Stevioside - Sirshendu De - ebook

Stevioside ebook

Sirshendu De

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Stevioside is one of the naturally occurring sweeteners,belonging to the diterpene glycoside family, which can be widelyapplied in food, drinks, medicine and consumer chemicals. It is agood dietary supplement, being non-calorific, thermally stable,non-toxic, with a sugar-like taste profile, and suitable fordiabetics, phenylketonuria patients and the obese. It is alsonon-fermentable, and exhibits anti-carcinogenic, antioxidant andanti-hyperglycemic properties. Stevioside tastes about 300 timessweeter than 0.4% sucrose solution. Thus, it offers a reasonablyrare combination of health benefits and taste benefits, making theextraction of Stevioside is an area of active research for the foodindustry as well as academic food scientists.With the rapidincrease in the popularity of Stevioside as a sugar substitute,particularly with regard to its associated health benefits, thereis a need for more efficient and feasible extraction processes forstevioside in the near future. This book offers an in-depth look at perhaps the major"sugar alternative of the future", up-to-date and inline with the latest global legislation regarding its suitabilityfor consumption and its scope for application.

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Authors’ biographies



List of figures

List of tables

1 Introduction to stevioside

1.1 History of Stevia

1.2 Composition of Stevia

1.3 Source of stevioside

1.4 Physicochemical and biological properties of steviol glycosides

1.5 Analysis of stevioside (steviol glycosides) and Stevia extract


2 Health benefits and pharmacological effects of steviol glycosides

2.1 Effect of stevioside in absorption, distribution, metabolism and excretion

2.2 Antihyperglycaemic effect

2.3 Antihypertensive effect

2.4 Anti-inflammatory effect

2.5 Anticarcinogenic antitumour effects

2.6 Antioxidant activity

2.7 Antimicrobial and antidiarrhoeal effects

2.8 Effect on renal function


3 Applications of stevioside


4 Conventional extraction processes of stevioside

4.1 Ion exchange

4.2 Solvent extraction

4.3 Extraction by chelating agents

4.4 Adsorption and chromatographic separation

4.5 Ultrasonic extraction

4.6 Microwave-assisted extraction

4.7 Supercritical fluid extraction


5 Brief introduction to pressure-driven membrane-based processes

5.1 Advantages of the membrane-based process

5.2 Classification of the processes

5.3 Characterisation of membranes

5.4 Membrane modules (Bungay et al. 1986; Ho and Sirkar 1992; Rautenbach and Albrecht 1986)

5.5 Limitations

5.6 Quantification of concentration polarisation

5.7 Applications of membrane-based processes


6 State of the art of stevioside processing using membrane-based filtration

6.1 Clarification and purification

6.2 Concentration by nanofiltration

6.3 Limitations


7 Detailed membrane-based technologies for extraction of stevioside

7.1 Outline of processing

7.2 Optimisation of water extraction process

7.3 Optimisation of primary clarification (centrifugation or microfiltration)

7.4 Selection of membrane

7.5 Optimisation of operating conditions

7.6 Mechanism of flux decline

7.7 Ultrafiltration of primary clarified Stevia extract

7.8 Concentration by nanofiltration


8 Performance modelling of stevioside separation using membrane processing

8.1 Modelling of stirred ultrafiltration

8.2 Modelling of cross-flow ultrafiltration

8.3 Steady state

8.4 Transient state



9 Enhancement of stevioside recovery by diafiltration

9.1 Multiple stage diafiltration


10 Economics of the process


Supplemental Images


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Library of Congress Cataloging-in-Publication Data

De, Sirshendu.Stevioside : technology, applications, and health / Sirshendu De, Sourav Mondal and Suvrajit Banerjee, IIT Kharagpur. pages cm Includes bibliographical references and index.

