Skip to main content

Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Enzyme-Assisted Three-Phase Partitioning


In this study, enzyme-assisted three-phase partitioning (EATPP) was used to extract oil from flaxseed. The whole procedure is composed of two parts: the enzymolysis procedure in which the flaxseed was hydrolyzed using an enzyme solution (the influencing parameters such as the type and concentration of enzyme, temperature, and pH were optimized) and three-phase partitioning (TPP), which was conducted by adding salt and t-butanol to the crude flaxseed slurry, resulting in the extraction of flaxseed oil into alcohol-rich upper phase. The concentration of t-butanol, concentration of salt, and the temperature were optimized to maximize the extraction yield. Under optimized conditions of a 49.29 % t-butanol concentration, 30.43 % ammonium sulfate concentration, and 35 °C extraction temperature, a maximum extraction yield of 71.68 % was obtained. This simple and effective EATPP can be used to achieve high extraction yields and oil quality, and thus, it is potential for large-scale oil production.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Rizwan, S., Naqshbandi, A., Farooqui, Z., Khan, A. A., & Khan, F. (2014). Protective effect of dietary flaxseed oil on arsenic-induced nephrotoxicity and oxidative damage in rat kidney. Food and Chemical Toxicology, 68, 99–107.

    CAS  Article  Google Scholar 

  2. 2.

    Goyal, A., Sharma, V., Upadhyay, N., Gill, S., & Sihag, M. (2014). Flax and flaxseed oil: an ancient medicine & modern functional food. Journal of Food Science and Technology-Mysore, 51, 1633–1653.

    CAS  Article  Google Scholar 

  3. 3.

    Oomah, B. D. (2001). Flaxseed as a functional food source. Journal of the Science of Food and Agriculture, 81, 889–894.

    CAS  Article  Google Scholar 

  4. 4.

    Bozan, B., & Temelli, F. (2002). Supercritical CO2 extraction of flaxseed. Journal of the American Oil Chemists Society, 79, 231–235.

    CAS  Article  Google Scholar 

  5. 5.

    Gandhi, A., Joshi, K., Jha, K., Parihar, V., Srivastav, D., Raghunadh, P., Kawalkar, J., Jain, S., & Tripathi, R. (2003). Studies on alternative solvents for the extraction of oil‐I soybean. International Journal of Food Science and Technology, 38, 369–375.

    CAS  Article  Google Scholar 

  6. 6.

    Ferreira-Dias, S., Valente, D. G., & Abreu, J. M. (2003). Comparison between ethanol and hexane for oil extraction from Quercus suber L. fruits. Grasas Y Aceites, 54, 378–383.

    CAS  Google Scholar 

  7. 7.

    Vidhate, G. S., & Singhal, R. S. (2013). Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning. Journal of Food Engineering, 117, 464–466.

    CAS  Article  Google Scholar 

  8. 8.

    Yu, X., Deng, Q. C., Xu, J. Q., Huang, F. H., Huang, Q. D., Yin, Z. H., & Yang, J. N. (2012). Effects of cold-pressed and vitamin E-enriched flaxseed oils on lipid profile and antioxidant status in high-fat fed rats. European Journal of Lipid Science and Technology, 114, 461–468.

    CAS  Article  Google Scholar 

  9. 9.

    Khattab, R. Y., & Zeitoun, M. A. (2013). Quality evaluation of flaxseed oil obtained by different extraction techniques. LWT--Food Science and Technology, 53, 338–345.

    CAS  Article  Google Scholar 

  10. 10.

    Sharma, A., & Gupta, M. N. (2004). Oil extraction from almond, apricot and rice bran by three-phase partitioning after ultrasonication. European Journal of Lipid Science and Technology, 106, 183–186.

    CAS  Article  Google Scholar 

  11. 11.

    Li, Z., Jiang, F., Li, Y., Zhang, X., & Tan, T. (2013). Simultaneously concentrating and pretreating of microalgae Chlorella spp. by three-phase partitioning. Bioresource Technology, 149, 286–291.

    CAS  Article  Google Scholar 

  12. 12.

