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CHAPTER 1
INTRODUCTION
1.1 PROPERTIES OF STEVIOL GLYCOSIDES
1.1.1 Physical properties of Steviol Glycosides
The herb Stevia rebaudiana (bertoni) accumulates several sweet
tasting diterpene glycosides in its leaves. The commercial importance of
Stevia rebaudiana stems from the use of these glycosides as sugar substitutes.
There are eight known glycosides in the plant (Figure 1.1) (Kennelly 2002;
Starrat et al 2002).
Steviol glycosides are high-intensity sweeteners ranging from 50-
300 times sweeter than sugar, with low water solubility and high melting
points (Table 1.1) (Crammer and Ikan 1987).
These molecules in solution are highly stable at broad pH and
temperatures. Prolonged heating of a stevioside solution results in a decrease
by 16.7 % after 12 hours at 100 °C in neutral condition and 46.0-53.8 % after
4 hours in acidic environment (Chang and Cook 1983). Significant decrease
in stevioside concentration at 80°C was found only at highly acidic conditions
(pH 1) in solution and at 140°C as a solid (Th.Kroyer 1999).
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Figure 1.1 The structure of the steviol glycoside backbonePosition 19 and 13 are marked on the backbone. R1, except in the case of
rebaudioside B is a glucose unit attached to the carboxyl group. R2 contains
various numbers of glucose residues that are attached to C13 depending
upon the specific glycoside.
Table 1.1 Physical properties of different steviol glycosides The molecular weight, melting point and solubility in water of the different
steviol glycosides are given. RS of the molecules is with respect to a 0.4% w/v
solution of sucrose.
Compound Molecular weight
Melting point (0C)
Solubility in water (%)
RS (Relative Sweetness)
Stevioside 804 196-198 0.13 143
Rebaudioside A 966 242-244 0.80 242
Rebaudioside B 804 193-195 0.10 300
Rebaudioside C 958 215-217 0.21 50
Rebaudioside D 1128 283-286 1.00 221
Rebaudioside E 966 205-207 1.70 174
Steviolbioside 642 188-192 0.03 125
Dulcoside A 788 193-195 0.58 50
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The advantages of stevioside as a dietary supplement for human
subjects are manifold: it is stable, it is non-calorific, it maintains good dental
health by reducing the intake of sugar and could be a good substitute for
diabetic and phenylketonuria (PKU) patients and obese persons (Jan 2003).
The omission of excessively added sugar in the food is beneficial to diabetics
by lowering the blood sugar content. Steviol glycosides are also safe for PKU
patients as no aromatic amino acids are involved. Obese persons might lose
weight by the fact that excessive sugar in the food is replaced by Stevia.
Omitting the added sucrose in foods increases the relative proportion of
polymeric carbohydrates. This has a beneficial effect for balanced food intake
and for human health.
1.1.2 Stevioside Biosynthesis pathway
Steviol glycosides have the ent-kaurene backbone similar to the
Gibberellic acids (GA). With glycosylation at C13 and C19, the different
steviol glycosides differ in the number of saccharide units added at C19
position. The saccharide units are added by various glycosyltransferases
(Shibata et al 1991; Richman et al 2005). The precursor of the backbone,
steviol, which was originally thought to be synthesized by the mevalonate
pathway, was later proved to be synthesized by the Methyl-Erythritol-
Phosphate (MEP) pathway (Totte et al 2000). The end product of the MEP
pathway, Iso-Pentenyl Di-Phosphate (IPP) is converted to Geranyl Geranyl
Di-Phosphate (GGDP) before being cyclised into Copalyl Di-Phosphate
(CDP). The CDP is then converted to kaurene by Kaurene synthase (KS).
Both CDP synthase (CDPS) and Kaurene synthase have been characterized in
stevia (Richman et al 1999). Kaurene oxidase (KO), a cyt-p450
Monoxygenase, then converts kaurene to Kaurenoic acid(Bennett et al 1967).
Both KS and KO are duplicated in the stevia genome (Humphrey et al 2006).
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This overexpression of the KS and KO genes should cause an increase in flux
through the GA biosynthesis pathway, but stevia does not have increased
concentrations of either GA or Kaurenoic acid in its tissues.