ISBN 978-1-118-35066-9 (cloth : alk. paper) 1. Stevioside. I. Mondal, Sourav. II. Banerjee, Suvrajit. III. Title. TP425.D427 2013 664′.5–dc23


A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover images: Stevia plant © Le Moal; Sugar background ©; Stevia © design by Steve Thompson

Authors’ biographies

Dr Sirshendu De is a professor in the Department of Chemical Engineering, at the Indian Institute of Technology (IIT) Kharagpur, India. He obtained his Bachelor of Technology (1990), Master of Technology (1992) and PhD (1997) degrees from the Indian Institute of Technology Kanpur. His major field of interest is membrane-based separation processes: design, modelling and fabrication of flat-sheet and hollow-fibre membranes. He has already authored six books, ten book chapters, five patents and more than 190 publications in national and international journals of repute. He has also transferred two technologies for commercialisation. His other fields of research are adsorption, transport phenomena, modelling of flow through microchannel, etc. He has been the recipient of several prestigious awards, including the Shanti Swarup Bhatnagar Award in 2011 for fundamental contribution and innovation in basic science and technology (Engineering Science category). He also received the Herdillia Award in 2010 for excellence in basic research in chemical engineering, the VNMM Award in 2009 for excellence in innovative applied research, the Young Engineer Award from the Indian National Academy of Engineering in 2001 for excellence in engineering research, and the Amar Dye Chem Award in 2000 for excellence in chemical engineering research. He is a fellow of the Indian National Academy of Engineering, New Delhi, and the Indian National Academy of Science, Allahabad, for his contribution to engineering and research.Sourav Mondal received his undergraduate degree in chemical engineering from Jadavpur University in 2010 and a Master’s degree in chemical engineering from the Indian Institute of Technology Kharagpur. Currently he is pursuing doctoral research in the Department of Chemical Engineering at the Indian Institute of Technology Kharagpur. He has co-authored one book, one book chapter and 18 publications in international journals of repute, and presented four papers in national and international conferences. He is also involved in a project for computer simulation of a magnesium reactor, development of a ceramic membrane module and molecular dynamics-based simulation of self-assembled structures in an aqueous environment.Suvrajit Banerjee obtained his Bachelor of Technology in chemical engineering from West Bengal University of Technology, India, in 2010. He gained his Master of Technology in chemical engineering in 2012 from the Indian Institute of Technology Kharagpur, India, where he worked extensively in the field of membrane separation processes. Suvrajit Banerjee is a novel separations enthusiast. Presently, he is a PhD student in chemical and biological engineering and graduate research assistant at the Center for Biotechnology and Interdisciplinary Studies at Rensselaer Polytechnic Institute, Troy, NY, USA. His present research focuses on the use of molecular dynamics simulations to gain insights into protein–ligand binding during chromatographic separation processes and the role of water molecules and mobile phase modifiers in these processes.


Stevioside is one of the naturally occurring sweeteners, belonging to the diterpene glycoside family, which can be widely applied in food as a dietary supplement, in soft drinks, medicine and daily chemicals. It is a good dietary supplement, non-calorific, thermally stable and non-toxic, with a sugar-like taste profile and suitable for diabetic and phenylketonuria patients and obese persons. It is also non-fermentable and exhibits anticarcinogenic, antioxidant, antihyperglycaemic, antihypertensive, antidiarrhoeal, anti-inflammatory and anticariogenic properties.

Stevioside tastes about 300 times sweeter than 0.4% sucrose solution. Thus, extraction and purification of stevioside is an area of active research. Stevioside has a greater presence in the extract of Stevia rebaudiana leaves compared to the other glycosides, namely rebaudioside A, B, D and E, dulcoside A and B. Current sweetener extraction techniques involve many unit operations, including solvent extraction (methanol and ethanol), ion exchange, etc. Solvent extraction may not be suitable for human consumption and ion exchange is not economic. In this regard, membrane-based processes can offer an attractive alternative. This book provides a detailed understanding of the design and modelling characteristics at various levels of processing using membranes.

With the rapid increase in popularity of stevioside as a sugar substitute and its associated health benefits, there is need for an efficient and feasible extraction process for stevioside in the near future. Since no other book exists on this topic, the proposed book covers the state of the art of stevioside extraction with an emphasis on membrane technology. Thus, it is envisaged that the significance of this book will be remarkably high in this context. Apart from extraction aspects, the book also presents an account of the history, medicinal values and other applications in some detail.