    Li, Z., Li, Y., Zhang, X., & Tan, T. (2015). Lipid extraction from non-broken and high water content microalgae Chlorella spp. by three-phase partitioning. Algal Research-Biomass Biofuels and Bioproducts, 10, 218–223.

    Google Scholar 

  13. 13.

    Alvarez-Guerra, E., & Irabien, A. (2015). Ionic liquid-based three phase partitioning (ILTPP) systems for whey protein recovery: ionic liquid selection. Journal of Chemical Technology and Biotechnology, 90, 939–946.

    CAS  Article  Google Scholar 

  14. 14.

    Alvarez-Guerra, E., Irabien, A., Ventura, S. P. M., & Coutinho, J. A. P. (2014). Ionic liquid recovery alternatives in ionic liquid-based three-phase partitioning (ILTPP). Aiche Journal, 60, 3577–3586.

    CAS  Article  Google Scholar 

  15. 15.

    Kurmudle, N. N., Bankar, S. B., Bajaj, I. B., Bule, M. V., & Singhal, R. S. (2011). Enzyme-assisted three phase partitioning: a novel approach for extraction of turmeric oleoresin. Process Biochemistry, 46, 423–426.

    CAS  Article  Google Scholar 

  16. 16.

    Vetal, M. D., & Rathod, V. K. (2014). Three phase partitioning a novel technique for purification of peroxidase from orange peels (Citrus sinenses). Food and Bioproducts Processing, 94, 284–289.

    Article  Google Scholar 

  17. 17.

    Wang, H. H., Chen, C. L., Jeng, T. L., & Sung, J. M. (2011). Comparisons of alpha-amylase inhibitors from seeds of common bean mutants extracted through three phase partitioning. Food Chemistry, 128, 1066–1071.

    CAS  Article  Google Scholar 

  18. 18.

    Gagaoua, M., Hoggas, N., & Hafid, K. (2015). Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes. International Journal of Biological Macromolecules, 73, 245–252.

    CAS  Article  Google Scholar 

  19. 19.

    Garg, R., & Thorat, B. N. (2014). Nattokinase purification by three phase partitioning and impact of t-butanol on freeze drying. Separation and Purification Technology, 131, 19–26.

    CAS  Article  Google Scholar 

  20. 20.

    Roy, I., & Gupta, M. N. (2002). Three-phase affinity partitioning of proteins. Analytical Biochemistry, 300, 11–14.

    CAS  Article  Google Scholar 

  21. 21.

    Sharma, A., & Gupta, M. N. (2001). Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor. Process Biochemistry, 37, 193–196.

    CAS  Article  Google Scholar 

  22. 22.

    Sharma, A., Sharma, S., & Gupta, M. N. (2000). Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode. Bioseparation, 9, 155–161.

    CAS  Article  Google Scholar 

  23. 23.

    Gaur, R., Sharma, A., Khare, S. K., & Gupta, M. N. (2007). A novel process for extraction of edible oils Enzyme assisted three phase partitioning (EATPP). Bioresource Technology, 98, 696–699.

    CAS  Article  Google Scholar 

  24. 24.

    Shah, S., Sharma, A., & Gupta, M. (2004). Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning. Industrial Crops and Products, 20, 275–279.

    CAS  Article  Google Scholar 

  25. 25.

    Dutta, R., Sarkar, U., & Mukherjee, A. (2015). Process optimization for the extraction of oil from Crotalaria juncea using three phase partitioning. Industrial Crops and Products, 71, 89–96.

    CAS  Article  Google Scholar 

  26. 26.

    Jung, S., Maurer, D., & Johnson, L. (2009). Factors affecting emulsion stability and quality of oil recovered from enzyme-assisted aqueous extraction of soybeans. Bioresource Technology, 100, 5340–5347.

    CAS  Article  Google Scholar 

  27. 27.

    Pare, A., Nema, A., Singh, V. K., & Mandhyan, B. L. (2014). Combined effect of ohmic heating and enzyme assisted aqueous extraction process on soy oil recovery. Journal of Food Science and Technology-Mysore, 51, 1606–1611.

    CAS  Article  Google Scholar 

  28. 28.

