Google Scholar
  8. 8.
    Sugano, M., A. Tsujita, M. Yamasaki, M. Noguchi, and K. Yamada, Conjugated Linoleic Acid Modulates Tissue Levels of Chemical Mediators and Immunoglobulins in Rats, Lipids 33:521–527 (1998).CrossRefGoogle Scholar
  9. 9.
    Ha, Y.L., J.M. Storkson, and M.W. Pariza, Inhibition of Benzo(a)pyrene-Induced Mouse Forestomach Neoplasia by Conjugated Dienoic Derivatives of Linoleic Acid, Cancer Res. 50:1097–1101 (1990).Google Scholar
  10. 10.
    Park, Y., K.J. Albright, J.M. Storkson, W. Liu, M.E. Cook, and M.W. Pariza, Change in Body Composition in Mice During Feeding and Withdrawal of Conjugated Linoleic Acid, Lipids 34:243–248 (1999).CrossRefGoogle Scholar
  11. 11.
    Park, Y., K.J. Albright, J.M. Storkson, W. Liu, and M.W. Pariza, Evidence That the trans-10,cis-12 Isomer of Conjugated Linoleic Acid Induces Body Composition Changes in Mice, Lipids 34:235–241 (1999).CrossRefGoogle Scholar
  12. 12.
    De Deckere, E.A., J.M. Van Amelsvoort, G.P. McNeill, and P. Jones, Effect of Conjugated Linoleic Acid (CLA) Isomers on Lipid Levels and Peroxisome Proliferation in the Hamster, Br. J. Nutr. 82:309–317 (1999).Google Scholar
  13. 13.
    Nagao, K., Y.-M. Wang, N. Inoue, S.-Y. Han, Y. Buang, T. Noda, N. Kouda, H. Okamatsu, and T. Yanagita, The 10trans,12cis Isomer of Conjugated Linoleic Acid Promotes Energy Metabolism in OLETF Rats, Nutrition 19:652–656 (2003).CrossRefGoogle Scholar
  14. 14.
    Nagao, K., N. Inoue, Y.-M. Wang, J. Hirata, Y. Shimada, T. Nagao, T. Matsui, and T. Yanagita, The 10trans,12cis Isomer of Conjugated Linoleic Acid Suppress the Development of Hyperttension in Otsuka Long-Evans Tokushima Fatty Rats, Biochem. Biophys. Res. Commun. 306:134–138 (2003).CrossRefGoogle Scholar
  15. 15.
    Haas, M.J., K.G. Kramer, G. McNeill, K. Scott, T.A. Foglia, N. Sehat, J. Fritsche, M.M. Mossoba, and M.P. Yurawecz, Lipase-Catalyzed Fraction of Conjugated Linoleic Acid Isomers, Lipids 34:979–987 (1999).CrossRefGoogle Scholar
  16. 16.
    McNeill, G.P., C. Rawlins, and A.C. Peilow, Enzymatic Enrichment of Conjugated Linoleic Acid Isomers and Incorporation into Triglycerides, J. Am. Oil Chem. Soc. 76:1265–1268 (1999).Google Scholar
  17. 17.
    Nagao, T., Y. Shimada, Y. Yamauchi-Sato, T. Yamamoto, M. Kasai, K. Tsutsumi, A. Sugihara, and Y. Tominaga, Fractionation and Enrichment of CLA Isomers by Selective Esterification with Candida rugosa Lipase, J. Am. Oil Chem. Soc. 79:303–308 (2002).Google Scholar
  18. 18.
    Nagao, T., Y. Yamauchi-Sato, A. Sugihara, T. Iwata, K. Nagao, T. Yanagita, S. Adchi, and Y. Shimada, Purification of Conjugated Linoleic Acid Isomers Through a Process Including Lipase-Catalyzed Selective Esterification, Biosci. Biotechnol. Biochem. 67:1429–1433 (2003).CrossRefGoogle Scholar
  19. 19.
    Yamauchi-Sato, Y., T. Nagao, T. Yamamoto, T. Terai, A. Sugihara, and Y. Shimada, Fractionation of Conjugated Linoleic Acid Isomers by Selective Hydrolysis with Candida rugosa Lipase, J. Oleo Sci. 52:367–374 (2003).Google Scholar
  20. 20.
    Shimada, Y., Y. Hirota, T. Baba, S. Kato, A. Sugihara, S. Moriyama, Y. Tominaga, and T. Terai, Enzymatic Synthesis of l-Methyl Esters in Organic Solvent-Free System, J. Am. Oil Chem. Soc. 76:1139–1142 (1999).Google Scholar
  21. 21.
    Kobayashi, T., T. Nagao, A. Kawashima, Y. Watanabe, and Y. Shimada, Synthesis of Polyunsaturated FattyAcid l-Methyl Esters Through Lipase-Catalyzed Esterification in an Organic Solvent-Free System, J. Oleo Sci. 53:309–312 (2003).Google Scholar
  22. 22.
    Chang, R.C., S.J. Chou, and J.F. Shaw, Multiple forms and Functions of Candida rugosa Lipase, Biotechnol. Appl. Biochem. 19:93–97 (1994).Google Scholar

Copyright information

© AOCS Press 2006

Authors and Affiliations

  • Takashi Kobayashi
    • 2
  • Toshihiro Nagao
    • 2
  • Yomi Watanabe
    • 2
  • Yoshie Yamauchi-Sato
    • 1
  • Satoshi Negishi
    • 1
  • Vuji Shimada
    • 2
  1. 1.Nisshin OilliO Group, Ltd.Japan
  2. 2.Osaka Municipal Technical Research InstituteOsakaJapan

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