Stevioside

Plant Foods Hum Nutr DOI 10.1007/s11130-017-0598-1

ORIGINAL PAPER

Rapid Solid-Liquid Dynamic Extraction (RSLDE): a New Rapid and Greener Method for Extracting Two Steviol Glycosides (Stevioside and Rebaudioside A) from Stevia Leaves Monica Gallo 1 & Manuela Vitulano 2 & Anna Andolfi 2 & Marina DellaGreca 2 & Esterina Conte 2 & Martina Ciaravolo 2 & Daniele Naviglio 2

# Springer Science+Business Media New York 2017

Abstract Stevioside and rebaudioside A are the main diterpene glycosides present in the leaves of the Stevia rebaudiana plant, which is used in the production of foods and low-calorie beverages. The difficulties associated with their extraction and purification are currently a problem for the food processing industries. The objective of this study was to develop an effective and economically viable method to obtain a highquality product while trying to overcome the disadvantages derived from the conventional transformation processes. For this reason, extractions were carried out using a conventional maceration (CM) and a cyclically pressurized extraction known as rapid solid-liquid dynamic extraction (RSLDE) by the Naviglio extractor (NE). After only 20 min of extraction using the NE, a quantity of rebaudioside A and stevioside equal to 1197.8 and 413.6 mg/L was obtained, respectively, while for the CM, the optimum time was 90 min. From the results, it can be stated that the extraction process by NE and its subsequent purification developed in this study is a simple, economical, environmentally friendly method for producing steviol glycosides. Therefore, this method constitutes a valid alternative to conventional extraction by reducing the extraction time and the consumption of toxic solvents and favouring the use of the extracted metabolites as food additives and/or nutraceuticals. As an added value and of local interest, the experiment was carried out on stevia leaves from the Benevento area (Italy), where a high content of

* Monica Gallo [email protected]

1

Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini, 5, 80131 Naples, Italy

2

Department of Chemical Sciences, University of Naples Federico II, via Cintia, 80126 Naples, Italy

rebaudioside A was observed, which exhibits a sweet taste compared to stevioside, which has a significant bitter aftertaste. Keywords Steviol glycosides . Extraction . Maceration . RSLDE . Food additives . Nutraceuticals

Introduction Stevia, scientifically known as Stevia rebaudiana Bertoni, is a perennial plant native to the Rio Monday Valley in the highlands of Paraguay. This plant possesses an extraordinary sweetening power thanks to the presence of diterpene glycosides, known as steviol glycosides, which have a sweetness capacity approximately 200–300 times greater than that of sucrose but has zero calories. The main components of steviol glycosides are stevioside and rebaudioside A, which represent a viable alternative to sugar and other artificial sweeteners [1, 2]. The total amount of steviol glycosides obtainable from the Stevia leaves is generally 8 to 10% of the dry weight. Percentages vary according to cultivar, cultivation methods and other extrinsic factors such as the composition of the soil, weather conditions, and irrigation, among others [3]. In particular, stevioside and rebaudioside A are more than 70% of the mixture of steviol glycosides present in the leaf, and in almost all varieties, stevioside is the main component [4, 5]. Typical proportions on a dry weight basis for the four major glycosides found in the leaves of wild Stevia plants are 0.3% dulcoside, 0.6% rebaudioside C, 3.8% rebaudioside A and 9.1% stevioside [6]. The sweetener properties of these glycosides differ from each other. Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A is between 150 and 320 times sweeter, rebaudioside C is 40 to 60 times sweeter, and dulcoside A is 30 times sweeter than sucrose

