• P. Vázquez-Villegas Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey
  • E. Acuña-González Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey
  • L.A. Mejía-Manzano Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey
  • M. Rito-Palomares Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey
  • O. Aguilar Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey
Keywords: alfalfa, aqueous two-phase system (ATPS), central composite design, leaf protein concentrate


There is a growing interest in producing protein from green crops, being alfalfa (Medicago sativa) one of the more studied models. In this work we develop an aqueous two-phase systems (ATPS) process to obtain a chlorophyl-free leaf protein concentrate. A two-level factorial experimental design in order to study five factors that interferes with recovery of total protein in ATPS is performed, employing two model proteins (BSA and Lyz). The factors of polymer concentration and total protein concentration were statistically significative. From here, we applied steepest descent and central composite design (CCD) methods in order to optimize chlorophyl-free, total protein recovery from alfalfa (Medicago sativa) green tissue. An extraction of more than 80% of total protein was reached for bottom phase LPC and 51% of protein in powder from the lyophilized LPC.


Abranches, R., Marcel, S., Arcalis, E., Altmann, F., Fevereiro, P. and Stroger, E. (2005). Plants as bioreactors: a comparative study suggests that Medicago truncatula is a promising production system. Journal of Biotechnology 120, 121-134.

Aguilar, O., Glatz, C. and Rito-Palomares, M. (2009). Characterization of green tissue protein extract from alfalfa (Medicago sativa), exploiting a 3-D technique. Journal of Separation Science 32, 3223-3231.

Aguilar, O., Rito-Palomares, M. and Glatz, C.E. (2010) Coupled application of aqueous twophase partitioning and 2D-electrophoresis for characterization of soybean proteins. Separation Science and Technology 45, 2210- 2225.

Aguilar, O. and Rito-Palomares, M. (2014). Aqueous two-phase system strategies for the recovery of proteins from plants. In: Methods in Molecular Biology, (N.E. Labrou, ed.), Pp. 89-100. Humana Press, Totowa.

Andrews, B.A., Schmidt, A.S. and Asenjo, J.A. (2005). Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. Biotechnology and Bioengineering 90, 380-390.

Archanaa, S., Moise, S. and Suraishkumar, G.K. (2012). Chlorophyll interference in microalgal lipid quantification through the Bligh and Dyer method. Biomass and Bioenergy 46, 805-808.

Arnon, D. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24, 1- 15.

Benavides, J. and Rito-Palomares, M. (2008). Practical experiences from the development of aqueous two-phase processes for the recovery of high value biological products. Journal of Chemical Technology and Biotechnology 83, 133-142.

Berges, J.A., Fisher, A.E. and Harrison, P.J. (1993). A comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonana. Marine Biology 115, 187-193.

Bernardo, S.C., Azevedo, A.M. and Aires-Barros M.R. (2014). Integrated platforms for the clarification and capture of monoclonal antibodies. Revista Mexicana de Ingeniería Química 13, 349-357.

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, 248-254.

Brodzik, K.R., Steplewski, Z. (2009) Production of antibodies in plants: approaches and perspectives. Current Topics in Microbioly and Immunology 332, 55-78.

Chen, Z., Liu, J., Zeng, M., Wang, Z., Yu, D., Yin, C., Jin, L., Yang, S. and Song, B. (2012). Dot immunobinding assay method with chlorophyll removal for the detection of southern rice blackstreaked dwarf virus. Molecules 17, 6886-6900.

Colas, D., Doumeng, C., Pontalier, P.Y. and Rigal, L. (2013). Twin-screw extrusion technology, an original solution for the extraction of proteins from alfalfa (Medicago sativa). Food and Bioproducts Processing 91, 175-182.

Diederich, P., Amrhein, S., Hämmerling, F. and Hubbuch, J. (2013). Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques. Chemical Engineering Science 104, 945-956.

Espitia-Saloma, E., Vazquez-Villegas, P., Aguilar, ´ O., Rito-Palomares, M. (2014). Continuous aqueous two-phase systems devices for the recovery of biological products. Food and Bioprodicts Processing 92, 101-112.

Eze, J.M.O. and Dumbroff, E.B. (1982). A comparison of the Bradford and Lowry methods for the analysis of protein in chlorophyllous tissue. Canadian Journal of Botany 60, 1046- 1049.

Ferreira, L., Fan, X., Mikheeva, L.M., Madeira, P.P., Kurgan, L., Uversky, V.N. and Zaslavsky, B.Y. (2014). Structural features important for differences in protein partitioning in aqueous dextran-polyethylene glycol two-phase systems of different ionic compositions. BBA - Proteins and Proteomics 1844, 694-704.

Ferreira, L., Madeira, P.P., Mikheeva, L., Uversky, V.N. and Zaslavsky, B. (2013). Effect of salt additives on protein partition in polyethylene glycol-sodium sulphate aqueous two-phase systems. BBA - Proteins and Proteomics 1834, 2859-2866.

Fiorentini, R., Galoppini, C. (1983). The proteins from leaves. Plant Foods for Human Nutrition 32, 335-350.

Gachovska, T.K., Ngadi, M., Oluka, S. and Raghavan, V. (2013). Electro-plasmolysis of alfalfa mash. Pulsed Power Conference, 19th IEEE 1, 16-21.