Composition, source, various physical and chemical properties and methods of analysis are covered in Chapter 1. Steviol glycosides have numerous health benefits. Various facets of this aspect are presented in Chapter 2. Applications of stevioside in different sectors, therapeutics, food, drink, etc., are discussed in Chapter 3. The conventional extraction process of stevioside includes a number of unit operations and the state of the art of these conventional extraction processes is presented in Chapter 4. The fundamentals of membrane-based processes, including advantages, features, classifications, modelling approaches, modules, limitations and applications, have been outlined in Chapter 5. Chapter 6 deals with the development of applications of membrane-based operations for stevioside processing in different steps: clarification, purification, concentration, etc. A description of membrane processes for extraction of stevioside is presented in Chapter 7. In this chapter, optimisation of water extraction, comparison between centrifuge and microfiltration as the primary clarification, use of ultrafiltration for main clarification, optimised selection of membranes and operating conditions, identification of flux decline mechanism, various modes of ultrafiltration (unstirred, stirred and cross-flow) and concentration by nanofiltration are discussed in detail. Relevant modelling for scaling up of membrane-based systems is covered with full details in Chapter 8. Enhancement of stevioside purity by using diafiltration is discussed in Chapter 9 and the economics of the process is presented in Chapter 10.

We believe this book will have two fold impacts. First, its academic value is high, since it deals with extraction of an upcoming bioproduct using membrane-based processes. Second, it will have a substantial impact on the scaling up of such systems in actual industrial scale from laboratory data. This book can be used as a reference for courses involving membrane and food technology and food processing taught in postgraduate level. And of course, it would be an extremely useful reference book for students and professionals in the fields of chemical engineering, food technology, biotechnology, bioengineering, agricultural engineering, industrial engineering and pharmaceutical engineering.

We have tried our best to make this book comprehensive and hope that it will ignite further research interest and industrial development in the relevant and associated fields of engineering. We hope that readers will benefit from the applicability and significance of membrane-based technology for food processing in general presented through this book. Although we have put our best efforts into organising all possible information regarding processing of Stevia extract, readers’ comments and suggestions for improvement will be gratefully acknowledged.

Sirshendu DeSourav MondalSuvrajit Banerjee


We extend our gratitude to Mr Dhruba Sakha, Department of Chemical Engineering, Indian Institute of Technology Kharagpur, for his invaluable efforts in helping us to determine the concentration of steviol glycosides by high-performance liquid chromatography. We also express our thanks to Dr Chhaya for her co-operation in conducting the membrane separation experiments.

Finally, it is our pleasure to thank all those who made this book possible, and to acknowledge the good wishes, blessings and whole-hearted support of our near and dear ones, to whom we are really indebted.

List of figures

List of tables


Major historical developments in the discovery and use of stevioside as a sweetener and dietary supplement


List of all the chemical constituents of S. rebaudiana leaves (excluding oil)


Amino acid, vitamin and fatty acid contents of Stevia leaves


World’s leading stevioside manufacturing companies in different countries


Current status of usage of stevioside in different countries


Physical properties of steviol glycosides present in S. rebaudiana


Proximate analysis of dry S. rebaudiana leaves


Different physiological effects of stevioside consumption


Maximum level of usage of different sweeteners in food products


Ethnomedical uses of Stevia in different countries


Properties of some supercritical fluids


Reverse osmosis membrane module comparison


Pretreatment methods used in sea water and brackish water desalination


Comparative performance of clarification by chelating agents and UF + DF


Summary of the flux decline phenomena of different membranes and operating conditions


Experimental range and levels of the independent variables


Experimental conditions and responses for three variables (in coded level) for stevioside extraction process


The regression coefficients of the second-order polynomial model for the response functions (stevioside and colour) in coded level


Experimental range and levels of the independent variables for centrifugation of stevioside extract


Experimental conditions and responses for two variables (in coded level) for centrifugation of stevioside extract


The regression coefficients of the second-order polynomial model for the response functions (colour, clarity, total solids and stevioside) in coded level


Properties of Stevia extract clarified by microfiltration process


Comparison of the properties of the extract clarified by two different clarification methods (centrifugation and microfiltration)


