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An improvised process of isolation, purification

of steviosidesfrom Stevia rebaudiana Bertoni
leaves and its biological activity

Article in International Journal of Food Science & Technology June 2012

DOI: 10.1111/j.1365-2621.2012.03134.x

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International Journal of Food Science and Technology 2012 1

Original article
An improvised process of isolation, purification of steviosides
from Stevia rebaudiana Bertoni leaves and its biological activity

Adari B. Rao1*, Goka R. Reddy1, Prasad Ernala1, Sundergopal Sridhar2 & Yerrapragada V. L. Ravikumar2
1 Organic Chemistry Division, Indian Institute of Chemical Technology, Hyderabad-7, India
2 Chemical Engineering Division, Indian Institute of Chemical Technology, Hyderabad-7, India
(Received 18 January 2012; Accepted in revised form 2 June 2012)

Summary The food industry has traditionally used sugar (sucrose) as a sweetening agent; however the dietary and
health demands are continuing to expand the market for sweeteners as an alternative to sucrose. The
leaves of Stevia rebaudiana are rich source of glycosides that have sweet taste with low caloric value.
The study highlights extraction of steviosides from leaves using pressurised hot water extractor,
followed by purication and concentration of the sweet glycosides through ultra (UF) and nano (NF)
ltration membrane in obtaining high purity steviosides. After the nal purication process the stevio-
side content was 9.05 g per 100 g and rebaudioside A 0.2 g per 100 g stevia leaf, with total purity of
stevioside 97.66% by HPLC at total operation time of 7 h. This process also improved the potency of
sweetness and palatability prole when compare with other commercially available steviosides. Thus,
the methodology developed establishes simple, in-expensive and eco-friendly process in obtaining pure
steviosides.
Keywords After-taste bitterness, antioxidant, membrane-ltration, natural-sweetener, stevio-glycosides.

The steviol-glycosides (steviosides) are diterpene

Introduction
Sweetness is one of the six basic taste sensations
known in humans for centuries. Natural sweeteners
such as honey, table sugar, maple sugar etc., we eat
and drink, consist of primarily sucrose, glucose and
fructose (Crammer & Ikan, 1977). The major
drawback of these natural sweeteners is, they are
known for high caloric value, which is undesirable to
health and body weight of the humans. The increase
in the number of diabetic patients and health
conscious individuals has pushed forward the need for
alternative sweeteners of low caloric value. There are
many synthetic and natural sources of sweeteners
available in the market for example saccharin,
aspartame, cyclamates etc., the use of some of these
sweeteners are prohibited or limited as they have
shown disadvantageous/toxic eects in clinical trials
(Weihrauch & Diehl, 2004; Anton et al., 2010). Thus,
there is a continuing search for non-nutritive, high
intensity sweeteners as an alternate to sucrose, with
non-toxic nature and low caloric value.
*Correspondent: Fax: +91 040 27160512;
e-mail: adarirao2002@yahoo.co.in
Indianpatent No: 0033NF dated 09-02-2011.
glycosides known as natural sweeteners obtained from
the leaves of Stevia rebaudiana Bertoni (family
Asteraceae) commonly called as sweet herb and
these glycosides are also known as sweeteners of the
future (Fig. S1) (Esmat et al., 2010; Brahmachari
et al., 2011; Lemus-Mondaca et al., 2012). Steviosides
are 300 times sweeter than sucrose and are widely
used in food, beverage, medicine, wine making,
cosmetics, household chemical industry and other
food industries (Gregersen et al., 2004; Chatsudthi-
pong & Muanprasat, 2009; Wolwer-Rieck et al.,
2010; Stoyanova et al., 2011). The stevia leaves accu-
mulate a mixture of at least eight dierent steviol gly-
cosides and the patterns of glycosylation heavily
inuence the taste perception of these intensely sweet
compounds. Despite the wide therapeutic application
of these sweet glycosides, the human consumption of
these steviosides are restricted because of its after
taste bitterness, astringency and grassy taste. This
may be because of the presence of saccharides,
non-glycosidic and un-identied diterpene/alkaloids
present in the leaf extracts, thus limiting the applica-
tion of steviosides in food and pharmaceutical indus-
tries (JECFA, 2007). The edulcorant properties of the
steviol-glycosides dier from one another in terms of

