Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of <i>Stevia rebaudiana</i> Bertoni

Abstract

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.

Chemistry and analytical techniques for ent-kaurene-glycosides of Stevia rebaudiana Bertoni - A review

“…Water extraction Abou et al, 2010;Inamake et al, 2010;Rao et al, 2012;Afandi et al, 2013;Deshmukh and Kedari, 2014;Gonzalez et al, 2014 Methanol extraction Liu et al, 1997;Pol et al, 2007;Deshmukh andKedari, 2014 Ethanol extraction Erkucuk et al, 2009;Deshmukh and Kedari, 2014 Pressurized fluid extraction (PFE) Pol et al, 2007 Microwave-assisted extraction (MAE) Jataik et al, 2009;Teo et al, 2009;Javad et al, 2014 Ultrasonic-assisted Extraction (UAE) Zlabur et al, 2015 Pressurized hot water extraction(PHWE) Teo et al, 2009;Tiwari et al, 2014 Supercritical fluid extraction (SFE) Pasquel et al, 2000;Choi et al, 2002;Yoda et al, 2003;Pol et al, 2007;Erkucuk et al, 2009Enzymatic extraction Puri et al, 2012Rao et al, 2015 Kolb et al, 2001;Woelwer et al, 2010;Hoekstra et al, 2009;Kumari et al, 2016HPLC C18 Vanek et al, 2001Abou et al, 2010;Afandi et al, 2013;Gonzalez et al, 2015;Gonzalez et al, 2014;Kubica et al, 2015;Samah et al, 2013;Javad et al, (Woelwer, 2012).…”

“…While, patent has claimed that CO 2 could be used as a solvent and methanol, ethanol and acetone as a cosolvant for steviol glycosides extraction (Kienle, 1990). Extractions of sweet glycosides using various enzymes have been also used successfully (Puri et al, 2012;Rao et al, 2015). Puri et al (2012) revealed a novel enzyme-mediated extraction (EME) method for stevioside extraction from leaves of S. rebaudiana.…”

“…Enzymes used for stevioside extraction in both studies were cellulase, hemicellulase and pectinase. Rao et al (2015) extracted stevioside through treatment of dry leaves of stevia with the help of hydrolytic enzymes aided by transition metal salts (FeCl 3 ). The metal salt assisted enzyme extraction of stevioside resulted in increased stevioside yield to 72 per cent with 98 per cent purity.…”

Natural and low‐caloric rebaudioside A as a substitute for dietary sugars: A comprehensive review

“…Rao et al. (2015) used hydrolytic enzymes aided by transition metal salts (FeCl 3 ) to pretreat dry stevia leaves, followed by PHWE to release stevioside. Cellulase pretreated with metal salt enhanced the yield of stevioside to about 72%, with 98% purity.…”

“…Enzyme-mediated extraction EME, a "green" method, is a practical alternative approach for recovering valuable compounds from stevia leaves (Rao et al, 2015). In this process, enzymes can help break down the plant's cell wall, thereby facilitating the recovery of target compounds in the cell.…”