Characteristics of the membranes used (temperature 30 ± 2 °C and transmembrane pressure range 276–690 kPa)


Properties of the permeate with the operating conditions under stirred continuous mode of ultrafiltration using 30 kDa membrane


(a) Statistical parameters for the fitting of the characteristic complete pore blocking equation with the experimental data(b) Statistical parameters for the fitting of the characteristic intermediate pore blocking equation with the experimental data(c) Statistical parameters for the fitting of the characteristic standard pore blocking equation with the experimental data(d) Statistical parameters for the fitting of the characteristic gel layer equation with the experimental data


Various statistical parameters of the cubic-square polynomial surface fit of the ratio of Rc/Rm varying MWCO and time


Properties of the stevioside permeate


Various properties of the ultrafiltered liquor at different operating conditions under total recycle mode of operation


Various properties of permeate of nanofiltration at the end of the experiment (feed is ultrafiltration permeate at 552 kPa and 100 L/h)


Values of distribution coefficient with transmembrane pressure drop


Details of the parameters estimated


Values of the partition coefficient with pressure in total recycle as well as batch mode


Comparison of the stevioside recovery (in percentage) values


Properties of permeate (colour, clarity and total solids) for the experimental conditions: TMP 690 kPa and cross-flow rate 50 L/h


Properties of permeate (colour, clarity and total solids) for the experimental conditions: TMP 414 kPa and cross-flow rate 50 L/h


Properties of permeate (colour, clarity and total solids) for the experimental conditions: TMP 690 kPa and cross-flow rate 100 L/h


Properties of permeate (colour, clarity and total solids) for the experimental conditions: TMP 414 kPa and cross-flow rate 100 L/h


Summary of the total energy required for hot water extraction (air velocity in the range of 0–15 cm/s)


Energy required in the centrifugation process


Energy requirement to produce unit m3 of permeate with different membrane surface areas


Energy per unit m3 of product for different diafiltration stages with varying areas of filtration


Energy requirement (kWh/m3) of evaporation using multi-effect evaporators with backward feed


Operating cost estimation of stevioside processing


Introduction to stevioside

In the past couple of decades, use of sweeteners as food additives has attracted considerable interest. The global market for high-potency sweeteners during 2010 was reported to be $1.146 billion (Leatherhead Food Research 2011). The market demand for stevioside in comparison to other sweeteners is presented in Figure 1.1. Among the sugar substitutes, artificial sweeteners saccharin and aspartame are quite popular because of their high sweetness potency (Mitchell 2006; Nabors 2011; Wilson 2007). However, the sweet herb Stevia rebaudiana Bertoni, belonging to the family Asteraceae within the tribe Eupatoricae (King and Robinson 1987), has sweet-tasting diterpenoid glycosides in its leaves (Bertoni 1905; Gosling 1901), which have high sweetness potency (Geuns 2003), with the added advantage that Stevia sweeteners are natural plant products (Kim and Dubois 1991). Stevia sweeteners are unique in having zero Glycaemic Index effect, zero carbohydrate and zero calories (O’Donnell and Kearsley 2012), compared to other conventional sweeteners. It is the world’s only natural sweetener in this category. The sweet part of the Stevia herb is extracted and then blended with other all-natural ingredients to create a delicious and healthy sweetener.

Stevia rebaudiana is native to Paraguay and is widespread in its country of origin. The natural habitat of Stevia rebaudiana is subtropical grasslands (mesothermal-humid climatic zone) at altitudes of about 200–600 m above sea level, in the Amambay Cordillera, a mountain range of north-eastern Paraguay (Katayama et al. 1976). It usually grows in semi-dry mountainous terrain, and its habitat ranges from grasslands, scrub forests, forested mountain slopes and conifer forests to subalpine vegetation (Kinghorn 2001).

Stevia rebaudiana is a New World genus distributed from the South American Andes to the southern United States, through Argentina, the Brazilian highlands and Central Mexico (Grashoff 1972). It is a 30–60 cm tall herbaceous plant with perennial rhiozomes, simple, opposite and narrowly elliptic to oblanceolate leaves, trinerved venation, paniculate-corymbose inflorescences with white flowers, and achenes bearing numerous, equally long pappus awns (Robinson 1930). A picture of the plant is shown in Figure 1.2.

