Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of Stevia rebaudiana Bertoni


Stevioside, extracted from the leaves of Stevia rebaudiana Bertoni, is a natural, high intensity, lowcaloric sweetener with wide therapeutic activities. Conventional stevioside extraction methodologies involve the use of non-green solvents, supercritical fluids, microwaves, etc., however, all these processes are expensive, time-consuming and eco-unfriendly. Therefore an alternative process is desired for the isolation of stevioside. In this study, a novel enzyme-mediated extraction (EME) method has been developed. The dry stevia leaves were pre-treated with hydrolytic enzymes aided by transition metal salts (FeCl3). This was followed by pressurized hot water extraction (PHWE) to release stevioside. The crude extract was purified and clarified through multi-stage membrane filtration. The results confirm that metal salt-assisted cellulase pre-treatment enhanced the yield of stevioside to 72%, with 98% purity, which was higher in comparison to existing methods. Thus, the methodology developed establishes a simple, “green”, enzyme-mediated process for the efficient isolation of stevioside under economical and eco-friendly conditions.

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Rao, A. , George, S. , Alavala, S. , Meshram, H. and Shekar, K. (2015) Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of Stevia rebaudiana Bertoni. Advances in Bioscience and Biotechnology, 6, 734-743. doi: 10.4236/abb.2015.612075.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Yoo, S., Nicklas, T., Baranowski, T., Zakeri, I.F., Yang, S.J., Srinivasan, S.R. and Berenson, G.S. (2004) Comparison of Dietary Intakes Associated with Metabolic Syndrome Risk Factors in Young Adults: The Bogalusa Heart Study. American Journal of Clinical Nutrition, 80, 841-848.
[2] Anton, S.D., Martin, C.K., Han, H., Coulon, S., Cefalu, W.T., Geiselman, P., et al. (2010) Effects of Stevia, Aspartame, and Sucrose on Food Intake, Satiety, and Postprandial Glucose and Insulin Levels. Appetite, 55, 37-43.
[3] Basu, S., Yoffe, P., Hills, N. and Lustig, R.H. (2013) The Relationship of Sugar to Population-Level Diabetes Prevalence: An Econometric Analysis of Repeated Cross-Sectional Data. PLoS ONE, 8, Article ID: e57873.
[4] Geuns, J.M.C. (2003) Molecules of Interest: Stevioside. Phytochemistry, 64, 913-921.
[5] Takahashi, K., Matsuda, M., Ohashi, K., Taniguchi, K., Nakagomi, O., Abe, Y., et al. (2001) Analysis of Anti-Rotavirus Activity of Extract from Stevia rebaudiana. Antiviral Research, 49, 15-24.
[6] Jayaraman, S., Manoharan, M.S. and Illanchezian, S. (2008) In-Vitro Antimicrobial and Antitumor Activities of Stevia rebaudiana (Asteraceae) Leaf Extracts. Tropical Journal of Pharmaceutical Research, 7, 1143-1149.
[7] Cekic, V., Vasovic, V., Jakovljevic, V., Mikov, M. and Sabo, A. (2011) Hypoglycaemic Action of Stevioside and a Barley and Brewer’s Yeast Based Preparation in the Experimental Model on Mice. Bosnian Journal of Basic Medical Sciences, 11, 11-16.
[8] Paul, S., Sengupta, S., Bandyopadhyay, T.K. and Bhattacharyya, A. (2012) Stevioside Induced ROS-Mediated Apoptosis Through Mitochondrial Pathway in Human Breast Cancer Cell Line MCF-7. Nutrition and Cancer, 64, 1087-1094.
[9] Kutowy, O., Zhang, Q.S. and Kumar, A. (1999) Extraction of Sweet Compounds from Stevia rebaudiana Bertoni. US Patent No. 5972120.
[10] Moraes, E. and Machado, N.R. (2001) Clarification of Stevia rebaudiana (Bert.) Bertoni Extract by Adsorption in Modified Zeolites. Acta Scientiarum, 23, 1375-1380.