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[6]. The quality of taste is expressed in the percentage of rebaudioside A; the higher the percentage (> 80%), the better the taste [7]. The use of these substances was approved in the US in 2008 when a committee of FAO/WHO experts for safety on Food Additives (JECFA) established an ADI of 4 mg/kg body weight [8], and later in 2011 in Europe following the positive EFSA opinion, which is a prerequisite for the European Commission to propose legislation that allows the use and marketing of these substances. Already established by the JECFA, the EFSA also reconfirmed that, following the isolation of these compounds, the final sweetener mixture should not contain less than 95% steviol glycosides and that stevioside and/or rebaudioside A must represent the highest percentage [9]. The extraction process is a key point in the production of steviol glycosides. To date, numerous procedures are known, and the oldest and most common involves an extraction with hot water or by boiling; other procedures, to improve the extraction efficiency, require the use of solvents, ultrasound, supercritical fluids [10–12] or other systems that require a considerable expenditure of energy and increase the risk of finding traces of potentially harmful organic compounds [13, 14]. Currently, the need for greener, sustainable, and viable processes has led the food industries and food scientists to develop new processes in full correspondence with the green extraction concept. In a recent paper by CarbonellCapella et al. [15], they investigated the potential use of nonconventional technologies (microwaves, ultrasounds, pulsed electric fields and high voltage electrical discharges) to enhance the extraction of high value added compounds (mainly stevioside and rebaudioside A) and antioxidants from Stevia rebaudiana leaves, and the results showed the advantages of these nonconventional technologies. In a review by Koubaa et al. [16], the authors showed that some nonconventional and emerging technologies could constitute a potential alternative to the conventional methods or even be combined to obtain a synergistic effect [16]. The study of the effects of emerging technologies, such as high voltage electrical discharges (HVED) and pulsed electric fields (PEF) and ultrasounds (US), on the intensification of the extraction of valuable compounds from stevia leaves has shown that HVED, PEF and US treatments improved both the kinetics and extraction yield of soluble matter [17]. An exhaustive review by Barba et al. [18] described the actual trend and the future applications of thermal and non-thermal technologies, as well as the classical techniques in order to improve the extraction of steviol glycosides from Stevia rebaudiana leaves. In a review by RosellóSoto et al. [19], the authors described the use of ultrasoundassisted extraction on the recovery of polyphenols, carotenoids and chlorophylls from vegetal and algae matters. In a work by Barba et al. [20], the authors evaluated the preservative potential of a high-pressure processing treatment (HPP) in the presence of Stevia leaf infusions to guarantee food safety while maintaining maximum retention of the nutritional properties.

In a recent review, some of the most renowned traditional and emerging pulsed electric fields (PEF) for different applications in food industry were described [21]. In the context of greener technologies, this paper has described a new solid-liquid extraction method for the principle sweeteners of Stevia, which is an alternative to conventional maceration, and employs the Naviglio extractor that uses a modern technology based on the principle of pressure/depression and provides water as the solvent at room temperature [22–24]. The most common use of Stevia leaves is the extraction and purification of steviol glycosides to obtain a non-caloric natural sweetener as a sugar substitute or as an alternative to artificial sweeteners [25–27]. The Stevia leaves can differ in quantities of glycosides due to several factors such as weather conditions, soil type, brightness, irrigation method, system of cultivation, processing and storage. Moreover, they contain an important amount of antioxidant compounds (vitamin C, polyphenols, chlorophylls, and carotenoids) and other important macro- and micronutrients such as folic acid and all of the essential amino acids except tryptophan. Therefore, given the commercial importance of this plant, in the present work we investigated a new variety of S. rebaudiana cultivated in Benevento area (Italy), where has been shown that the Stevia leaves grown in this area have a high content of rebaudioside A. This variety was selected for local interest and for the enhancement of the territory. The sweetening properties of glycosides differ from one another. However, whereas stevioside exhibits a significant bitter aftertaste, rebaudioside A has a sweet taste, which has been attributed to the presence of an extra glucose moiety in the rebaudioside A structure [5].

Materials and Methods Stevia rebaudiana Bertoni Cultivation In 2014, an experiment was conducted in order to verify the possibility of cultivating Stevia plants in soils in the province of Benevento (Italy). Three experimental fields were prepared at one hectare each. In each field, 35,000 plantlets were transplanted at a spacing of 90 cm and 35 cm between and along the rows, respectively. The plantlets were obtained from seeds sown in 220 hole polystyrene containers grown in a floating system. Sowing was performed in the last 10 days of March to allow plantlets to reach a height of 15 cm by early June. Prior to transplanting, ploughing, hoeing and a supply of 6 quintals of organic-mineral fertilizer were carried out. After the crop was planted, the following practices were performed: sprinkling irrigation after transplanting, one fertilization, and mechanical weed removal between the rows and manual weed control along the rows during the spring-summer period. No pesticide applications were needed. Only one harvest was carried out in the second half of October by cutting the plants with a mower and manually loading them on a trailer with a pitchfork. Crop

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purchased from Carlo Erba (Milan, Italy). The solvents and reagents used in this study were of analytical grade for HPLC. Ion exchange resins of food grade were purchased from Atlas Filters (Padova, Italy).