Figure 1.1 Market potential of Stevia and related products in the world. (a) Percentage compound annual growth rate (CAGR) of different sweeteners in the projected years 2011–15. (b) Global Stevia market in different regions of the world in 2010. (c) Annual global Stevia production in the last 5 years. For a colour version of this figure, see Plate 1.1.

The following chemical description of steviol glycoside is taken from the original JECFA monograph (WHO 2000):

‘Stevioside is a glycoside of the diterpene derivative steviol (ent-1 3-hydroxykaur-1 6-en-19-oic acid). Steviol glycosides are natural constituents of the plant Stevia rebaudiana Bertoni. The leaves of S. rebaudiana Bertoni contain eight different steviol glycosides, the major constituent being stevioside (triglucosylated steviol), constituting about 5–10% in dry leaves. Other main constituents are rebaudioside A (tetraglucosylated steviol), rebaudioside C, and dulcoside A. S. rebaudiana is native to South America and has been used to sweeten beverages and food for several centuries. The plant has also been distributed to South-east Asia. Stevioside has a sweetening potency 250–300 times that of sucrose and is stable to heat. In a 62-year-old sample from a herbarium, the intense sweetness of S. rebaudiana was conserved, indicating the stability of stevioside to drying, preservation, and storage (Soejarto et al. 1982; Hanson and de Oliveira 1993).’

Figure 1.2Stevia rebaudiana Bertoni plant. For a colour version of this figure, see Plate 1.2.

1.1 History of Stevia

Stevia rebaudiana Bertoni is one of 154 members of the genus Stevia and one of only two that produces sweet steviol glycosides (Robinson 1930; Soejarto et al. 1982, 1983). Stevia was first brought to the attention of Europeans in 1887 (Bertoni 1899) when M.S. Bertoni learned of its unique properties from the Paraguayan Indians and Mestizos (Lewis 1992). Various reports cited by Lewis (1992) indicate that it was long known to the Guarani Indians of the Paraguayan highlands who called it caá-êhê, meaning ‘sweet herb’. Stevioside, the most abundant sweet constituent present in the leaves of Stevia rebaudiana, was first isolated in impure form in the first decade of the 20th century (Bertoni 1905, 1918) but the final chemical structure was determined 60 years later by Mosettig et al. (1963). The second major sweet diterpene glycoside from S. rebaudiana was identified around 1970 (Kohda et al. 1976). Further, six less abundant sweet component glycosides were isolated from the species, namely rebaudioside B–E, dulcoside A and steviolbioside (Kobayashi et al. 1977; Tanaka 1982; Yamasaki et al. 1976).

The first reports of commercial cultivation in Paraguay were in 1964 (Katayama et al. 1976; Lewis 1992). A large effort aimed at establishing Stevia as a crop in Japan was begun by Sumida (1980). Since then, Stevia has been introduced as a crop in a number of countries including Brazil, Korea, Mexico, United States, Indonesia, Tanzania and, since 1990, Canada (Brandle and Rosa 1992; Donalisio et al. 1982; Fors 1995; Goenadi 1983; Lee et al. 1979; Saxena and Ming 1988; Schock 1982). Currently, Stevia production is centred in China and the major market is in Japan (Kinghorn and Soejarto 1985). Milestones of discovery and various uses of stevioside are presented in Table 1.1.

In 1999, the Joint Food and Agriculture Organisation (FAO)/World Health Organisation (WHO) Expert Committee on Food Additives (JECFA) and the EU Scientific Committee for Food reviewed stevioside and concluded that it was unacceptable for use as a sweetener, on the basis of the data available at that time. In 2004, the JECFA reviewed stevioside again and granted a temporary maximum usage level of 2 mg/kg body weight for steviol glycosides.