doi:10.1111/j.1365-2621.2012.03134.x
2012 The Authors. International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
2 Steviosides sweeteners membrane technology A. B. Rao et al.
sweetness and quality of the taste, therefore, the
organoleptic property of the steviosides depend on
the ration of the steviosides present on dierent
extraction and purication methodologies. There is
abundant published/patented literature available with
respect to extraction and isolation of steviosides from
the stevia leaves. (Giovanetto, 1990; Choi et al., 2002;
Pol et al., 2007; Jaitak et al., 2009; Huang et al.,
2010; Teo et al., 2010; Puri et al., 2012). Traditional
procedures of extraction of natural products from
plant or microbial fermentation process involve sol-
vent/enzymatic/supercritical uid methods followed by
purication of the active constituents involve column/
liquid-liquid/ion-exchange chromatography, UF/NF
membrane ltration, fractional crystallisation/distilla-
tion process. Despite remarkable advancement in
extraction and purication of natural products, it is
still a challenging task in isolation of water soluble
compounds from plants without impurities. The exist-
ing process of extraction of steviosides from leaves of
S. rebaudiana Bertoni involve many conventional pro-
cess (chemical/physical) including long procedures in
isolation and ion exchange purication steps, thus
leaving noxious residues and impurities in the nal
product, which are responsible for the quality and
taste of the sweet glycosides (Kutowy et al., 1999;
Zhang et al., 2000; Midmore & Rank, 2006; Silva
et al., 2007; JECFA, 2007; Abelyan et al., 2010).
Attempts were made to improve the quality of the
sweetness of stevioside by adding at least one natural
sweetener for example sucrose, glucose and fructose
by glycosylation of steviosides, or by supercritical
uid extraction or through UF/NF membrane separa-
tion to obtain high purity steviosides of better taste
and quality. (Fuh & Chiang, 1990; Tanaka, 1997;
Zhang et al., 2000; Yoda et al., 2003 Li & Chase,
2010; Chhaya et al., 2012). The main object of our
study is to develop a simple, inexpensive process of
isolation of steviosides by minimising unit operations
and further additional improvement of the taste pro-
le of the nal product of the stevio- glycosides.
Materials and methods
Materials and chemicals
Leaves: S. rebaudiana Bertoni leaves were obtained
from Sigma-Aldrich Chemicals, St.Louis, MO, USA
S5381-batch #100F0417. The standard stevioside 97%
(HPLC) was purchased from Sigma chemicals. All the
chemicals and solvents used in the studies were of
commercial grade and were glass distilled before use.
The hollow bre ultraltration (HF-UF) membrane of
30 000 molecular weight cut o (MWCO) the length
of spiral wound module was twenty-one inches and
diameter of 2.5 inches. The hydrophilised polyamide
International Journal of Food Science and Technology 2012
International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
(HPA) nanoltration (NF) membrane of 200250
MWCO and each eective area 2.5 m2 used in the
study were procured from M/s Permionics Membranes
India Ltd. (Vadodara, India).
Analysis
The purity of steviosides and related steviol-glycosides
isolated from the crude extract of stevia, were quanti-
ed by HPLC and structure of the puried stevioside
were conrmed by 1HNMR and LC-Mass spectral
studies and compared with standard stevioside.
HPLC analysis
HPLC analyses were carried out using Waters spheri-
sorb NH2 analytical column (250 9 4.6 mm, particle
size 5 lM), elution solvent used was acetonitrile:
0.1 mM Na-phosphate buer pH-5 (80:20) in isocratic
mode; with ow rate of 1.0 mL min 1, column
temperature maintained at 2728 C and UV detection
was adjusted at 210 nm. (Gardana et al., 2010). In
LC-Mass analysis isocratic solvent system acetonitrile:
0.1 mM Na-phosphate buer pH-5 (60:40) with ow
rate of 1.0 mL min 1 was used.