Fig. 1 Schematic drawing of the Naviglio extractor

biomass was transferred from the field to the farm centre where it was laid on the ground in a 10 cm layer under a polyethylene tunnel for drying (with no overturning). After this step, it was packed in cartons. The marketable biomass are the leaves, which must be separated from the shoots. This practice was performed manually to get the necessary amount needed for the trial; so far, no mechanical separation tests have been performed. Approximately 1200 kg of dry biomass was obtained per hectare from the harvest, of which the leaves accounted for 500 kg (50%). Sample Preparation The leaves of S. rebaudiana Bertoni were provided by the farms Politano, Lemmo and Maio (Benevento, Italy). These plants were cultivated in different areas of the province of Benevento to verify the rooting, production capacity and quality of the finished product. The leaves were harvested and dried in the sun. Before testing, the samples were comminuted, weighed, placed in bags for food use, vacuum heat-sealed and stored at room temperature. Solvents and Reagents The standards of stevioside (purity ≥95%) and rebaudioside A (purity ≥96%), acetonitrile and calcium hydroxide were purchased from Sigma-Aldrich (Milan, Italy). MilliQ water and ferric chloride were

Extraction Procedures To evaluate the extractive efficiency of the NE on the final yield of steviol glycosides, the experiments were conducted under various conditions of time and material concentrations. The dried and crushed leaves (10, 30 and 50 g) were included in a filter bag (Ø 5 μ) and inserted into the Naviglio extractor series Lab. 500 cm3 (Fig. 1). All extractions were conducted at a pressure of 8 to 9 bars, and the extract samples were collected after 30, 60, 90 and 120 min, using 550 mL of distilled water at room temperature for each extraction. Contextually, the same amount of material was used for the comparative analysis with conventional maceration (CM), which was placed in a batch at 70 °C with constant shaking of the extract while taking samples at the same times. HPLC-DAD Analysis The quantification of steviol glycosides was performed using a multi-solvent delivery system (Waters, Milford, MA, USA) equipped with a pump (Model 600E) and a multiple UV wavelength photodiode array detector (Model 996). The analysis was performed by injecting 20 μL of appropriately diluted sample that was filtered (PTFE, Ø 0.45 μm) using a Phenomenex Luna C18 column (250 mm × 4.60 mm). The mobile phase was formed from the solvent A (water) and B (acetonitrile) with the following elution gradient: 0–20 min, from 95% to 60% of A; 20–25 min, 60% of A; and 25–30 min from 60% to 5% of A, with a flow of 0.70 mL/min and a wavelength of UV detector at 210 nm. Comparison between the Varieties of Stevia The stevia leaves cultivated in the Benevento area were compared with different varieties to assess quality based on the principle sweetener contents (stevioside and rebaudioside A); specifically, the varieties were S. rebaudiana cv. Morita, Criollo, Eirete and Bertoni. For each variety, 10 g of leaves were

Fig. 2 Kinetics of cold extraction by NE and hot extraction in BATCH for rebaudioside A (blue) and stevioside (grey). Values are mean ± standard deviation; n = 3

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Fig. 3 Kinetics of cold extraction by NE and hot extraction in BATCH for rebaudioside A (light blue) and stevioside (orange). Values are mean ± standard deviation; n = 3

extracted with 600 mL of distilled water in a batch at 70 °C for 45 min. Each aliquot was subjected to chromatographic analysis under the previously described conditions. Stevia Extract Purification The stevia leaves extracts obtained with CM and NE were subjected to a purification process to isolate the two major steviol glycosides. In the first step, 200 mL of each extract were flocculated with inorganic salts (calcium hydroxide [28] and ferric chloride (ratio 1:1)) to obtain a first clarification. In the second step, the filtrate was collected and passed through a mixed bed ion exchange column at a flow of 1 mL/min to remove further unwanted coloured substances and any ions formed in the previous phase. The colourless eluate obtained was lyophilized, and the white powder obtained was subjected to chromatographic analysis to verify the yield of the process and the degree of purity of the steviol glycosides. Statistical Analysis All analyses were carried out in triplicate and the resulting data were evaluated by analysis of variance (ANOVA) using XLSTAT 2012.1 (Addinsoft, Paris, France).