Spatial and temporal separation of the key enzymes in steviol
biosynthesis from the GA biosynthetic pathway is one reason for keeping the
GA concentrations under check (Davidson et al 2005). Work on duplicating
the CPS and KS genes in Arabidopsis thaliana also proved that regulation of
GA concentrations in tissues was further downstream from these enzymes, at
the dioxygenase, which is the first committed step to GA biosynthesis (Fleet
et al 2003). Arabidopsis was able to accumulate surprisingly high
concentrations of the Kaurenoic acid without any ill effects.
In stevia, increased flux of Kaurenoic acid could be handled by the
presence of the sink pathway leading to steviol glycoside biosynthesis.
Kaurenoic acid -13-hydroxylase is the first committed step to steviol
glycoside biosynthesis (Figure1.2). At this point GA precursors are
hydroxylated at C7. This enzyme has immense applications in biotechnology
and there is some controversy regarding reported sequences of this enzyme
(Kim et al 1996; Brandle and Telmer 2007).
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Figure 1.2 Stevioside biosynthesis pathway CDPS - Copalyl Di Phosphate Synthase, KS - Kaurene Synthase, KO
- Kaurene Oxidase, KAH - Kaurenoic acid Hydroxylase, UGT -
UDP-Glycosyl Transerase. (Adapted from Brandle and Telmer,
2007)
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1.1.3 Phytochemical constituents of Stevia leaves
The interest in use of the plant for sweetening purposes has lead to
extensive investigation of the constituents of the plant material. The leaves of
the plant accumulate the diterpene glycosides responsible for the sweet taste
and other phytochemicals. The nutritional composition of the leaves was
analyzed and reported by many workers (Savita et al 2004; Dr.Duke’s
phytochemical and ethnobotanical databases, accessed on 22.07.2008;
Tadhani and Subhash 2006).
Stevia leaves have been reported to contain tannins. Tannins are
synthesized in a wide variety of plants and trees (Haslam 1981; Okuda et al
1990; Kumar and Vaithiyanathan 1990) which can accumulate in the wood,
bark, leaves or galls of the plants. Gallo and ellagitannins or mixtures of both,
classified as hydrolysable tannins, are found in stevia leaves. Hydrolysable
tannins consist of central glucose attached to many gallic or ellagic acid units.
Complex reaction products of this core structure leads to many
uncharacterized tannins in plants (Nonaka 1989). There are many methods
available for analysis of tannins (Irene 2001). Analytical methods for tannins
are dependant on sample preparation, storage and extraction techniques.
Mixtures of tannins may be analyzed by general tannin assays such as
precipitation with metals or proteins, and, by colorimetric assays for total
phenols. While colorimetric assays are widely used for tannin analysis, TLC
and HPLC provide more sensitive methods for differentiating and quantifying
tannins. The high polyphenolic concentrations in stevia leaves are significant
because of their sensory properties and anti-oxidant activity (Zhang and Lin
2008; Beninger and Hosfield 2003; Amarowicz et al 2000). Antioxidants are
free radical scavengers, inhibitors of lipid peroxidation and chelating agents.
Phenolic compounds like gallic acid, ellagic acid and their derivatives,
flavonoids and even vitamins are classified as primary anti-oxidants or chain-
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breakers. These compounds act by oxidizing the free radicals which cause
oxidative stress.
Pigmented tannin-like polyphenols cause astringency of wines
(Vidal et al 2004). Tannins also have anti-protein activity but, the major factor
acting against their presence in food is their contribution to taste. Astringency
and bitterness of tannins are a function of their molecular weights, with low
molecular weight tannins being astringent and high molecular weight tannins
being bitter.
1.1.4 Sweetness perception of Steviol glycosides
The sensory attributes of stevia extracts are, therefore, due to a
complex interaction between the various constituents of the leaf. Sweet and
bitter taste properties are found in most chemical classes of sweeteners, and a
close relationship between sweet and bitter tastes is found in many structural
categories (Schiffman et al 1991). A single compound can have both sweet
and bitter qualities. The interdependence of sweet and bitter tastes comes
from psychophysical taste experiments using mixtures containing varying
amounts of sweet and bitter components. This phenomenon called mixture
suppression, suggests a possible relationship between the sweet and bitter
components in a mixture.