Isolation and purication of steviosides
The air dried leaves of S. rebaudiana (5 kg) were
initially treated/soaked in hexane to remove unwanted
colour pigments and other waxy materials present on
the surface of stevia leaves. Later the dried leaves were
crushed to obtain ne leaf powder ranging between 20
and 30 mm mesh sizes. All the experimental parame-
ters represented in the study such as pH, temperature,
pressure, duration and UF/NF membrane operating
conditions were selected based on the optimisation
experiments carried out in isolation of steviosides pres-
ent from S. rebaudiana Bertoni leaves. The powdered
leafy material soaked in aqueous water solution pH 5,
maintained at 80 C, with agitation for 23 h (leaf:
water ratio being 1:10), later the extract was subjected
to pressurised hot water extractor (PHWE) working
under optimised operating conditions of 100 kPa pres-
sure, 120 rpm, temperatures of 100110 C for
10 min, for extraction of steviosides. The soaking of
dried plant material in minimum amount of water
would have softened the plant tissue, which further
facilitated easy extraction of steviosides on PHWE
process. On completion of the water extraction pro-
cess, the crude extract was initially cloth ltered and
later the ltered solution was passed through spiral
wound ultraltration (UF) membrane of 30 kDa,
molecular weight cut-o (MWCO), operating at a feed
pressure of 200500 kPa, at room temperature, to
remove leaf cell debris and other unknown impurities.
This process helps in clarication of the crude leaf
2012 The Authors

extract and in obtaining a clear solution (straw) con-
taining rich in sweet stevio-glycosides. The UF mem-
brane unit operation results in retaining leaf
carotenoid pigments (7080%); quantied through
spectrophotometer by absorbing optical density at
446 nm, as described in AOAC, 2000. (Horwitz,
2000). Thus obtained ltrate was passed through NF
membrane namely HPA membrane with a MWCO
ranging from 200250 kDa, operated at a trans mem-
brane pressure of 1500 kPa at room temperature. This
process removed 8090% of water as permeate, thus
concentrating the product steviosides in the retentate.
The aqueous retentate containing steviosides were
extracted thrice with organic solvent butanol. The
organic layer was separated and washed with basic/
neutral aqueous solution to remove molecular impuri-
ties and solvent medium was ltered through active
animal charcoal and celite to obtain golden yellow
colour solution containing steviosides, which on con-
centration and crystallisation by (polar solvent) etha-
nol gave a white stevioside powder. For comparison
of the extraction process between simple aqueous
extraction process and UF/NF membrane ltration
process, the reference experiment using simple aqueous
solution followed by solvent extraction of the stevia
leaves has been carried out to obtain steviosides
(experiment-1).
The concentration of sweet glycosides stevioside and
rebaudioside present during the extraction and
purication process was calculated by HPLC. The
structure of steviosides were conrmed by NMR and
mass spectral studies (LC-Mass chromatogram) and
compared with standard stevioside.
Ultra and nano membrane ltration procedures
The purication of steviosides present in the crude leaf
extract was carried out by UF/NF membrane ltration
studies. The ultralltration membrane HF-UF 30 kDa
made up of hydrophilised version of polyether sulfone
was used in the study, the membrane gave high ux
without compromising the quality of the clarication
of the permeate (Vanneste et al., 2011). The crude ste-
via extract (30 L) obtained on PHWE was passed
through UF membrane for clarication of feed and
stevioside rich permeate (23 L) was recovered. A per-
meate enriched in the stevio-glycosides (20 L) was feed
to HPA- 250 NF membrane to collect enrich stevioside
in the reject and the permeate (12 L) free from sweet
glycosides. Thus, the principle followed in both UF
and NF membrane ltration was the same except that
of the enriched terpenoid glycosides was present in the
permeate of UF membrane, whereas in case of NF
membrane ltration the permeate was concentrated to
obtain reject containing enriched steviosides and per-
meate free of stevioside. The enriched stevioside reject
was extracted with organic solvent, followed by
2012 The Authors
International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
Steviosides sweeteners membrane technology A. B. Rao et al. 3
crystallisation of stevio- glycoside by adding polar sol-
vent (ethanol).
