Revista Mexicana de Ingeniería Química, Vol. 22, No. 3 (2023), Alim2340

Sunflower seed oil: enzymatic aqueous extraction, oil recovery by green solvent and chemical composition

D. S. Aquino, N. Stevanato, D. T. Raspe, C. Silva



The combination of Alcalase® 2.4L FG (ALC) and Celluclast® 1.5L (CEL) enzymes in the sunflower seed oil extraction were evaluated, using ethyl acetate in the oil recovery step. The influence of the enzyme on the oil composition (minor compounds and fatty acid profile) was determined. Soxhlet extraction with ethyl acetate was also performed, as well as extraction with each enzyme isolated. Oil yield (Yo) after 4 cycles with the reuse of ALC and CEL enzymes was verified. The CEL/ALC extraction obtained the highest Yo (43.11 wt%) among the evaluated enzymatic extractions, which represents 76.65% of extraction efficiency. Oleic and linoleic acids constitute a significant portion of the oil's composition, totaling about 87% of the fatty acids. In the phytosterols composition, β-sitosterol represented the highest value and α-tocopherol showed a higher amount in extractions with the combination of enzymes. Squalene concentration was similar in the obtained oils. The reuse of ALC and CEL enzymes proved to be viable, as after the 4 cycles the Yo reduced about 9.50% and 11.33%, respectively.

Keywords: fatty acids, squalene, Alcalase® 2.4L FG, Celluclast® 1.5L.