In June 2008, the JECFA concluded that steviol glycosides are safe for use in foods and beverages and established an acceptable daily intake (ADI) of 4 mg/kg body weight (FAO 2008; FAO/WHO 2009). The JECFA established specifications for the identity and purity of steviol glycosides, requesting a minimum content of 95% of the sum of the seven steviol glycosides, which are stevioside, rebaudioside A, rebaudioside C, dulcoside A, rubusoside, steviolbioside and rebaudioside B (WHO 2008; 2009).

Food Standards Australia New Zealand (FSANZ) completed its evaluation of the use of steviol glycosides in foods in 2008 and recommended that the Australia and New Zealand Food Regulation Ministerial Council allow the use of steviol glycosides in food (FSANZ 2008). In 2008, Switzerland approved the use of Stevia as a sweetener, citing the favourable actions of the JECFA. Subsequently, France published its approval for the food uses of rebaudioside A with a purity of 97% (AFSSA 2009). In December 2008, the US Food and Drug Administration (FDA) stated it had no objection to the conclusion of expert panels that Stevia containing a minimum of 95% rebaudioside A is generally recognised as safe (GRAS) for use as a general-purpose sweetener in foods and beverages.

In September 2009, based on a review of the international regulation of Stevia rebaudiana and the clinical evidence for safety and efficacy, the Natural Health Products Directorate, Health Canada recommended an acceptable daily intake of 4 mg steviol/kg body weight established by WHO (2008), for consumption of Stevia and steviol glycosides in natural health products (NHPs). In Japan, China, Korea, Brazil, Paraguay and several other countries worldwide, steviol glycosides are considered natural food constituents and, as such, are implicitly accepted for food use.

Table 1.1 Major historical developments in the discovery and use of stevioside as a sweetener and dietary supplement

Chronological sequence of events


Report of sweetness of

S. Rebaudiana

leaves from Paraguay published in a major scientific paper

Gosling 1901

First chemical report on the sweet constituents of Stevia

Bertoni 1905

Realisation that stevioside is a form of glycoside

Dieterich 1908

Production of steviolbioside from stevioside

Wood et al. 1955

Evidence that stevioside is a sophoroside

Vis and Fletcher 1956

Final structures of steviol and isosteviol confirmed

Mosetigg et al. 1963

Steviol chemically synthesised

Cook and Knox, 1970; Mori and Matsui 1970

S. rebaudiana

from Brazil cultivated experimentally in Japan

Sumida 1973

Isolation and characterisation of rebaudioside A

Kohda et al. 1976


S. rebaudiana

leaf diterpene glycosides obtained

Yamasaki et al. 1976; Kobayashi et al. 1977

Advent of extensive use of

S. rebaudiana

extracts for sweetening and flavouring of foods and beverages in Japan

Abe and Sonobe 1977; Akashi 1977

From 1982 onwards, large-scale cultivation of

S. rebaudiana

in mainland China and nearby islands

Kinghorn and Soejarto 1991

Demonstration of mutagenic activity of metabolically activated steviol in a forward mutation test

Pezzuto et al. 1985

First approval of

S. rebaudiana

products in Brazil

Schwontkowski 1995

During the 1980s,

S. rebaudiana

leaves become a popular herbal tea in the USA

Blumenthal 1995

First approval of stevioside in South Korea

Korea National Institute of Health 1996

Import ban on

S. rebaudiana

into USA by FDA (1991)

Blumenthal 1995

FDA import ban on

S. rebaudiana

leaves rescinded in 1995

Blumenthal 1995

Long-term toxicity test, showing lack of any carcinogenic effects by stevioside, conducted in rats of both sexes in Japan

Toyoda et al. 1997

In USA, rebaudioside A and stevioside are considered as generally recognised as safe (GRAS) products

Curry 2010

Steviol glycosides are permitted as food additive by European Union in December 2011

Stones 2011

In Europe, steviol glycosides have recently been approved for use as a sweetener. The European Food Safety Authority (EFSA) has conducted a general safety assessment for the approval of steviol glycosides as a sweetener in foodstuffs and for use as a flavour enhancer. A positive scientific opinion from the EFSA is a prerequisite to the European Commission proposing legislation for the authorisation and marketing of this substance in the EU. In light of the JECFA’s 2008 findings and in response to a June 2008 request by the European Commission regarding the safety of steviol glycosides as a sweetener for use in the food, the EFSA re-examined the safety of steviol glycosides (EFSA 2010). After careful consideration of the data on stability, degradation products, metabolism and toxicology, the EFSA panel established an acceptable daily intake for steviol glycosides, which is similar to the JECFA’s determination.