The eect of pressure varying from 1350 to
4100 kPa in relation of concentration of stevioside was
studied. In UF and NF membrane ltration processes,
the separation and performance of the membrane was
calculated as described in our earlier studies (Sridhar
et al., 2000).
Sensory (taste) evaluation
On conformation of the absolute purity of the isolated
stevioside (9098%) and also in absence of residual
solvents, the isolated steviosides were taken up for
taste sensory evaluation studies. The tasting panels
consisting of ten human volunteers ve men and ve
women in an age group between 20 and 25 years were
volunteered in this study and to compare the relative
sweetness, standard sucrose was taken for calculation.
Each volunteer was advised to taste it thrice in an
hour to get mean reading of the same, after every
expectoration the members were asked to rinse oral
cavity with sucient water to avoid overlapping of
tasting sense of the tongue. On the basis of their
mouth feel eect, felt by the volunteers, they were
asked to rate the sample from 0 to 6. Points were
allotted for each taste, like sweet, bitter, palatable and
also its avour (DuBois et al., 1981; Cardello et al.,
1999).
Determination of antioxidant activity
Free radical scavenging activity was carried out by
DPPH (2, 2-Diphenyl-1-picrylhydrazyl) modied
procedure (Lu et al., 2011). Standard quantities of
steviosides were added to 3 mL tubes containing
solution of 6 9 10 5 M DPPH, after incubating for
30 min the decrease in the absorbance at 520 nm was
measured against blank. The concentrations of stevio-
sides required for 50% inhibition of free radical (IC-
50) values were calculated to determine the antioxidant
potential of the puried steviosides.
Statistical analysis
All tests were conducted in triplicates (n = 3) and data
were expressed as means with standard deviations. The
P-values were calculated and a signicant dierence
was considered at P < 0.01. (version 11.5 for windows;
SPSS Inc., Chicago, IL, USA).
Results and discussions
The study highlights the development of a simple and
eective methodology in extraction of sweet glyco-
sides, steviosides and rebaudioside A, from S. rebau-
diana leaves. The process involves combined use of
aqueous extraction using autoclave (PHWE), followed
International Journal of Food Science and Technology 2012
4 Steviosides sweeteners membrane technology A. B. Rao et al.
by UF and NF to obtain high quality steviosides
from the leaves of stevia. The initial treatment of ste-
via leaves with hexane helps in removing plant pig-
ments (carotenoids) and wax material present on
surface of the plant leaf material; this step determines
in subsequent purication process of steviosides.
(JECFA, 2007) Total leaf pigments (carotenoids)
extracted from dry stevia leaves was found to be 19
22 mg per 100 g of dry stevia leaves.(Schertz, 1928)
The stevia leaves were powdered and subjected to
water extraction in a pressure reactor to obtain stevi-
oside rich extract. Thus, obtained crude steviol leaf
extract was passed through to UF/NF multi stage
membrane purication process, followed by n-butanol
extraction and purication to remove suspended par-
ticles, colour pigments and high molecular weight
impurities and unidentied bitter taste glycosides/
alkaloids as conrmed by HPLC chromatogram and
NMR spectral studies. The total operation time of
7 h taken for complete process of extraction and
purication of steviosides in obtaining total isolated
stevioside yield was 9.05 g per 100 g of stevia leaves
from 5 kg S. rebaudiana. (Lit. reported yields 2.84 g
per 100 g Huang et al., 2010; 10.5 g per 100 g
Zuoqing et al. 1995; Chhaya et al., 2011).
