Sucrose has traditionally been the most commonly used sweetening agent in the food and beverage industries. However, the consumption of high calorie sugars has resulted in various nutritional and clinical problems including obesity, type 2 diabetes mellitus and metabolic syndrome in humans [1] - [3] . Therefore, the food industry has witnessed a rise in demand, especially from diet-conscious people, for a natural, high intensity, low caloric, non-nutritive and stable sweetener that can be used as a sugar (sucrose) substitute in food and nutraceuticals.

Steviol-glycosides are natural diterpene glycosides present in the leaves of Stevia rebaudiana, which are 250 - 300 times sweeter than sucrose at a concentration of 0.4% (w/v), with low calorific value [4] . These steviol- glycosides also possess wide therapeutic properties such as anti-diabetic, anti-cancer, anti-ulcer, antiviral and diuretic activities, thus enhancing their market value [5] -[8] .

Although abundant literature on the isolation of stevioside using conventional solvent based extraction methods, microwaves, supercritical fluids, ultrasound, etc., is available, these methodologies have disadvantages such as use of non-green solvents, prolonged extraction time and requirement of multiple purification steps resulting in low yields. In addition, the processes generate noxious wastes and by-products which are toxic to health and the environment [9] -[13] . The potential wide spread use of the steviol-glycosides necessitates the development of a simple and effective extraction methodology, which has high efficiency and specificity, requires shorter time,lesser solvents and results in better yield and quality of the extracted compounds. Enzyme-based extraction of plant bioactives is a potential alternative to conventional solvent based extraction methodologies [14] - [23] .

The objective of the present study is to design a state-of-the-art, metal salts-assisted enzymatic (EAE) process for the efficient extraction of stevioside from the leaves of Stevia rebaudiana, followed by a multi-stage filtration/purification step for the isolation of stevioside, with high yield and purity.

Stevioside is the most abundant, sweet-tasting diterpene glycoside present in the leaves of Stevia rebaudiana, and has high commercial value as a natural, non-calorific alternative to sucrose in the food industry. The steviol- glycosides isolated by conventional chemical or solvent based extraction processes however, leave a bitter after-taste after consumption; hence there is need to develop high yielding and environmentally friendly technologies which may assist in developing larger food based applications. In this study an improved methodology for the extraction and purification of stevioside was designed (Figure 1). The optimized process involves the initial removal of plant pigments and waxes by treating with non-polar solvent (hexane). After drying, the stevia leaves were enzymatically pre-treated in an aqueous phosphate buffer pH 4.6 in the ratio of 1:15 with 1% of enzyme + 0.1 M FeCl₃, at 55˚C for 2 hours, followed by pressurized hot water extraction (PHWE).The filtered solution was passed through microfiltration using a micron cartridge (5 µm pore size). Later, the MF permeate was fed to a spiral wound UF membrane module (10 kDa MWCO) operated at a feed pressure of 7 kg/cm² and temperature of 28˚C. The UF permeate was then fed to a series of five diafiltration (DF) units, for the further recovery of the stevioside and to remove leaf cell debris and other unknown impurities (Figure 2). The permeate from each diafiltration step was collected and processed using hydrophilized polyamide NF membrane (MWCO 250Da) at a feed pressure of 18 kg/cm² and temperature of 30˚C until 80% - 90% of water was removed to get concentrated stevioside solution. Thus the NF membrane process showed higher retention of stevioside from 80% - 95%. The crude stevioside-rich NF retentate was basified (to minimize the impurities―alkaloids, from the crude extract) and then extracted into the organic solvent (n-butanol), to isolate stevioside. The separated organic

layer was concentrated and crystallized with ethanol to obtain a yield of 72% of stevioside with 98% purity, in comparison to other known extraction methods (Figure 3). The structure of stevioside was confirmed by analytical data obtained by ¹H-NMR, Mass and IR spectroscopy, which were compared with literature values (Supplementary Figures 4-9).