Figure 1.3 Structures of the sweet-tasting glycosides isolated from S. rebaudiana.

1.2 Composition of Stevia

A number of natural products can be derived from the plant Stevia rebaudiana. However, the best known are the diterpenoid glycosides, comprising stevioside, rebaudioside A and C–E and dulcoside A. The structures of the sweet-tasting components are illustrated in Figure 1.3.

The yield of stevioside from the dried leaves of S. rebaudiana can vary from 5% to 20%, depending upon the cultivation (Kim and Dubois 1991). In addition to the diterpenoid glycosides, several other components such as flavonoids, labdane, oils, etc., are present in varied amounts. A complete list of the components (except the volatile oils) is presented in Table 1.2.

Table 1.2 List of all the chemical constituents of S. rebaudiana leaves (excluding oil)

A number of labdane-type diterpenes can also be identified from S. rebaudiana, along with the glycosides (see Figure 1.4). Besides jhanol and asutroinulin which were isolated using methanol extraction (Sholichin et al. 1980), eight novel labdane type diterpenoids, sterebins A–H, have been identified using spectroscopic and nuclear magnetic resonance (NMR) techniques (Oshima et al. 1986, 1988).

Figure 1.4 Structures of different labdane type glycosides isolated from S. rebaudiana.

The triterpenoids and sterols primarily constitute β-sitosterol (39.4%) and sigmasterol (45.8%) of the total sterol fraction (D’Agostino et al. 1984). A lupeol ester, lupeol 3-palmitate and β-amyrin acetate were obtained from methanolic extraction by gas chromatography-mas spectrometry (GC-MS) (Sholichin et al. 1980; Yasukawa et al. 1993), as shown in Figure 1.5.

Figure 1.5 Structures of different triterpenoids and sterols from S. rebaudiana.

In the estimation of flavonoid constituents, six standard flavonoids were extracted in ethyl acetate fraction and two from chloroform as shown in Figure 1.6 (Rajbhandari and Roberts 1983). These compounds were determined using a combination of ultraviolet (UV) and proton nuclear magnetic resonance (1H-NMR) spectroscopy, and mass spectroscopy.

The essential oil components include a number of alkanols, aldehydes, aromatic alcohols, monoterpenes and sesquiterpenes (Fujita et al. 1977; Martelli et al. 1985). Apart from this, other phytochemicals such as chlorophylls, β-carotene, organic acids, etc. are also present (Cheng and Chang 1983). Among the minerals, potassium is the major proportion, while other metals such as zinc, calcium, iron, etc. are also present in trace amounts.

The protein content represented by amino acids in Stevia leaves (Abou-Arab et al. 2010; Mohammad et al. 2007), water-soluble vitamins (Kim et al. 2011) and fatty acids (Tadhani and Subhash 2006) are reported in Table 1.3.

Figure 1.6 Flavonoids structures isolated from S. rebaudiana.

1.3 Source of stevioside

The sweet-tasting glycosides have been reported to be present in the leaves, flowers and stems but not in the roots of S. rebaudiana (Tanaka 1982).The primary source of stevioside and rebaudioside A is the leaves (5–20% w/w). They are also found in the flowers at lower concentrations, around 0.9–1% (w/w) (Darise et al. 1983). Because of the economic importance of steviol glycosides, synthetic methods of synthesis have also been attempted (Mori and Matsui 1965, 1966, 1970; Mori et al. 1970a,b; Nakahara 1982; Nakahara et al. 1971; Ziegler and Kloek 1971, 1977). Conversion of steviol glycosides to stevioside and rebaudioside A was also reported (Kaneda et al. 1977; Ogawa et al. 1978, 1980). However, extraction of glycosides using solvent (refer to Chapter 4) and membrane separation (Chapter 6–9) is feasible for large-scale production of stevioside as a dietary supplement and sweetener.