    Gai, Q.-Y., Jiao, J., Wei, F.-Y., Luo, M., Wang, W., Zu, Y.-G., & Fu, Y.-J. (2013). Enzyme-assisted aqueous extraction of oil from Forsythia suspense seed and its physicochemical property and antioxidant activity. Industrial Crops and Products, 51, 274–278.

    CAS  Article  Google Scholar 

  29. 29.

    Jin, G., Yang, F., Hu, C., Shen, H., & Zhao, Z. K. (2012). Enzyme-assisted extraction of lipids directly from the culture of the oleaginous yeast Rhodosporidium toruloides. Bioresource Technology, 111, 378–382.

    CAS  Article  Google Scholar 

  30. 30.

    J-j, L., Y-j, F., Y-g, Z., Li, J., Wang, W., C-b, G., & Luo, M. (2011). Ultrasound-assisted extraction of flaxseed oil using immobilized enzymes. Bioresource Technology, 102, 9991–9996.

    Article  Google Scholar 

  31. 31.

    Zhang, S., Zu, Y.-G., Fu, Y.-J., Luo, M., Liu, W., Li, J., & Efferth, T. (2010). Supercritical carbon dioxide extraction of seed oil from yellow horn (Xanthoceras sorbifolia Bunge.) and its anti-oxidant activity. Bioresource Technology, 101, 2537–2544.

    CAS  Article  Google Scholar 

  32. 32.

    Gai, Q.-Y., Jiao, J., Mu, P.-S., Wang, W., Luo, M., Li, C.-Y., Zu, Y.-G., Wei, F.-Y., & Fu, Y.-J. (2013). Microwave-assisted aqueous enzymatic extraction of oil from Isatis indigotica seeds and its evaluation of physicochemical properties, fatty acid compositions and antioxidant activities. Industrial Crops and Products, 45, 303–311.

    CAS  Article  Google Scholar 

  33. 33.

    Dennison, C., & Lovrien, R. (1997). Three phase partitioning: concentration and purification of proteins. Protein Expression and Purification, 11, 149–161.

    CAS  Article  Google Scholar 

  34. 34.

    Harde, S. M., & Singhal, R. S. (2012). Extraction of forskolin from Coleus forskohlii roots using three phase partitioning. Separation and Purification Technology, 96, 20–25.

    CAS  Article  Google Scholar 

  35. 35.

    Tan, Z.-J., Wang, C.-Y., Yi, Y.-J., Wang, H.-Y., Zhou, W.-L., Tan, S.-Y., & Li, F.-F. (2015). Three phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction. Process Biochemistry, 50, 482–486.

    CAS  Article  Google Scholar 

  36. 36.

    Aysu, Ş., Melike, E., Bayraktar, H., & Seçil, Ö. (2011). Purification of α-galactosidase from pepino (Solanum muricatum) by three-phase partitioning. Separation and Purification Technology, 83, 130–136.

    Article  Google Scholar 

  37. 37.

    Shanmugaprakash, M., Jayashree, C., Vinothkumar, V., Senthilkumar, S., Siddiqui, S., Rawat, V., & Arshad, M. (2015). Biochemical characterization and antitumor activity of three phase partitioned l-asparaginase from Capsicum annuum L. Separation and Purification Technology, 142, 258–267.

    CAS  Article  Google Scholar 

  38. 38.

    Mulchandani, K., Kar, J. R., & Singhal, R. S. (2015). Extraction of lipids from Chlorella saccharophila using high-pressure homogenization followed by three phase partitioning. Applied Biochemistry and Biotechnology, 176, 1613–1626.

    CAS  Article  Google Scholar 

Download references


This work was financially supported by the National Natural Science Foundation of China (21406262) and the Agricultural Science and Technology Innovation Program (ASTIP-IBFC07).

Author information



Corresponding authors

Correspondence to Zhi-jian Tan or Chao-yun Wang.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tan, Zj., Yang, Zz., Yi, Yj. et al. Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Enzyme-Assisted Three-Phase Partitioning. Appl Biochem Biotechnol 179, 1325–1335 (2016).

Download citation


  • Enzyme-assisted three-phase partitioning
  • Flaxseed oil
  • Enzyme kinetics
  • GC–MS analysis