González-Valdez, J., Mayolo-Deloisa, K., González-González, M. and Rito-Palomares, M. (2014). Trends in Bioseparations. Revista Mexicana de Ingeniería Química 13, 19-27.

Gu, Z. (2014). Recovery of recombinant proteins from plants using aqueous two-phase partitioning systems: an outline. In: Methods in Molecular Biology, (N.E. Labrou, ed.), Pp. 77-87. Humana, Totowa.

Haghtalab, A., Mokhtarani, B. and Mauer, G. (2003). Experimental results and thermodynamic modeling of the partitioning of lysozyme, bovine serum albumin and α-amylase in aqueous two phase system of PEG and (KH2PO4 or Na2SO4). Journal of Chemical Engineering Data 48, 1170-1177.

Hua, X., Yang, J., Wang, L., Fang, O., Zhang, G. and Liu, F. (2012). Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products. PLoS ONE 7, e53099.

Ibarra-Herrera, C.C., Aguilar, O. and RitoPalomares, M. (2011). Application of an aqueous two-phase system strategy for the potential recovery of a recombinant protein from alfalfa (Medicago sativa). Separation and Purification Technology 77, 94-98.

Karg, S.R. and Kallio, P.T. (2009). The production of biopharmaceuticals in plant systems. Biotechnology Advances 27, 879-894.

Kerfai, S., Mathe, S., Alfenore, S. and Fernández, A. (2011). Intensive ion exchange process for soluble proteins recovery from industrial alfalfa green juice. Chemical Engineering Transactions 25, 1061-1066.

Knuckles, B.E. and Kohler, G.O. (1982). Functional properties of edible protein concentrates from alfalfa. Journal of Agricultural and Food Chemistry 30, 748-752.

Kohler, G.O. and Bickoff, E.M. (1975). Industrial production of leaf protein in the USA. In: Food protein sources (N.W. Pirie, ed.), Pp. 141. Cambridge University Press, Cambridge.

Levitt, J. (1980). Responses of Plants to Environmental Stresses. Academic Press, New York.

Melnik, S. and Stoger, E. (2013). Green factories for biopharmaceuticals. Current Medicinal Chemistry 20, 1038-1046.

Ordiales, E., Martín, A., Benito, M.J., Hernández, A., Ruiz-Moyano, S. and Córdoba, M.G. (2012). Technological characterization by free zone capillary electrophoresis (FCZE) of the vegetable rennet (Cynara cardunculus) used in “Torta del Casar” cheese-making. Food Chemistry 133, 227-235.

Perez, B., Pellegrini-Malpiedi, L., Tub´ıo, G., Nerli, B. and Pessoa-Filho, P.A. (2013). Experimental determination and thermodynamic modeling of phase equilibrium and protein partitioning in aqueous two-phase systems containing biodegradable salts. Journal of Chemical Thermodynamics 56, 136-143.

Schillberg, S., Raven, N., Fischer, R.M., Twyman, R. and Schiermeyer, A. (2013). Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Current Pharmaceutical Design 19, 5531-5542.

Sharma, A.K. and Sharma, M.K. (2009). Plants as bioreactors: Recent developments and emerging opportunities. Biotechnology Advances 27, 811- 832.

Solano-Castillo, C. and Rito-Palomares, M. (2000). Kinetics of phase separation under different process and design parameters in aqueous twophase systems. Journal of Chromatography B 743, 195-201.

Stochmal, A., Piacente, S., Pizza, C., De Riccardis, F., Leitz, R. and Oleszek, W. (2001). Alfalfa (Medicago sativa L.) Flavonoids. 1. Apigenin and Luteolin Glycosides from Aerial Parts. Journal of Agricultural and Food Chemistry 49, 753-758.

Walker, J.M. (2002). Protein Protocol handbook. Humana Press, Totowa.

Wang, J.C. and Kinsella, J.E. (1976). Functional properties of novel proteins: Alfalfa leaf protein. Journal of Food Science 41, 286-292.

Wu, Y.T., Lin, D.Q., Zhu, Z.Q. (1998) Thermodynamics of aqueous two-phase systems-the effect of polymer molecular weight on liquid-liquid equilibrium phase diagrams by the modified NRTL model. Fluid Phase Equilibria 147, 25-43.

Yari, M., Valizadeh, R., Naserian, A.A., Ghorbani, G.R., Rezvani-Moghaddam, P., Jonker, A. and Yu, P. (2012). Protein molecular structures in alfalfa hay cut at three stages of maturity and in the afternoon and morning and relationship with nutrient availability in ruminants. Animal Feed Science and Technology 172, 162-170.
How to Cite
Vázquez-Villegas, P., Acuña-González, E., Mejía-Manzano, L., Rito-Palomares, M., & Aguilar, O. (2020). PRODUCTION AND OPTIMIZATION OF A CHLOROPHYL-FREE LEAF PROTEIN CONCENTRATE FROM ALFALFA (Medicago sativa) THROUGH AQUEOUS TWO-PHASE SYSTEM. Revista Mexicana De Ingeniería Química, 14(2), 383-392. Retrieved from