Elsevier

The Journal of Supercritical Fluids

Volume 95, November 2014, Pages 422-430
The Journal of Supercritical Fluids

Subcritical water extraction of steviol glycosides from Stevia rebaudiana leaves and characterization of the raffinate phase

https://doi.org/10.1016/j.supflu.2014.10.017Get rights and content

Highlights

  • Stevia rebaudiana leaves were subjected to subcritical water.

  • Yielding 38.67 mg stevioside and 35.68 mg rebaudioside A per g dry leaves.

  • Raffinate phase was analyzed for total chlorophyll, carotenoid and dietary fibers.

  • Yielding 31.91 mg/100 g, 5.71 mg/100 g and 4.98%, respectively.

  • S. rebaudiana leaves can be utilized as sources of natural sweeteners, fibers and coloring agents in the industry.

Abstract

The objectives of this work were to obtain steviol glycosides of S. rebaudiana leaves, possessing natural and noncaloric sweetener properties, using subcritical water extraction; to assess optimum extraction conditions; to determine biological activities of Stevia extracts and to characterize the raffinate phase. A Box–“Bhenken” statistical design was used to evaluate the effects of various values of temperature (100–150 °C), time (30–60 min) and flow rate (2–6 ml/min) at a pressure of 230 bar applying a solid/liquid ratio of 1:10 (m:v). The most effective parameter was temperature (p < 0.005). Optimum extraction conditions were elicited as 125 °C, 45 min, 4 ml/min flow rate which yielded 38.67 mg/g stevioside and 35.68 mg/g rebaudioside A. The total phenolic, flavonoid contents and DPPH free radical scavenging activity were found as 48.63 mg gallic acid/g extract, 29.81 mg quercetin/g extract and 92.50%, respectively. After extraction, total chlorophyll, carotenoid contents and dietary fibers were quantified as 31.91 mg/100 g, 5.71 mg/100 g and 4.98% in the raffinate phase. Hence, both extract and raffinate phases of S. rebaudiana leaves can be utilized as sources of natural sweeteners, fibers and coloring agents in the industry.

Introduction

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteracease (Compositae) family native to certain regions of South America (Paraguay and Brazil). It is often known as “the sweet herb of Paraguay” [1], [2]. Steviol glycosides, namely steviol, steviolbioside, rebaudioside A, B, C, D, E, and F, and dulcoside A are known to accumulate in the leaves. Among these, stevioside and rebaudioside A are present in higher concentrations. Stevioside is 110–270 times and rebaudioside A is 150–320 times sweeter than sucrose [3], [4]. In addition to its natural, noncaloric sweetening properties, leaf extracts of stevia are reported to possess anti-hypertensive, anthyperglycemic, antioxidant, anti-tumor, anti-diarrheal, anti-inflammatory, immunomodulatory, and anti-viral effects [5]. Moreover, toxicological studies have shown that stevioside does not have mutagenic, teratogenic or carcinogenic effects. Likewise, allergic reactions have not been observed when it is used as a sweetener [6].

Extracted steviol glycosides, stevioside and rebaudioside A are approved as food additives in many countries throughout the world. In USA, highly purified steviol glycosides received the GRAS (Generally Recognized As Safe) status in both 2008 and 2009 [7]. The introduction of steviol glycosides on the European market as food additives with a purity of more than 95% is imminent after EFSA issued a positive opinion on their safety and raised the acceptable daily intake of steviol glycosides to 4 mg/kg expressed as steviol equivalents, in March 2010 [8]. Final approval was given on 11 November 2011.

Being heat and pH stable it has a wide range of applications in food industries. The steviol glycosides are currently in use as sweeteners in a number of industrial foods, such as soft drinks or fruit drinks, desserts, cold confectionery, sauces, delicacies, sweet corn, breads, biscuits, table-top sweetener and replaced saccharose, for example in ready-to-eat cereals, pickles, yoghurt, candies, soju, soy sauce and seafoods [9]. These showed the advantages of stevia over other artificial sweeteners as an ingredient for the food industry, thereby making Stevia a more suitable substitute for saccharose in different drinks, beverages and bakery products.

In the extraction of bioactive compounds from botanicals, modern extraction techniques such as pressurized hot water extraction (PHWE), supercritical fluid extraction, microwave assisted extraction (MAE) and ultrasonically assisted extraction (UAE) have become more popular than the conventional methods such as reflux, Soxhlet and boiling [6], [10], [11], [12], [13], [14], [15], [16], [17]. Among various extraction methods of steviosides from stevia leaves, hot water extraction process is safer from the health point of view as no chemicals are involved [18]. To reduce or minimize the usage of organic solvents, subcritical water extraction (SBWE) offers a feasible green option for the extraction of biocompounds in botanicals [10]. Water can be used effectively as a solvent in SBWE due to the pysico-chemical properties which can be readily altered through changes in temperature and pressure [19]. The term “subcritical water” is used to denote the region of condensed phase of water between the temperature ranges from the boiling point, 100 °C to the critical point of water, 374 °C [11]. Subcritical water can be maintained in the liquid form until a temperature of 374 °C and a pressure of 221 bar are reached after which, it becomes supercritical water. The technique could achieve the extraction of polar, moderately polar and nonpolar organics selectively by altering extraction parameter, such as temperature and pressure [20].

As far as our literature search could ascertain, no systematic optimization study of the subcritical water extraction of Stevia rebaudiana leaves existed. Hence, the objective of this study was to optimize the conditions in SBWE and to compare the glycoside contents with that of traditional hot water extractions of S. rebaudiana. In addition, the biological activities of Stevia extracts were investigated in order to interpret the results from the perspective of human welfare and the raffinate phase after extraction was characterized to determine the utilization possibilities in industry with a holistic engineering approach which can be regarded as the originality of this work.

Section snippets

Plant materials

S. rebaudiana leaves were supplied by Ocal Organic Agriculture in Manisa, Turkey. Prior to the extraction processes, the plant material was ground using a Waring laboratory scale blender and sifted using a 30 mesh-sieve (590 μm, average). Powdered plant material was then packed in plastic bags and stored at +4 °C.

Materials and reagents

HPLC standards, stevioside and rebaudioside A (98%) were purchased from Extra synthese (France). Ethanol, acetonitrile and methanol were of HPLC grade and purchased from Merck (Darmstadt,

Optimization of subcritical water extraction

Second-order polynomial equations were used to express the extraction yield of stevioside (mg/g dry leaves) (Y1) and rebaudioside A (mg/g dry leaves) (Y2) as a function of the coded independent variables (Table 1), where A, B, C represents the code of temperature, time and flow rate, respectively.Y1mg/g=+30.748.30×A1.35×B0.95×C0.96×A×B+1.52×A×C0.45×B×C8.81×A24.50×B26.80×C2Y2mg/g=+34.4910.77×A1.58×B+1.77×C2.43×A×B+1.41×A×C+1.56×B×C12.48×A20.57×B24.96×C2

Simultaneous optimizations of

Conclusion

The following recommendations were listed for practical applications:

  • (i)

    water should be used as an extracting solvent to obtain stevioside and rebaudioside A for human consumption;

  • (ii)

    response surface methodology coupled with numerical optimization undertaken in this study can be useful for maximizing the extraction of steviol glycosides from any variety of stevia leaves in aqueous medium.

  • (iii)

    the total phenolic and flavonoid contents along with high DPPH radical scavenging activity indicate that S.

Acknowledgements

This study was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK) 2210-C National Graduate Scholarship Programme. The authors are thankful to the Natural Product Chemistry Laboratory of Bioengineering Department for access to the facilities.

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