[11] Yoda, S.K., Marqes, M.O.M., Petentate, A.J. and Meireles, M.A.A. (2003) Supercritical Fluid Extraction from Stevia rebaudiana Bertoni Using CO2 and CO2+ Water: Extraction Kinetics and Identification of Extracted Components. Journal of Food Engineering, 57, 125-134.
[12] Jaitak, V., Badna, B.S. and Kaul, V.K. (2009) An Efficient Microwave-Assisted Extraction Process of Stevioside and Rebaudioside-A from Stevia rebaudiana (Bertoni). Phytochemical Analysis, 20, 240-245.
[13] Abelyan, V.H., Ghochikyan, V.T., Markosyan, A.A., Adamyan, M.O. and Abelyan, L.A. (2010) Extraction Separation and Modification of Sweet Glycosides from Stevia rebaudiana Plant. US Patent No. 7838044 B2.
[14] Delgado-Vargas, F. and Paredes-Lopez, O. (1997) Effects of Enzymatic Treatments on Carotenoid Extraction from Marigold Flowers (Tagetes erecta). Food Chemistry, 58, 255-258.
[15] Santamaria, R.I., Reyes-Duarte, M.D., Barzana, E., Fernando, D., Gama, F.M., Mota, M., et al. (2000) Selective Enzyme Mediated Extraction of Capsaicinoids and Carotenoids from Chili Guajillo puya (Capsicum annuum L.) Using Ethanol as Solvent. Journal of Agricultural and Food Chemistry, 48, 3063-3067.
[16] Ruiz-Teran, F., Perez-Amador, I. and Lopez-Munguia, A. (2001) Enzymatic Extraction and Transformation of Gluco-Vanillin to Vanilla Pods. Journal of Agricultural and Food Chemistry, 49, 5207-5209.
[17] Liu, L., Sun, J., Li, M., Wang, S., Pei, H. and Zhang, J. (2009) Enhanced Enzymatic Hydrolysis and Structural Features of Corn Stover by FeCl3 Pretreatment. Bioresource Technology, 100, 5853-5858.
[18] Yadav, S. and Kumar, A. (2012) Enzymatic Extraction of Curcumin from Curcuma longa Rhizome. American Journal of PharmTech Research, 2, 898-905.
[19] Puri, M., Sharma, D., Barrow, C.J. and Tiwary, A.K. (2012) Optimisation of Novel Method for the Extraction of Steviosides from Stevia rebaudiana Leaves. Food Chemistry, 132, 1113-1120.
[20] Wang, W., Yuan, T.Q. and Cui, B.K. (2013) Fungal Treatment Followed by FeCl3 Treatment to Enhance Enzymatic Hydrolysis of Poplar Wood for High Sugar Yields. Biotechnology Letters, 35, 2061-2067.
[21] Shen, Z., Jin, C., Pei, H., Shi, J., Liu, L. and Sun, J. (2014) Pretreatment of Corn Stover with Acidic Electrolyzed Water and FeCl3 Leads to Enhanced Enzymatic Hydrolysis. Cellulose, 21, 3383-3394.
[22] Zhang, Y., Mo, L., Chen, F., Lu, M., Dong, W., Wang, Q., et al. (2014) Optimized Production of Vanillin from Green Vanilla Pods by Enzyme-Assisted Extraction Combined with Pre-Freezing and Thawing. Molecules, 19, 2181-2198.
[23] Zhang, Y.D., Li, W., Lu, T. and Xia, Y.M. (2015) The Effect of Microwave Irradiation on Transglycosylation Pathway of Stevioside with Starches or Cyclodextrins Catalyzed by a Cyclodextrin Glucanotransferase. Journal of Molecular Catalysis B: Enzymatic, 120, 151-157.
[24] Rao, A.B., Reddy, G.R., Prasad, E., Sridhar, S. and Ravikumar, Y.V.L. (2012) An Improvised Process of Isolation, Purification of Steviosides from Stevia rebaudiana Bertoni Leaves and Its Biological Activity. International Journal of Food Science and Technology, 47, 2554-2560.
[25] Rao, A.B., Prasad, E., Roopa, G., Sridhar, S. and Ravikumar, Y.V.L. (2012) Simple Extraction and Membrane Purification Process in Isolation of Steviosides with Improved Organoleptic Activity. Advances in Bioscience and Biotechnology, 3, 327-335.
[26] Liu, J., Li, J.W. and Tang, J. (2010) Ultrasonically Assisted Extraction of Total Carbohydrates from Stevia rebaudiana Bertoni and Identification of Extracts. Food and Bioproducts Processing, 88, 215-221.
[27] Ladisch, M.R., Lin, K.W., Voloch, M. and Tsao, G.T. (1983) Process Considerations in the Enzymatic Hydrolysis of Biomass. Enzyme and Microbial Technology, 5, 82-102.

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