Although sweet taste is usually regarded as pleasant and bitter taste
as unpleasant, there is much evidence to suggest that the two tastes are quite
closely related with respect to their transduction mechanisms (DuBios et al
1991). Biochemical studies suggest that the degree of interdependence of
bitter and sweet ratings may be compound specific and related to the
transduction mechanisms involved in inducing the taste sensations (Walters
et al 1991). Generally, with increasing concentration, high-potency
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sweeteners including acesulfame-K, neohesperidin dihydrochalcone, sodium
saccharin, rebaudioside-A and stevioside tend to become bitter. Low-potency
sweeteners including fructose, sucrose, and lactitol tend to become less bitter
with increasing concentration (Schiffman et al 1995).
Sweetness enhancement by odour and colour associations plays a
major role in taste perception (Joseph and Harry 1998). In fruits, ripening is
often associated with a transition from green to red colour, and this often
parallels a major increase in the sugar content of fruits. If green colour in
fruits represents unripeness or a preponderance of acid (sour) taste over
sweetness, then an association of lighter green colour with less acid and
sweeter taste seems reasonable as well. An association of certain acceptable
colours with foods is thought to begin early in our cognitive development and
to stay with us as we age. It is also possible due to a cohort effect that depends
on their specific experiences with some commercial products. For example,
orange coloured soft drinks are associated with acidic taste of orange fruit.
Statistically, colour and odour have a stronger influence on sensory
perception in adults than children. A control over colour and odour thereby
influences sensory perception of specific attributes (Clydesdale 1993).
It is due to the significant contributions of colour and odour on
sweetness perception of stevia, that purification becomes necessary.
Phytochemical synergy is important in determining the health benefits of any
herbal food ingredient. Although the main use of stevia is the sweet taste, the
associated free radical scavenging activity is also important. Furthermore,
processing is an expensive step towards producing a commercially acceptable
sweetener product. When minimal processing is sought to be introduced, the
product must have commercially acceptable organoleptic properties.
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The sweetness of the steviol glycosides varies significantly with
small variations in structure. The magnitude of sweetness and quality of taste
improves according to the number of glucosyl units attached to the C13 to
some extent. Cyclomaltodextrin glucanotransferases (CGTases) were used to
treat stevioside in the presence of starch as a donor. Significant improvements
in the taste profile were observed in the products which were mono and di-
glycosylated at the C13 position (Fukunaga et al 1989). Other factors
affecting taste of the glycosides need to be studied.
1.2 LITERATURE REVIEW
1.2.1 Analytical Methods for Steviol Glycosides
The glycosides of the plant, Stevia rebaudiana, accumulate in all
the photosynthesizing parts of the plant. In wild cultivars, stevioside
accumulates between 8-10% on a dry weight basis in the leaves and
rebaudioside A between 1 and 3%. Selective breeding has developed many
varieties with altered steviol glycoside composition. Many analytical
techniques have also been developed for detection and quantification of these
glycosides in plant and food samples.
1.2.1.1 Thin Layer Chromatography
TLC-colorimetric methods were among the earliest to be
standardized and reported for steviol glycosides (Metivier and Viana 1979).
Silica gel (SG60) chromatoplates were used for TLC analyses of Stevia
sweeteners with isopropanol: ethyl acetate: acetone: water (30:53:2:15, v/v) as
mobile phase and orcinol 0.5 g% in methanol: sulphuric acid (95:5) as
revelator following heating at 10–58 ºC for 2–4 min (Antonio et al, 2005).
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A HPTLC method for steviol glycosides was most recently
described (Jaitak et al 2008). This method utilized pre-coated silica gel
HPTLC 60 F254 plates with a mobile phase consisting of ethyl acetate:
ethanol: water (80:20:12 v/v). Bands were visualized by spraying with acetic
anhydride: sulfuric acid: ethanol (01:01:10, v/v/v) followed by heating on
Camag HPTLC plate heater at 110°C for 2 min. This method provided a
lower detection limit of 80 ng/spot with linearity for quantification between
1-
1.2.1.2 Enzymatic method
An enzymatic method for quantification of stevioside was
standardized (Mizukami et al 1982). This method was based on the hydrolysis
of stevioside by a crude hesperidinase, which releases glucose. The released
glucose was followed by an enzyme based glucose detection system. The
enzyme was able to hydrolyze stevioside efficiently within two hours at 50°C,
but Rebaudioside A took more than 48 hours for complete hydrolysis. Hence,
the assay could be considered specific for stevioside under the conditions
prescribed. The specificity of the enzyme was questioned by the authors,
indicating that the presence of interfering oligosaccharides may give false
results. The advantage of the method was its simplicity and ability to use it in
analyzing large volumes of Stevia rebaudiana leaves.