A number of methods for the extraction of stevioside from stevia leaves are known, but all these techniques have several limitations. Supercritical CO₂ extraction was employed to isolate steviosides to obtain a yield of 1.6% of steviol-glycosides; thus only 50% of stevioside was extracted [11] . This technique is applicable only to compounds that are CO₂ soluble, thus despite being scalable it is inefficient in extracting the steviol-glycosides present in the stevia leaves and is also expensive. Ultrasonication of stevia leaves was performed for the extraction of steviosides, to obtain a yield of 43.62% of stevioside and 28.96% rebaudioside-A [26] . This technique is highly energy consuming, thus upscaling of the process would not be viable due to the large expenses involved. Microwave extraction of steviosides resulted in very low yields of stevioside (6.54%) in 1 minute, and as in the case of ultrasonication, the process was costly, thus this technique cannot be used at the commercial scale [12] .

Puri et al. (2012) [19] pre-treated the stevia leaves with cell wall degrading enzymes to isolate a mixture of steviol-glycosides (2.84 gm/100gm of stevia leaves). In our study, however, we were able to extract 98% pure stevioside at a yield of 72% (3.76 gm/100gm of stevia leaves) by using a combination of enzyme and transition metal (0.1 M FeCl₃), which synergistically enhanced the yields of stevioside in comparison to treatment with only cellulase. The hydrolytic enzymes have the ability to degrade or disrupt cell walls and membranes thus enabling the release of bioactives present in the stevia leaves, therefore enzyme-based extraction of bioactive compounds from plants is a potential alternative to conventional solvent-based extraction methods [27] .

An integrated membrane extraction process was employed to isolate stevioside from the crude extract, making the process economical since the membranes are reusable and have high half-lives. This overall process developed can be scaled up and used for commercial purposes.

As the pressure mounts on the food industry to identify cleaner routes for the extraction of natural compounds; the methodology developed demonstrates the feasibility of enzymatic pre-treatment, followed by pressurized hot water extraction, in combination with inexpensive multi-stage membrane based separation process that helps in removing impurities and obnoxious residues present in the extract, thus improving the yields and taste profile of the stevioside isolated. This simple, efficient, cost-effective and eco-friendly process thus has great potential in the food industry.

In this study, the isolation of stevioside from Stevia rebaudiana leaves using enzyme-assisted extraction was

successfully demonstrated. From the results, it was confirmed that the combination of metal salt (0.1 M FeCl₃) assisted enzymatic extraction with multi-stage membrane filtration (MF, UF and NF) enabled the enhancement of the isolated yields (72%) and purity of stevioside from the stevia leaves. This process also helps in removing the undesirable alkaloids thus improving the organoleptic properties. Thus it is concluded that this green process of extraction of stevioside from stevia leaves, using cellulase assisted by metal salts, is more feasible in comparison to known conventional physical and chemical processes, since it is simple, economical and eco-friendly.

The authors would like to thank the Council of Scientific and Industrial Research (CSIR), New Delhi for funding this work under XII Five year plan project (CSC-0133). SAG sincerely thanks CSIR for awarding the Project fellowship.

The authors do not declare any conflict of interest, whatsoever, either financially or otherwise.

A. BhaskarRao,Sara A.George,SateeshAlavala,H. M.Meshram,K. ChandraShekar, (2015) Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of Stevia rebaudiana Bertoni. Advances in Bioscience and Biotechnology,06,734-743. doi: 10.4236/abb.2015.612075

Experimental data:

The spectral data of isolated Stevioside: m.p 197˚C - 199˚C; LC-MS (m/z) 803 (M-H): 839 (M-Na); [α]_D -9.92 (H₂O, c 3.0; std. stevioside [α]_D -27.3 (H₂O, c 3.0); IR (KBr): 3388, 2937, 1739, 1658, 1458, 1372, 1075, 892 cm⁻¹; ¹HNMR (300 MHz, C₅D₅N): δ 0.9 (d, J = 13.4 Hz, C₁-H), 2.22 (s, 2H, C₂-H), 1.03 (d, J = 11.70 Hz, C₃-H), 1.05 (d, J = 12.4 Hz, C₅-H), 2.46 (d, J = 12 Hz, C₆-H), 1.30 (s, 2H, C₇-H), 0.88 (s, 1H, C₉-H), 5.13 (d, J = 7.55 Hz, C₁₁-H), 2.25 (s, 2H, C₁₂-H), 2.71 (d, J = 11.8 Hz, C₁₄-2H), 2.05 (s, 2H, C₁₅-H), 5.3 (d, J = 7.5 Hz, C₁₇-2H), 1.25 (s, 3H, C₁₈-H).