Table 1.3 Amino acid, vitamin and fatty acid contents of Stevia leaves

Steviol glycosides were first commercialised as a sweetener in 1971 by the Japanese firm Morita Kagaku Kogyo, a leading Stevia extract producer in Japan. It has been cultivated and manufactured by several companies in different parts of the globe. Some of the leading companies making stevioside products are mentioned in Table 1.4. Currently, China is the largest exporter of stevioside in the world.

Since the first commercialisation, the demand for Stevia for sweetening and flavouring purposes has increased enormously in Japan (Kinghorn et al. 2001). Cultivation of S. rebaudiana for the Japanese market mainly occurs in China, Taiwan, Thailand and some parts of Malaysia (Kinghorn and Soejarto 1991). Stevioside has also been consumed in Korea since 1995, with the majority of its use being in the sweetening of the beverage, soju (Kinghorn et al. 2001). Stevioside extract, containing 60% stevioside and free from steviol and isosteviol, is approved for use in foods, beverages, medicines, soft drinks, etc., in Brazil. In Brazil, production occurs in the southern province of the country for the local market (Oliveira Ferro 1997, personal communication). Significant amounts of cultivation and stevioside processing are also done in Canada (Brandle et al. 1998), the Czech Republic (Nepovim et al. 1998), India (Chalapathi et al. 1997) and Russia (Dzyuba and Vseross 1998). The current status of stevioside use in different countries is reported in Table 1.5.

Table 1.4 World’s leading stevioside manufacturing companies in different countries

Table 1.5 Current status of usage of stevioside in different countries




Widely used as a sweetener since 1970

South Korea

Consumption as sweetener in beverages from 1990 (presently constitutes more than 50% of the sweetener market in Korea)

Australia and New Zealand

All steviol glycosides are approved for use as food additives from 2008


Approved in 1986 for use as sweetener and food additive


Mixed steviol glycoside extracts as food additives, not as individual products (2009)

Hong Kong

Steviol glycosides as food additives from 2010


Steviol glycosides as food additives in 2012


Currently available in liquid form, for use in herbal tea

Russian Federation

Allowed in minimal dosage limit as food additive (2008)


Steviol glycoside as food additive in 2012, but the plant itself is banned


Steviol glycoside is a permitted sweetening agent in certain foods, since 2005


Available as dietary supplement

European Union (Europe)

Permitted use as food additive in 2011

United States

Stevia leaf and extracts available as dietary supplements in 1995. Rebaudioside available as dietary supplement in 2008


Currently, the largest exporter of stevioside in the world. Cultivated and produced in large scale from 1990

Argentina, Chile, Malaysia, Vietnam, Thailand, Indonesia, Uruguay, UAE, Taiwan, Peru, Philippines, Turkey, Columbia and India

Steviol glycosides were approved for use as sweetener in food after the FDA’s approval in 2008

1.4 Physicochemical and biological properties of steviol glycosides

The sweetness potency of stevioside has been rated to be 300 times the relative sweetness intensity of 0.4% sucrose solution. The compound exhibits a slightly menthol-like bitter after-taste (Bakal and Nabors 1986). The sweetness intensities (i.e. sweetening power relative to sucrose, which is taken as 1) of the other S. rebaudiana sweet components have been determined as: dulcoside A, 50–120; rebaudioside A, 250–450; rebaudioside B, 300–350; rebaudioside C (previously known as dulcoside B), 50–120; rebaudioside D, 250–450; rebaudioside E, 150–300; and steviolbioside, 100–125 (Crammer and Ikan 1987). Also, stevioside has been found to be synergistic with aspartame, acesulfame-K and cyclamate, but not with saccharin (Bakal and Nabors 1986).

Table 1.6 Physical properties of steviol glycosides present in S. rebaudiana

The solubility of stevioside in aqueous systems is fairly low but the second most abundant component in S. rebaudiana leaves, rebaudioside A, which has a more pleasant taste than stevioside, is 6–7 times more soluble in water, since it contains an additional glucose unit in its molecule (Kinghorn and Soejarto 1991; Kohda et al. 1976). Table 1.6 details the physical properties and chemical index for all the glycosides.