1.2.1.3 High Performance Liquid Chromatography
High-Performance Liquid Chromatography has been used for
analysis of steviol glycosides. Hutapea et al (1999) developed a method using
a C-18 column with water: acetonitrile linear gradient and measurement at
210 nm. This method was capable of separation and quantitation of steviol
glcosides and their metabolite
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and steviol, found in blood, feces and urine. Kolb et al (2001) used an NH2
column with isocratic acetonitrile/water elution and detection at 210 nm for
separation and quantitation of these glycosides in leaf extracts. Vanek et al
(2001) used a C-18 reverse phase column and a linear gradient of
water/acetonitrile ending in acetonitrile/water in 30 minutes. Detection was at
205 nm with a Photodiode array UV detector. Samples analyzed included leaf
and fruit teas commercially available. The method proposed by Hutapea et al
mL mL. Isocratic elutions are
faster than gradient elutions while providing similar efficiency of separation.
NH2-bonded silica is the most common packing material in the columns used.
The sensitivity and robustness of HPLC methods are their advantage.
Single column HPLC combined with ESI-MS was employed for
optimizing conditions for supercritical extraction of steviosides from the leaf
(Young et al 2002). Negative ion method was found to be more reliable for
stevioside when compared to positive ion and UV mode. Detection limits for
positive and negative ionization modes were 10 ng and 1 ng respectively.
Stevioside also lacks any chromophores hence UV absorption was not
considered a sensitive method for detection.
A two dimensional Liquid Chromatography method combined with
Time of Flight Mass Spectrometry (TOF-MS) was employed for analysis of
leaf extracts (Jaroslav et al 2007). Two dimensional LC provides a high
separation power because of the use of two columns with varying separation
mechanisms. LCxLC combined with ESI-TOF-MS was capable of resolving
the mixture of glycosides and characterizing them. A combination of C-18
and NH2 bonded columns in that order gave the best possible separation.
Single column HPLC was not capable of separating all the eight known
glycosides as reported by these workers.
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1.2.1.4 Capillary Electrophoresis
Capillary electrophoresis (CE) or capillary zone electrophoresis
(CZE) can be used to separate ionic species by their charge and frictional
forces. CE was designed to separate species based on their size to charge ratio
in the interior of a capillary filled with electrolyte. CE offers unparalleled
sensitivity to resolution of analytes with little physical differences. CE in the
micellar mode is a valuable alternative to HPLC. It allows separation of non-
ionic analytes, including plant secondary metabolites. CE was used for the
separation and analysis of steviol glycosides from the plant material (Mauri et
al, 1996). The system used has a linear response to concentrations between
0.2 and 5.0 mg/mL. Although CE is a viable alternative to HPLC in analysis
of steviol glycosides, it is not widely used.
1.2.1.5 Near Infra Red Reflectance Spectroscopy
Near Infrared spectroscopic analysis of stevia leaves for estimating
the content of stevioside in the leaves was proposed (Nishiyama et al 1992).
Near Infrared radiations are capable of analyzing bulk material with little or
no sample preparation due to their ability to penetrate the material deeper. But
analytical techniques based on NIR spectroscopy lack sensitivity. In the
analysis of stevioside, there was an overestimation of the stevioside content.
The proposed method was also sensitive only to the stevioside concentration.
Since NIR spectroscopic analysis was a rapid, high-throughput, non-
destructive technique, it has immense applications in continuous monitoring
during storage and for development of better varieties of the plant. But NIR
spectroscopy has not been widely reported to be used in stevioside analysis.
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1.2.1.6 Advantages of targeting glucose
The proposed assay method targets glucose released from the
glycosides by alkaline hydrolysis. There are multiple methods available for
analysis of glucose in clinical samples. Glucose monitoring is important for
diabetic patients. Hence, methods of assay of glucose have been developed
for increased accuracy, sensitivity, range of sample conditions and robustness.