The UF process removed more than 90% of the
plant pigments present in the stevia extract where as
by nanolltration more than 90 lg mL 1 of steviosides
in the retentate with product purity of 8090% and
permeate free of stevioside (Table S1).The membrane
ltration has shown reasonably high permeation rates
and no signicant fouling of UF/NF membranes was
observed during the process of ltration. The obtained
aqueous retentate containing steviosides was extracted
with butanol and the separated organic layer was
washed with alkaline/neutral aqueous solution to
remove high molecular impurities. The butanol solu-
tion containing steviol glycosides was concentrated
and crystallised by adding ethanol to obtain pure
white product of stevio-glycosides. The conceptual
ow diagram Fig. S2 shows the improvised process of
isolation and purication process of steviosides with
recovery yield of sweet glycosides in the range of 85
98%, of which steviosides-A (8595%) and rebaudio-
side-A (38%) and the purity and the chemical
structure of the stevioside conrmed through HPLC,
LC-Mass and NMR spectral analysis and compared
with literature data. (Kedik et al., 2003; Chaturvadula
et al., 2011; Puri et al., 2012) The melting point of the
isolated white stevioside powder was 197199 C and
observed spectral data of the product was 1H NMR
(300 MHz) d0.98 (s, 3H, C20CH3), 1.21 (s, 3HC18-
CH3), 4.86 (S, 1H, C17-H) 5.21 (S, 1H, C17-H) 4.59
(D, J = 7.9 Hz, 1H) 5.38 (D, J = 7.9 Hz, 1H);
+ESIMS (M + Na) + m/z 827.3891 (calcd. for
C38H60O18Na:827.3891). The Fig. S3ac shows the
International Journal of Food Science and Technology 2012
International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
HPLC chromatogram prole of the stevioside isolated
in dierent extraction steps. The purity of the product
stevioside and rebaudioside-A was conrmed by stan-
dard samples. The chromatogram Fig. S3a,b depicts
the HPLC prole showing stevioside and rebaudioside-
A of step-1 and step-2 where as the Fig. S3c show the
improvement of the purity of steviosides of the nal
product where less number impurities; this is further
conrmed by LC- mass and NMR studies as shown in
Fig. S4S6. The step wise extraction and isolation pro-
cess of stevioside from the leaves of S. rebaudiana
show that the initial concentration stevioside and re-
baudioside A was 10.9 g per 100 g and 2.6 g per 100 g
present in the stevia leaves respectively, whereas on
nal purication process the stevioside content was
9.05 g per 100 g and rebaudioside A was 0.2 g per
100 g with total purity of stevioside 97.66% by HPLC
(Table S3).
The results of the sensory perception show
(Table S2), that the selective multistage membrane
ltration of stevia extract improved sweetening
potency and taste prole when compared with stan-
dard steviosides. This may be because of the removal
or minimised alkaloids and other impurities of bitter
taste present in the steviol extract by UF/NF mem-
brane purication process (Zhang et al., 2000; JECFA,
2007). It is also well known that the stevia extracts
promote wide range of physiological activities like
cardiovascular, anti cancer, hyperglycemia and also
inuence in reducing hypertension. These pharmaco-
logical activities were known to correlate with
antioxidant activity (scavenging super oxide radical) of
the glycosides present in the extract of stevia. The
antioxidant activity of the steviosides show the
concentration required for 50% inhibition (IC-50) of
DPPH radical was calculated. From the results it was
observed that the crude stevia has shown IC-50.
135 2.5 mg g 1, whereas IC-50 values of the
isolated pure steviosides obtained from leaves of the
stevia was 75 3.2 lg mL 1 *extraction -1, and
21 1.3 lg mL 1 for **extraction-2 respectively,
whereas the standard IC-50 values for stevioside
obtained from sigma was 24 2.2 lg mL 1 and
standard BHA (butylated hydroxyanisole) has shown
47 3.5 lg mL 1 of antioxidant activity.