  • Adeleke, B. S. and Babalola, O. O. (2020) Oilseed crop sunflower (Helianthus annuus) as a source of food: Nutritional and health benefits. Food Science & Nutrition 8, 4666–4684.
  • Aquino, D. S., Roders, C., Vessoni, A. M., Stevanato, N. and Silva, C. (2022) Assessment of obtaining sunflower oil from enzymatic aqueous extraction using protease enzymes. Grasas y aceites 73, e452.
  • Aquino, D. S., Fanhani, A., Stevanato, N. and Silva, C. (2019) Sunflower oil from enzymatic aqueous extraction process: Maximization of free oil yield and oil characterization. Journal of Food Process Engineering 42, e13169.
  • AOCS. American Oil Chemists´ Society. Official methods and recommended practices. 4th ed. Champaign, IL. 1993.
  • AOCS. American Oil Chemists´ Society. Official methods and recommended practices. 5th ed. Champaign, IL. 1998.
  • Becerra, E. H., López, E. J. P. and Sossa, J. W. Z. (2021) Recovery of Biomolecules from Agroindustry by Solid-Liquid Enzyme-Assisted Extraction: a Review. Food Analytical Methods 14, 1744–1777.
  • Belingheri, C., Giussani, B. and Rodriguez-Estrada, M. T. (2015) Ferrillo, A., Vittadinia, E. Oxidative stability of high-oleic sunflower oil in a porous starch carrier. Food Chemistry 166, 346-351.
  • Campbell, K. A., Vaca-Medina, G., Glatz, C. E. and Pontalier, P. Y. (2016) Parameters affecting enzyme-assisted aqueous extraction of extruded sunflower meal. Food Chemistry 208, 245-251.
  • Chen, B. K. and Diosady, L. L. (2003) Enzymatic Aqueous Processing of Coconuts. International Journal of Applied Science and Engineering 1, 55 – 61.
  • Cheng, M. H., Dien, B. S. and Singh, V. (2019) Economics of plant oil recovery: A review. Biocatalysis and Agricultural Biotechnology 18, 101-106.
  • Codex Alimentarius Commission. Standard for Named Vegetable Oils. CXS 210-1999. Codex Stan 2019, 210, 1–15.
  • FAO (2022) Food and Agriculture Organization of the United Nations. Available at: http://www. Accessed: January 11, 2023.
  • Gao, P., Liu, R., Jin, Q. and Wang, X. (2019) Comparison of solvents for extraction of walnut oils: Lipid yield, lipid compositions, minor-component content, and antioxidant capacity. LWT - Food Science and Technology 110, 346-352.
  • Gasparetto, H., Nunes, A. L. B., Castilhos, F. and Salau, N. P. G. (2022) Soybean oil extraction using ethyl acetate and 1-butanol: From solvent selection to thermodynamic assessment. Journal of Industrial and Engineering Chemistry 113, 450-460.
  • Huesca-Toral A., López-Hernández, A., Angulo-Guerrero, J. O., Hill, C. G. Jr and García, H. S. (2005) Sintesis de triacilgliceridos ricos en acido linoleico conjugado (CLA) mediante esterificacion enzimatica en un medio libre de solvente. Revista Mexicana de Ingenieria Quimica 4, 75-87.
  • Jesus, S. S. and Maciel Filho, R. (2020) Recent advances in lipid extraction using green solvents. Renewable and Sustainable Energy Reviews 133, 110289.
  • Kojima, M., Tachibana, N., Yamahira, T., Seino, S., Izumisawa, A., Sagi, N., Arishima, T., Kohno, M., Takamatsu, K., Hirotsuka, M. and Ikeda, I. (2010) Structured triacylglycerol containing behenic and oleic acids suppresses triacylglycerol absorption and prevents obesity in rats. Lipids Health Dis 9, 77.
  • Liu, J. J., Gasmalla, M. A. A., Li, P. and Yang, R. (2016) Enzyme-assisted extraction processing from oilseeds: Principle, processing and application. Innovative Food Science & Emerging Technologies 35, 184-193.
  • Liu, Z., Gui, M., Xu, T., Zhang, L., Kong, L., Qin, L. and Zou, Z. (2019) Efficient aqueous enzymatic-ultrasonication extraction of oil from Sapindus mukorossi seed kernels. Industrial Crops and Products 134, 124-133.
  • Lu, W., Wang, Z. and Yuan, Z. (2015) Characteristics of lipid extraction from Chlorella sp. cultivated in outdoor raceway ponds with mixture of ethyl acetate and ethanol for biodiesel production. Bioresource Technology 191, 433–437.
  • Mello, B. T. F., Stevanato, N., Cardozo-Filho, L. and Silva, C. (2021) Pressurized liquid extraction of radish seed oil using ethanol as solvent: Effect of pretreatment on seeds and process variables. Journal of Supercritical Fluids, 176, 105307.
  • Meng, X., Ge, H., Ye, Q., Peng, L., Wang, Z. and Jiang, L. (2018) Efficient and Response Surface Optimized Aqueous Enzymatic Extraction of Camellia oleifera (Tea Seed) Oil Facilitated by Concurrent Calcium Chloride Addition. Journal da American Oil Chemists Society 95, 29-37.
  • Micera, M., Botto, A., Geddo, F., Antoniotti, S., Bertea, C. M., Levi, R., Gallo, M. P. and Querio, G. (2020) Squalene: More than a step toward sterols. Antioxidant 9(8), 688.
  • Mohammadi, A., Jafari, S. M., Esfanjani, A. F. and Akhavan, S. (2016) Application of nano-encapsulated olive leaf extract in controlling the oxidative stability of soybean oil. Food Chemistry 190, 513-519.
  • Moradi, N. and Rahimi, M. (2018) Effect of ultrasound- and pulsed electric field-assisted enzymatic treatment on the recovery and quality of sunflower oil. Separation Science and Tecnology 54, 1043-1054.
  • Mwaurah, P. W., Kumar, S., Kumar, N., Attkan, A. K., Panghal, A., Singh, V. K. and Garg, M. K. (2020) Novel oil extraction technologies: Process conditions, quality parameters, and optimization. Food Science and Food Safety 19, 3-20.
  • Nguyen, H. C., Vuong, D. P., Nguyen, N. T. T., Nguyen, N. P., Su, C., Wang, F. and Juan, H. (2020) Aqueous enzymatic extraction of polyunsaturated fatty acid–rich sacha inchi (Plukenetia volubilis L.) seed oil: An eco-friendly approach. Food Science and Technology 133, 109992.
  • Núñez-Gastélum, J. A. (2023) Screening of stability of Moringa oleifera seed oil under extended frying conditions. Revista Mexicana de Ingeniería Química 22, 2975.
  • Oliveira Filho, J. G. and Egea, M. B. (2021) Sunflower seed byproduct and its fractions for food application: An attempt to improve the sustainability of the oil process. Journal of Food Science 86(5), 1497-1510.