High throughput automated methods for glucose analysis in plasma and urine
samples have become fairly routine in clinical labs. This ubiquitousness made
glucose an ideal target for assaying when trying to determine total level of
steviosides in the sample. The glucose oxidase based method for assay of
glucose was proposed in 1956 (in Fales 1963). But the peroxide coupling
reaction was reported to be prone to much interference from components
present in biological samples. A hexokinase coupled to Glucose-6-phosphate
hydrogenase system where the reduction of NAD was followed at 340nm was
later proposed and evaluated (Walter et al 1971). This method although
validated to be accurate and reliable is not quite as popular as the Glucose
oxidase based method for clinical samples.
The advantages of the glucose oxidase method are readily available
chemicals, stable solutions, specificity and the ability of the user to adjust the
sensitivity of the reaction over a wide range of glucose concentrations (John
and Kathie 1975). The reagents are not hazardous and are capable of being
adapted to the auto analyzer.
The glucose oxidase based system has been adapted to a sequential
injection system with spectrophotometric detection, where the enzyme is
immobilized in glass beads packed in a micro-column. This method has been
successfully applied to real world samples (Kanchana et al 2007). Sequential
injection analysis coupled with chemiluminscent detection was found to be
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advantageous since the enzyme need not be immobilized (Anastasias et al,
2006) and similarly glucose oxidase also formed the basis of a Lab-on-Chip
system for glucose detection (Vijay et al 2004) The ability to immobilize the
enzyme and couple it to a sensor was used to develop a needle type sensor for
monitoring blood glucose in fish (Hideaki et al 2006). Auto analyzers utilize
an oxygen electrode instead of the coupled dye method in the manual method.
Both methods are linear until 25mM glucose concentration after which it
becomes necessary to dilute the samples. (Violet and John 2000) The
increased sensitivity offered by the enzymatic method ensured that highly
turbid plasma samples could also be analyzed since small volumes of samples
were required. When turbidity is present, it is necessary to appropriately
dilute the samples. Triple enzyme electrodes for simultaneous detection of
glucose, L-Lactate and pyruvate were constructed by immobilizing the
respective enzyme detection systems on platinum disks (Toshio et al 2004).
It is not necessary for pure enzyme to be available. Microbial biosensors
working on the same principle of glucose oxidation have been developed
(Min et al 1997). Some adaptations of the glucose oxidase based method
include amperometric sensing using bilayer electrodes (Naruhide and
Hirokazu 2006).
Many more sensitive methods for analysis of glucose are available.
Post-column derivatisation of glucose by iodine and subsequent LC/MS based
detection have been proposed (Eduard et al 2007). The limit of quantitation
was approximately 50pg on the LC column. Non-enzymatic electrochemical
sensors are popular despite a number of problems including poisoning of the
electrode and irreproducible current responses. Boron doped diamond
electrodes were successfully applied to the detection of glucose at different
concentrations. These electrodes provide long term reliability and are easily
regenerated (Joowook and Su-Moon 2005). Capillary electrophoresis which
was successfully used for stevioside detection has also been applied for
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glucose detection and quantitation (Gang et al 2005). But among all the
systems proposed, enzymatic biosensors have been studied extensively and
adapted for a wide variety of applications. The review of literature suggested
an abundance of glucose assay systems which were sensitive, robust and
widely used.
1.2.1.7 Outline of work
There are both quantitative and qualitative methods available for
analysis of steviol glycosides. TLC provides an efficient method of separating
and identifying the glycosides quickly. The other chromatographic methods,
HPLC, HPTLC, GC and CE provide rapid and sensitive methods for detection
and quantification of the glycosides in plant material and food matrices.
All methods rely on the separation of the glycosides before
individual quantification. When expressing quality of leaf material or
concentration of a mixture of these glycosides in food, the quantities of
individual glycosides are usually added together. The above mentioned
methods utilize external standards of individual glycosides. While trying to
find the total glycoside concentrations in the plant material, all the known
glycosides have to be procured and used as standards. A new method to
overcome the cumbersome method of quantifying using individual standards
was felt.
In addition, leaf material had to be analyzed at source for
determining quality and commercial value. Laboratories equipped with the
expensive and sensitive instrumentation required for analysis of the leaf are
not commonly found in rural areas. Hence, a robust method that would work
on a ubiquitous assay platform would become widely accepted.
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The method outlined in this work addresses both these concerns by
targeting glucose, released from the glycosides by an alkaline hydrolysis step.