Results and Discussion Extraction Yields To evaluate the extracted yield, two different solid-liquid ratios were applied to CM and NE. The results related to the CM and NE extraction kinetics are shown in Table 1 Concentration range (CR, mg L−1) of the standards used to prepare the calibration curves, standard purity (SP, %), equations of the calibration curves, and the coefficients of determination (R2) Steviol glycosides

CR (mg L−1)

SP (%)

Equation of calibration curves

R2

Rebaudioside A Stevioside

0–450 0–400

> 96% > 95%

Y = 5914× + 14,107 Y = 5956× + 21,017

0.999 0.999

Figs. 2 and 3. In the first experiment, the usual conditions of hot maceration were applied; in particular, a solid-liquid ratio of 1:60 was used. In the second experiment, a solid-liquid ratio of 5:60 was used. As seen from the graphs, the optimum extraction time for the NE was reached after 120 min with a quantity of total steviol glycosides equal to 9.66%, while for the CM, the optimum time was 90 min with a total steviol glycosides content equal to 15.84%. After 30 min, the maximum yield for hot maceration was 15% total steviol glycosides, while for the same time of extraction by NE, the total concentration of steviol glycosides was equal to 7.32%. Only after 120 min of extraction the curve showed a constant trend with an amount of rebaudioside A and stevioside equal to 0.72 and 2.46%, respectively (0.97% total steviol glycosides). The CM extraction was decidedly better than that by NE; however, this result was reversed in favour of extraction by NE following the increase of the solid-liquid ratio by using 50 g Stevia leaves in 600 mL of distilled water (Fig. 3). In this case, the final yield at 120 min of steviol glycosides extracted by NE was higher than CM (16.30% against 14.59%). At this time, the composition of the two main steviol glycosides was equal to 75% for rebaudioside A and 25% for stevioside. From the results, it can be stated that extraction by NE is preferred to the conventional maceration because, in addition to a higher yield of steviol glycosides that allow savings in terms of the process, the extraction occurs without the need to use high temperature liquid or use a greater amount of material in a single

Table 2 Calibration curve data for rebaudioside A and stevioside

Rebaudioside A

Stevioside

mg/L 0.00 53 106 212 424

mg/L 0.00 50 100 200 400

uAu 0 327,520 653,108 1,278,151 2,513,704

uAu 0 340,908 627,696 1,198,952 2,404,491

Plant Foods Hum Nutr Fig. 4 Comparison of varieties on the content of the main steviol glycosides. Values are mean ± standard deviation; n = 3

extraction cycle. Furthermore with the CM, it is necessary to separate the solid material dispersed in the liquid, while with the NE, it is possible to obtain an extract ready for the purification step as the leaves remain enclosed in the filter bag. It is also important to note that the spent material waste from NE is more intact and stable, and therefore may be more easily destined for further use in agronomy and animal nutrition.

concentrations using weighted linear regression. Following the construction of the calibration curve, the extract samples were suitably diluted on the basis of the initial concentrations of dry material (1:20–1:50) and subjected to HPLC analysis. The steviol glycosides were identified according to the retention times of the corresponding standard reference mixture and by measuring the area of the peaks.