A mixture of diterpene glycosides extracted from S.
rebaudiana leaves by dierent extraction methods
known to contain the impurities (alkaloids and
glycosides) that are responsible for after taste and palat-
ability of the product steviosides. To remove the impuri-
ties present in the stevioside extract was passed through
ion exchange resins or adsorbent columns to remove the
impurities and to improve the yield and purity of the
steviosides, this process will not only increase the pro-
cessing time, no improvement in palatability was
observed and also aect the overall cost of production
2012 The Authors

of steviosides. (Toru, 1982; Fuh & Chiang, 1990; Giova-
netto, 1990; Payzant et al., 1999; Jonnala et al., 2006;
JECFA, 2007; Mondal et al., 2012).
This study describes a simple extraction process to
remove leaf colour pigments, cell debris and metal che-
lates from the crude leaf extract from the leaves of the
stevia by minimum solvent separation (hexane and
butanol) and in addition the use of UF and NF
membrane ltration of the stevia extract also helps in
removing the impurities of high molecular weight
compounds and some non- glycoside impurities. The
main advantage of this process is production of an
improvised and palatable stevioside by minimum
operation conditions and time taken for extraction of
steviosides on eco-friendly environment.
The membrane purification process of stevioside
Eect of feed (stevia extract) concentration
The eect of feed concentration on performance of
UF and NF membranes was displayed in Figs S7S9.
It can be observed from Fig. S7, that the ux reduced
from 30 to 7.5 L m 2 h 1; at the time of UF opera-
tion increased from 10 to 100 min. The decrease in
ux could be attributed to concentration polarisation
of solute molecules including sugar, chlorophyll, bio-
mass etc. at the membrane surface. However, in the
permeate concentration of stevioside increased from 55
to 126 lg mL 1, as more and more stevioside mole-
cules are available for permeation. The ultra ltration
membrane used in the study was HFUF membrane of
30 000 MWCO, which have anity towards low
molecular weight molecules, containing polar groups.
In the process of UF, an enrichment of steviosides in
permeate was observed because of preferential anity
brought about through hydrogen bonding and polar
interactions. The Fig. S8, highlights the rate of con-
centration of stevioside in feed on increase in permeate
conductivities time from 1.97 to 5.65 and 0.25 to
1.42 mS cm 1 respectively. This meant that the solutes
including part of the salts and most of the steviosides
would get concentrated because of the smaller pore
size of the NF membrane (0.52 nm) which has a
MWCO of 250. The increase in permeate conductivity
was attributed to an increase in the osmotic pressure
of the feed solution with time (Murthy et al., 2005).
The NF membrane purication resulted in increase
concentration of steviosides from 2 to 46 lg mL 1
proportionally with that of time taken for NF (Fig. S9)
and no steviosides in permeate was observed through-
out the study because of its higher molecular weight.
Eect of pressure
Results show increase of feed pressure by NF
membrane ltration, as observed from Fig. S10 pressure
vs. conductivity studies; the rise in feed pressure was
2012 The Authors
International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
Steviosides sweeteners membrane technology A. B. Rao et al. 5
13504100 kPa, and proportional increase in ux was
from 25.2 to 39.3 L m 2 h 1, whereas the permeate
conductivity reduced from 0.48 to 0.26 mS cm 1. The
recovery of water was constantly maintained at 60%
for all the operating pressures. The increase in pressure
would obviously exert greater driving force for
transport of the water molecules leading to a rise in
ux. Owing to solution diusion mechanism, the
hydrophilic NF membrane allows more and more
water molecules to pass through because of preferen-
tial sorption of water on the membrane surface. In
addition, water molecules have smaller molecular size
(MW = 18) and would therefore exhibit higher
diusivity compared with the much larger stevioside
molecules. Thus, the rejection of stevioside remains
100% throughout the range of feed pressure studies.