The hydrolysis step can be easily performed by using a hot plate and glucose
assay kits which are widely used for routine blood analysis. The ability to
work with interference from other components of the leaf material was
evaluated.
1.2.2 Purification Strategies for Steviol Glycosides
Aqueous and alcoholic extracts of stevia are associated with a
number of impurities, including plant pigments and phenolics which
contribute to colour, odour and taste that are not intrinsic to the molecule.
Brown to yellow colour is contributed by phenolics which also affect the
stability of any formulation made out of a semi-pure extract, since phenolics
are easily oxidisable. Green and yellow colour is contributed by chlorophylls
and xanthophylls which impart a leafy odor and taste to the extract. Odour is
contributed by volatile components which again contribute to herbal or leafy
sensations. Taste is affected by the presence of polyphenolic components.
Low molecular weight phenolics are astringent and high molecular weight
phenolics are bitter. Aqueous extracts of stevia typically contain polymeric
hydrolysable polyphenols contributing to a bitter taste, which acts to amplify
the inherent bitterness of the molecule. Purification was felt to be necessary
due to the above reasons.
1.2.2.1 Fractionation/Ion-exchange/Crystallization
Even though it has low solubility in water, stevioside is soluble in
dioxane, methanol and alcohols up to butanol, insoluble in chloroform,
hexane and other non-polar solvents. Choice of solvent, sequence of usage,
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temperature and pH in the case of aqueous extraction, influence the extraction
efficiency as well as the amount of impurities eluted out.
One of the earlier patents for purification of stevioside (US Patent
No: 3723410) involved defatting of coarsely ground leaves with chloroform
and subsequent extraction with dioxane or water in the presence of an alkali.
In this case the alkali served to precipitate out the phenolic acids by salting
them out of the organic phase. Ion-exchange resins were already being used
for purification of the molecule, the resin of choice being Amberlite IR-120
(Harry et al 1955). In both processes the final step was crystallizing out of the
molecule from cold methanol before recovery. Strong acidic and weak basic
ion exchange resins have been used on a decoction of stevia leaves, followed
by filtration to obtain pure products (US Patent No.: 4892938). This was also
one of the first patents to completely eliminate the use of organic solvents in
the processing.
One major modification in later methods involving solvent
extraction was the use of chelating agents which perform the function of the
ion-exchange resin in removing charged impurities (US Patent No:4599403).
The chelatant of choice was organic di or tricarboxylic acids. Phosphoric acid
was the only mineral acid capable of producing the desired reduction in color.
Phosphoric acid has the added advantage of contributing phosphate ions
which are nutritive when the residual sludge is used as animal feed. The acids
could remove metal ions, proteins and some phenolic acids from the extract.
Calcium oxide and hydroxide served to precipitate out impurities that are not
chelated by acidic precipitation. These include plant pigments contributing to
green and yellow colour. Subsequent fractionation of the molecule into a
water immiscible alkanol gave further purification. Addition of acids and
alkaline salts for clarification provided the advantage of cost, easy waste
handling and simple unit operations.
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Stevioside was reported to have a greater stability in methanol at
temperatures between 1200C and 1400C (Jaroslav et al 2007). In addition, the
authors also concluded that methanol is better for extraction in both static and
pressurized conditions. Water was concluded to provide a “green” alternative.
Supercritical fluid extraction has been proposed as a method increasing the
efficiency of extraction as well as affecting the composition of the final
product by adjusting the ratio of Stevioside and Rebaudioside A (Pasquel
et al 2000). A Japanese patent held by Tan (Japan Patent No.: JP-A-
62-000496) involves the use of acetone along with the aforementioned
solvents and further purification was achieved by adsorption. US Patent No.
5112610 simply described a method for using supercritical CO2 to remove
taste-impairing components without describing them. The gas was employed
in leaves, crude extracts, purified extracts in solid form as well as in liquid
extracts.
1.2.2.2 Adsorption Chromatography
Separation by column chromatography on a silica stationary phase
was easily accomplished. HPLC provided the advantage of greater separation
efficiency and faster flow rates. Derivatised silica, either Octadecylsilane
(Vanek et al 2001), glycerylpropyl (US Patent No: 4361697) or aminopropyl
(Kolb et al 2001), are commonly used. Cross linked starch and styrene gels
have also been used on HPLC systems (US Patent No.: 4171430). HPLC
though has the disadvantage of higher capital and operational costs.