  • Orsavova, J., Misurcova, L., Ambrozova, J., Vicha, R. and Mlcek, J. (2015) Fatty acids composition of vegetable oils and its contribution to dietary energy intake and dependence of cardiovascular mortality on dietary intake of fatty acids. International Journal of Molecular Sciences 16, 12871–12890.
  • Pal, U. S., Patra, R. K., Sahoo, N. R, Bakhara, C. K. and Panda, M. K. (2015) Effect of refining on quality and composition of sunflower oil. Journal Food Science Technology 52(7), 4613–4618.
  • Pilorge, E. (2020) Sunflower in the global vegetable oil system: situation, specificities, and perspectives. Oilseeds & fats Crops and Lipids 27, 34.
  • Peng, L., Ye, Q., Liu, X., Liu, S. and Meng, X. (2019) Optimization of aqueous enzymatic method for Camellia sinensis oil extraction and reuse of enzymes in the process. Journal of Bioscience and Bioengineering 6, 716-722.
  • Pérez-Salazar, Y. I., Peña-Montes, C., Moral, S. and Aguilar-Uscanga, M. G. (2022) Cellulases production from Aspergillus niger-ITV-02 using corn lignocellulosic residues. Revista Mexicana de Ingeniería Química 21, 2772.
  • Polmann, G., Badia, V., Frena, M., Teixeira, G. L., Rigo, E., Block, J. M. and Feltes, M. M. C. (2019) Enzyme-assisted aqueous extraction combined with experimental designs allow the obtaining of a high-quality and yield pecan nut oil. LWT – Food Science and Technology 113, 108283.
  • Ribeiro, S. A. O., Nicacio, A. E., Zanqui, A. B., Biondo, P. B. F., Abreu-Filho, B. A., Visentainer, J. V., Gomes, S. T. M. and Matsushita, M. (2016) Application of Enzymes in Sunflower Oil Extraction: Antioxidant Capacity and Lipophilic Bioactive Composition. Journal Brazilian Chemical Society 27(5), 834-840.
  • Rosa, A. C. S., Stevanato, N., Garcia, V. A. S. and Silva, C. (2020) Simultaneous extraction of the oil from the kernel and pulp of macauba fruit using a green solvent. Journal of Food Processing and Preservation 44, e14855.
  • Santos Júnior, O. O., Montanher, P. F., Bonafé, E. G., Prado, I. N., Maruyama, S. A., Matsushita, M. and Visentainer, J. V. (2014) A simple, fast and efficient method for transesterification of fatty acids in foods assisted by ultrasound energy. Journal of the Brazilian Chemical Society 25, 1712–1719.
  • Sadoudi, R., Ammouche, A. and Ali Ahmed, D. (2014) Thermal oxidative alteration of sunflower oil. African Journal of food science 8(3), 116 – 121.
  • Seiler, G. J. and Gulya, T. J. (2016) Sunflower: Overview. Encyclopedia of Food Grains 1, 247-253.
  • Silva, H. R. P., Iwassa, I. J., Marques, J., Postaue, N., Stevanato, N. and Silva, C. (2020) Enrichment of sunflower oil with β-carotene from carrots: Maximization and thermodynamic parameters of the β-carotene extraction and oil characterization. Journal of Food Process Engineering 44, e14399. 14399
  • Sitepu, E. K., Candra, A., Zaidar, E. F., Vika, A., Sebayang, F., Dewi, F. R., Karo-Karo, J. A. and Tarigan, J. B. R. (2022) Green solvent enhanced mechanical extraction of palm kernel oil. Rasayan Journal of Chemistry 15, 1063–1070.
  • Song, Y., Zhang, W., Wu, J., Admassu, H., Liu, J., Zhao, W. and Yang, R. (2019) Ethanol-Assisted Aqueous Enzymatic Extraction of Peony Seed Oil. Journal of the American Oil Chemists' Society 96, 595-606.
  • Stevanato, N. and Silva, C. (2019) Radish seed oil: Ultrasound-assisted extraction using ethanol as solvent and assessment of its potential for ester production. Industrial Crops and Products 132, 283-291.
  • Stevanato, N., Mello, B. T. F., Saldaña,  M. D. A., Cardozo-Filho, L. and Silva, C. (2022) Production of ethyl esters from forage radish seed: An integrated sequential route using pressurized ethanol and ethyl acetate. Fuel, 332(2), 126075.
  • Tirgarian, B., Farmani, J. and Milani, J. M. (2019) Enzyme‑assisted aqueous extraction of oil and protein hydrolysate from sesame seed. Journal of Food Measurement and Characterization 13, 2118–2129.
  • Velasco, L. and Ruiz-Méndez, M. V. (2015) Sunflower oil minor constituents, in: Martínez-Force, E., Dunford, N.T., Salas, J.J. (Eds.), Sunflower: Chemistry, Production, Processing, and Utilization. Academic Press and AOCS Press, Illinois, pp 297–329.
  • Vijayakumar, M., Vasudevan, D. M., Sundaram, K. R., Krishnan, S., Vaidyanathan, K., Nandakumar, S., Chandrasekhar, R. and Mathew, N. (2016) A randomized study of coconut oil versus sunflower oil on cardiovascular risk factors in patients with stable coronary heart disease. Indian Heart Journal 68, 498-506.
  • Wang, D., Fan, W., Guan, Y., Huang, H., Yi, T. and Ji,, J. (2018) Oxidative stability of sunflower oil flavored by essential oil from Coriandrum sativum L. during accelerated storage. LWT - Food Science and Technology 98, 268-275.
  • Wang, H., Geng, H., Tang, H., Wang, L., Yu, D., Wang, J. and Song, Y. (2019) Enzyme-assisted Aqueous Extraction of Oil from Rice Germ and its Physicochemical Properties and Antioxidant Activity. Journal of Oleo Science 68(9), 881-891.
  • Wen, S., Lu, Y., Yu, N., Nie, X. and Meng, X. (2022) Microwave pre-treatment aqueous enzymatic extraction (MPAEE): A case study on the Torreya grandis seed kernels oil. Journal of Food Processing and Preservation. 4, e17115.
  • Xu, D., Hao, J., Wang, Z., Yang, H., Gao, Q., Ma, N., Wang, J., Ma, Y. and Zhang, M. (2020) Aqueous enzymatic extraction of oil from rice bran and its quality evaluation. Authorea,
  • Yusoff, M. M., Gordon, M. H., Ezeh, O. and Niranjan, K. (2016) Aqueous enzymatic extraction of Moringa oleifera oil. Food Chemistry 211, 400-408.
  • Zeng, J., Xiao, T., Ni, X., Wei, T., Liu, X., Deng, Z. and Li, J. (2022) The comparative analysis of different oil extraction methods based on the quality of flaxseed oil. Journal of Food Composition and Analysis 107, 104373.