Membrane fouling and its control
The membrane would be especially prone to biological
fouling as it gathers fungal growth on storing. (Van
der Bruggen et al., 2008). The UF and NF membrane
used for clarication and purication of stevioside did
not show any membrane fouling during the process of
ltration and the membrane was nally stored in a 1%
(w/v) sodium metabisulte solution to prevent biologi-
cal fouling. The study establishes a simple advanta-
geous process in isolation and purication of
steviosides using UF and NF membranes operating
with higher ux and ow rates, at room temperatures.
The membrane show longer half life, without mem-
brane fouling when compared with other existing
methods available. Thus, the improvised process con-
rms the isolation of high purity steviosides free from
impurities of bitterness, enhanced palatability and anti
oxidant activity, in an eco-friendly process.
Conclusions
Sugar consumption may be one of the dietary causes
of metabolic disorders, such as obesity. Therefore,
substituting sugar with low calorie sweeteners like
stevioside from the leaves of stevia plant may be an
ecacious weight management strategy. The study
established a simple process in isolation of high purity
sweet glycosides using inexpensive membranes in
removing colour pigments, high molecular impurities
and obnoxious residues. An enrichment of the
steviosides could be obtained in permeate during
clarication by UF process which may have signicant
anity for stevia leading to its preferential separation
of stevioside (65.33%) into the permeate leaving high
molecular impurities in retentate. Whereas in the NF
membrane ltration 8090% of the water along with
alkaloids was ltered leaving stevioside 84.04% rich
retentate. There were no losses of steviosides in
permeate, and membrane fouling was observed during
International Journal of Food Science and Technology 2012
6 Steviosides sweeteners membrane technology A. B. Rao et al.
the NF membrane ltration process indicating 100%
rejection of the glycosides which meant that most of
the stevio-glycosides that was extracted from stevia
leaves could be recovered. On nal purication process
through organic/aqueous washings, the purity of the
stevioside was increased to 97.66% with total yield
steviosides was 9.05 g per 100 g stevia leaves. Thus,
the methodology developed has established a simple
multi-stage membrane based separation and purica-
tion process producing highly puried steviol
glycosides with improved taste, palatability and
antioxidant activity from the extracts of S. rebaudiana
in a viable, economical and eco-friendly process, this
methodology can be extended for commercial scale
production of steviosides.
Acknowledgments
The authors are grateful for the nancial support by
Department of the Biotechnology, (DBT) New Delhi,
Author G R R express sincere thanks to DBT for
awarding the research fellowship.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Structures of steviosides and related sweet
glycosides.
2012 The Authors
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Steviosides sweeteners membrane technology A. B. Rao et al. 7
Figure S2. Flow diagram shows clarication and
concentration of stevioside extract processes.
Figure S3. (A) HPLC spectra-after UF membrane
ltration. (B) HPLC spectra-after NF membrane ltra-
tion. (C) HPLC spectra-after crystallization.
Figure S4. ESI-Mass spectra-after NF membrane l-
tration.
Figure S5. ESI-LC Mass spectra- pure stevioside iso-
lated.
Figure S6. NMR spectra-pure stevioside isolated.
Figure S7. Variation of ux and permeate stevioside
concentration by ultraltration membrane (values
expressed were average of three determinations).
Figure S8. Variation of concentrate and permeate
conductivities with time by nanoltration (values
expressed were average of three determinations).
Figure S9. Variation of ux and stevioside concen-
tration in reject stream with time by nanoltration
membrane (values expressed were average of three
determinations).
Figure S10. Eects of feed pressure on permeate ux
and conductivity at constant water recovery by nano-
ltration membrane (values expressed were average of
three determinations).
Table S1. Comparative performance of UF and NF
membranes in concentration and purication of stevio-
sides (7-8%) present in Stevia rebaudiana.
Table S2. Comparative sweetness of steviosides gly-
cosides with sucrose in human volunteers.
Table S3. Step wise purity and yields of steviosides
isolated from the leaves of Stevia rebaudiana Bertoni.
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