Highly cross linked copolymer of ethyl styrene and divinylbenzene
with about 3% methyl-methacrylate has a high adsorption and desorption
capacity for steviol glycosides. Macroporous strongly basic anion exchange
resins with –N+(CH3)3 has a high decolourisation capacity. Shi et al (2002)
were able to synthesize a bifunctional polymeric adsorbent that combined
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both these activities by introducing the –N+(CH3)3 groups into the AB-8
adsorbent. Steviol glycosides could be desorbed from this polymer by
aqueous ethanol, whereas pigments required that the eluting solvent contain
sodium chloride or hydroxide. Based on the adsorption pattern, the authors
were able to conclude that steviol glycosides interact with the polymer based
on hydrophobic interactions, whereas the plant pigments had much more
complex mechanisms of interaction.
1.2.2.3 Filtration Techniques
Ultra and Nano filtration techniques provide means of separating
the steviol glycosides from the extract of leaves, with minimal use of solvents
and chemicals. There is also less generation of waste. Zhang et al (2000)
described a scheme for separation of the glycosides based on the use of ultra
and nanofiltration units. While pH did not affect the elution of the steviol
glycosides in the first stage of water extraction, lower pH reduced the amount
of coloured impurities. This was the basis of a patent (US Patent
No.:5972120). Suspended particles from the extraction step was removed by a
ceramic micro filter with a mean pore size of 0.3-
membrane with a cut-off size of 2.5-3.0KDa allowed the glycosides to pass
through while retaining the higher molecular weight impurities. Finally
nanofiltration allowed for concentration and removal of impurities which had
lower molecular weights. One additional step of ion-exchange resin based
clarification could allow for the production of high purity steviol glycosides.
A recent permutation of unit operations has allowed for supercritical
extraction to be used in conjunction with nanofiltration for increased
efficiency of separation (Sarrade et al 1998). This could find useful
applications in stevioside purification given our knowledge of supercritical
extraction of the glycosides.
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1.2.2.4 Evaluation of Process Choice
Optimum economic design of a process involves choosing the
process with least cost among many, provided the end results are the same. In
comparing different processes for phytochemical purification, the following
factors have to be considered (Peters and Timmerhaus 1991)
a. Technical – flexibility of process, yields, energy and
equipment requirement, health and safety hazards.
b. Raw materials – present and future availability, handling and
processing requirements.
c. Waste products – amount and value, method of disposal.
d. Location, time and cost evaluations
e. Technology availability.
In the case of extraction of stevia leaves, the end-use of the product
determines the processing method employed. In Japan, stevia based
formulations are mainly used in soy sauce, pickles, dried seafood and miso.
The sweetness of stevia is offset by its residual bitterness. Hence, in such
foods, stevia extracts are mixed with glycyrrhizin, for better sensory
attributes. Purification is not necessary for such an application and most
“stevia extracts” available in the Japanese market are aqueous decoctions
clarified using adsorbent resins (Mizutani and Tanaka, 2002). In Korea, the
major consumers are Soju manufacturers, who use 50% of the total
manufactured stevioside in the country (Kim et al 2002). Soju, a clear liquor,
therefore requires purified steviol glycosides. A laboratory scale process for
producing 63% pure steviol glycosides from stevia leaves was developed at
the Institute for Himalayan Bioresource Technology, Palampur, India.
This technology involves aqueous decoction, filtration and crystallization of
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the glycosides (Megeji et al 2005). Extraction efficiencies of the glycosides as
described in patents, increased with the progress of time (approximately 6.5%
in US Patent No. 3723410, 7.5% in US Patent No. 4599403, 8% in US Patent
No. 4892938). However all the reported processes, except that based on
membrane separation, generate large volumes of sludge, use significant
quantities of chemicals and have complex steps. But the membrane based
process has the disadvantage of increased capital cost including replacement
of fouled membranes periodically and energy costs of operations. The energy
required for evaporation of the extracting solvent plays a significant role in
operating costs. While water is preferred as a cheap, easy to handle and safe
solvent of choice, its latent heat of vaporization is 2257 kJ / kg which is
nearly double that of methanol (1100 kJ / kg). Hence, choice of extraction
solvent determines the energy cost to a large extent. Membrane processes
used for desalination typically provide pure water at 50% of the cost of
distillation units (Malek et al 1996). A survey of processing strategies
employed by companies involved in purification of steviol glycosides reveals
a preference to aqueous extraction and liquid-liquid partitioning for
purification. Recent advances in structure-activity relationship have made the
manufacture of modified glycosides by enzymatic transglycosylation more
lucrative. These products have steviol glycosides with glucose units added on
the C13 for better taste perceptions (Fukunaga et al 1989).