Identification and Quantification of Steviol Glycosides Chromatographic peaks of stevioside and rebaudioside A present in the extracts were identified and quantified by comparing retention times and UV spectra with the standards used for the construction of the calibration curve (Table 1). The construction of the calibration curve was prepared using scalar concentrations of 50, 100, 200 and 400 mg/L (Table 2). The two calibration curves were constructed by analysing the standard solutions for each concentration in triplicate and by estimating the peak areas against the corresponding

Qualitative Assessment of Stevia Cultivated in Benevento Area The comparative analysis showed that the stevia plant grown in Benevento area, despite containing a lower total amount of steviol glycosides than some of the other varieties, had a higher percentage of rebaudioside A (Fig. 4). In particular, Stevia cv. Morita was the variety with the highest content at 17.91 g/100 g of dry leaves in total, while in Stevia cv. Benevento, the total amount of steviol glycosides was equal to 12.41 g/100 g of dry leaves (Fig. 5). However, Fig. 6 shows significant differences between the samples of Stevia

Fig. 5 The relationship between rebaudioside A and stevioside present in the different varieties of stevia

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Fig. 6 Chromatograms of rebaudioside A and stevioside present in the extracted samples of Stevia rebaudiana var. Benevento (left) and Stevia rebaudiana var. Bertoni (right)

rebaudiana var. Benevento and Stevia rebaudiana var. Bertoni. In particular, the variety cultivated in the Benevento area presented a rebaudioside A/stevioside ratio superior to the other varieties. This result was very positive because, as reported in the literature, rebaudioside A is the preferable stevioside for sweetening foods and drinks due to its less bitter aftertaste and solubility in water [29]. For this reason, the development of new varieties of S. rebaudiana with a higher content of rebaudioside A is the goal of all farmers interested in the production and use of this sweetener plant. Purification and Isolation of Steviol Glycosides The extracts in NE and CM that underwent purification gave different results. Specifically, the extract obtained with NE was easily purified and made it possible to get a completely colourless product (Fig. 7), while that in the CM kept a dark yellow following the passage on resins. This can be attributed to the fact that the extraction in batch, where the product had higher results in terms of the amounts of steviol glycosides, conversely also produced a high amount of total solids due to the high temperatures applied during the step of extraction, thus making the extracts more difficult to purify. These results demonstrated, as already verified by Deshmukh and Kedari

Fig. 7 Extracts obtained by NE (left) and in BATCH (right) after passage over resins

[29], that the amount of pigments present in the initial extract adversely affects the clarification step. Therefore, the coldextraction in NE is preferable to the CM in order to avoid further steps in the clarification and purification process that would involve a greater loss of steviol glycosides and additional production costs. The analyses carried out to check the degree of purity of isolated steviol glycosides following the process of purification of the extracts in NE showed that in all cases, the values were higher than 95%. Therefore, these metabolites could be used as food additives and/or nutraceuticals, among other applications [30]. Finally, several clinical studies have provided evidence that purified rebaudioside A has no effects on either blood pressure or glucose homeostasis; therefore, it is safe for human consumption under its intended conditions of use as a general purpose sweetener [31].

Conclusions The RSLDE used in this study allowed the extraction of these glycosides in shorter times compared to other currently existing extractive techniques. Moreover, by performing the extraction at room temperature and exploiting a pressure

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increase in the extractant liquid on the solid matrix to be extracted, it avoided thermal stress charged to thermolabile substances. Therefore, these metabolites could be used as food additives and/or nutraceuticals. In addition, whereas traditionally the solid/liquid extractions are performed with systems that require the use of solvents, NE represents a new way to extract. The development of solvent-free techniques is of great interest today, with the aim of modernizing the conventional processes and making them more Bgreen^ and Bcleaner^ to lower the environmental impact and the risk to human health, as well as to make them safer and easier to perform in the industrial field. In addition, the results of this study have shown that the extracts obtained from the Stevia leaves cultivated in Benevento area had a characteristically high percentage of rebaudioside A compared to the other varieties already known. Among other things, while stevioside has a liquorish aftertaste, rebaudioside A has no liquorish aftertaste despite having a sweetness flavour comparable to sugar. Acknowledgements This study was supported by the Campania Region that funded the project DoDiS (Sweet Stevia of Sannio) and acknowledges Coldiretti Benevento as the leader of the project (PSR Campania 2007-2013, Misura 124 Progetto BDo.di.S - Dolci di Stevia del Sannio^ DRD n.99 del 17/06/2014, CUP: B76G14000290006).

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Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Human or Animal Studies This article does not contain any studies with human or animal subjects.

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