Electrocoagulation for removing the pigments provides an
interesting method of reducing solvent usage to remove chlorophylls
(Jumpatong et al 2006). This process needs further study before it can be used
industrially. Flavonoid components of stevia leaves have been characterized
(Rajbhandari and Roberts 1983; Putieva and Saatov 1997). But tannins, which
comprise nearly 7.8% w/w of stevia leaves, have not been characterized yet.
These tannins contribute substantially to the colour and knowledge of their
chemistry will help in fine tuning the current processing methods available.
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1.2.2.5 Free Radical Scavenging Assays
A number of radical scavenging capacity (RSC) assays have been
established and widely used for the rapid screening and evaluation of novel
antioxidant preparations using peroxyl, hydroxyl, cation ABTS (2,2'-azino-
bis(3-ethylbenzthiazoline-6-sulphonic acid)), peroxide anion, and
2,2-diphenyl-1-picryhydrazyl (DPPH) radicals (Cheng et al 2006). The stable
DPPH and the chemically generated ABTS are still widely utilized in
antioxidant research due to their simple reaction systems, which involve only
the direct reaction between the radical and the antioxidants, and have no other
interference such as enzyme inhibition or the presence of multiple radicals,
although they are not physiologically relevant. In contrast to the chemically
generated ABTS, DPPH may be utilized in aqueous and nonpolar organic
solvents such as benzene and can be used to examine both hydrophilic and
lipophilic antioxidants. The DPPH scavenging capacity assay performed in
organic solvents may evaluate lipophilic antioxidants without any additional
solubilizing agents such as the â-cyclodextrin, required in the oxygen radical
absorbing capacity (ORAC) (Cao et al 1993) and peroxyl radical scavenging
capacity assays, which has been reported to have a strong interference in HO
scavenging capacity estimation. The DPPH scavenging capacity assay is
considered to be a valid and easy colorimetric method for antioxidant
property evaluation. This assay has been successfully utilized for
investigating antioxidant properties of wheat grain and bran, vegetables,
conjugated linoleic acids, herbs, edible seed oils, and flours in several
different solvent systems including ethanol, aqueous acetone, methanol,
aqueous alcohol, and benzene. Hence it was the method of choice in
following the free radical scavenging activity of the stevia extracts.
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1.2.2.6 Outline of Work
Many physical and chemical processes known for separation of
molecules have been employed and standardized for the purification of steviol
glycosides. All of these processes had the objective of complete purification.
Whole extracts of the plant contain components that have similar
solvation properties; hence multiple solvent fractionation steps are required
for purification. Chromatographic mobilities of these molecules are also
similar requiring stringent choice of adsorbents and mobile phases. Membrane
fouling by the organic components of these extracts increases operational
expenses either in pretreatment of the extracts or in replacement of
consumables.
The whole extracts of the plant contain phytochemicals which
possess qualities that are desirable. The polyphenols contribute to the anti-
oxidant activity of the extracts. In most of the foods that they are added to,
this activity is synergistic. Removal of these polyphenols is wasteful.
Hence a method for modifying or removing the pigments, that
cause the aforementioned problems while retaining the advantages of
phytochemical synergy, was needed. Biological systems provide the means to
selectively modify the colour and odour causing components in an extract
containing mixtures of molecules. Systems based on bioremediation are used
in treatment of industrial and municipal effluents. Similar fermentation based
on fungi was employed to evaluate the utility of this process in decolourising
extracts of Stevia rebaudiana.
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The efficiency of decolourising the extract was monitored
spectrophotometrically. Enzymes required for decolourising the extract were
profiled during the fermentation. The addition of dextrose and its influence in
decolourisation of the extract was established. The anti-oxidant activity of the
extract was followed to ensure the objective of retaining the beneficial
properties of the whole extract was achieved.