METHOD OF MANUFACTURING REBAUDIOSIDE A IN HIGH YIELD BY
RECYCLING BY-PRODUCTS PRODUCED FROM MANUFACTURING PROCESS FOR
REBAUDIOSIDE A
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present disclosure relates to a method of manufacturing
Rebaudioside A in a high yield and high purity by reusing by-
products produced from a manufacturing process for Rebaudioside
A. More particularly, the present disclosure provides
Rebaudioside A in a higher yield and higher purity than that of
the prior manufacturing method, by reusing by-products, which
are produced from a crystallization process of Rebaudioside A,
as a raw material.
More particularly, the present disclosure provides a
manufacturing method of Rebaudioside A, which comprises the
steps enhancing the purity of mother liquor of Rebaudioside A
crystals by eliminating materials besides steviol glycosides
such as minerals, ash, and other organic materials contained
therein through re-crystallization; and converting stevioside,
which is a major ingredient, into Rebaudioside A by a conversion
method using a microorganism or enzyme with ß-1,3-glucosyl
transactivation so that it can be reused as a raw material.
DISCUSSION OF THE BACKGROUND
Stevioside (ST), which is contained in a Stevia plant, is a
diterpene glycoside having steviol as aglycon, and besides
stevioside other sweetening ingredients are Rebaudiosides A, C,
D, E, and Dulcoside A. Such sweetening components are different
in their degrees of sweetness, and although a relationship
between sweetness and a characteristic in chemical structure has
not been clearly revealed, it has been known that sweetness and
quality of sweetness are largely affected by a glucose bonding
site of a glycoside, an arrangement of functional groups (in
particular, -OH), and a inter-distance of the arrangement
thereof. Stevioside and Rebaudioside A (ß-1,3-monoglucosyl
stevioside) are high intensity natural sweeteners having
sweetness of about 200 and 250 times higher than that of sugar,
respectively. Stevioside leaves a bit of bitter taste behind,
whereas Rebaudioside A has almost no bitter taste, and thus is
superior in its sweetening properties. Likewise, Rebaudioside
A, which is superior in sweetness and sweetening properties, is
drawing much attention as a high intensity natural sweetener
that can replace a current high intensity synthesized sweetener.
In particular, the US FDA has approved only stevioside that
contains 95% or more of Rebaudioside A as a high intensity
sweetener and that can be used as a food additive since 2008.
Thereafter, large food companies in the U.S.A. are actively
using Rebaudioside A, and Rebaudioside A has already been
commercially available in the trade name of PureVia and TRUVIA.
Among these products, a leading product in the stevia market,
TRUVIA is a product produced by Cargill and Coca-Cola, and
occupies 58% of the stevia market. Yet, the market share of
TRUVIA in the artificial sugar market is only 6%. This is
because the manufacturing cost for TRUVIA is high and TRUVIA
gives of a very unique flavor. Accordingly, many food companies
are making efforts to overcome such problems.
A conventional production process of Rebaudioside A can be
largely divided into two steps. The first step is the step for
obtaining a purified product of steviol glycoside with high
content, wherein the purified steviol glycoside in the first
step is conventionally used as a high intensity sweetener in the
Southeast Asia market. The first step produces the product by
the following processes: extracting a solution containing
steviol glycoside from dry stevia leaves by using a hydrothermal
fluid, ethanol, methanol, or polyalcohols; decolorizing a
pigment and so on that are contained in the extracted solution;
purifying the decolorized solution by desalinization,
microfiltration and adsorbing resin to give steviol glycoside in
a high purity; and spraying and drying, etc. the obtained
steviol glycoside, (see FIG. 1).
However, since a steviol glycoside content ratio in a

product produced from the first step remains the same as that in
the dry stevia leaves raw material, substantially, a Rebaudioside
A content ratio in a final product is as low as 20% or as high
as 60%, and the content ratio in the final product is
dependently varying according to a seed of stevia cultivated as a
raw material and a cultivation condition. Conventionally, a
high purity steviol glycoside product produced as described
above is directly used as a high intensity sweetener as itself,
or as an enzyme treated-product produced by purification through
a glucose-transferring enzymatic reaction.
The second step is a selective isolation and purification
step for enhancing the purity of Rebaudioside A. A high purity
product produced through the second step is conventionally
limitedly produced and sold for the purpose of sales in such as
the U.S.A. and Europe, where only a high purity product is
approved for use as a food additive. Such second step, a
process using a selective fractional crystallization principle
is performed using a high purity product of steviol glycoside
that has been produced (or sold) through the first step as a raw
material. According to a conventional example of the second
step, 50 to 60% of Rebaudioside A as a raw material in a mixed
solution including alcohols (EtOH) is collected through
crystallization preparing 80 to 85% of Rebaudioside A as a
primary crystallization product, and then the 80 to 85% of

Rebaudioside A is dissolved in a solvent having a higher alcohol
(EtOH) content than the above and then crystallized, thereby
finally producing 95% or higher of Rebaudioside A as a high
purity product (see FIG. 1).
A fractional crystallization process has an advantageous
feature of purifying a single material in order to produce a
high purity among other materials having similar properties.
However, it has a physical limitation in that the obtain-ratio
of target product obtainable from one-time crystal collection is
low. In particular, in the case of high purity Rebaudioside A,
due to the characteristic of the typical process using two-time
crystallization as a major process, it is difficult to increase
the common obtain-ratio of Rebaudioside A beyond 50%.
After all, during the process of producing a high purity
Rebaudioside A product as described above, more by-products are
obtained than the target product. The by-products are sold as a
first raw material for enzyme processed products at low product
value, and it can be found that the characteristic of the by-
product is a major factor limiting profitability in the overall
process steps to produce Rebaudioside A beginning with stevis
dry leaves.
In order to overcome such problems, there have been efforts
to cultivate a plant species having a high Rebaudioside A
content so as to produce Rebaudioside A in a great amount, and

seeds that were proven to be partially effective are disclosed
in various prior arts including Korean Patent No. 10-2008-0058236
(Title of the Invention: New Species of Stevia Plant containing
High Content Rebaudioside-A and Cultivation Method Thereof), and
overseas patents including PCT/JP2006/303992, US00PP10562P
(Title of the Invention: Stevia Plant Named "RSIT 94-1306",
US00PP10563P (Title of the Invention: Stevia Plant Named "RSIT
95-166-13", and US00PP10564P (Title of the Invention: Stevia
Plant Named "RSIT 97-751".)
However, a stevia plant requires as long as 5 to 6 months
from sowing to harvesting, and a wide area. In particular, its
yield is dependent upon weather conditions every year, and
production cost is determined according to a cultivation
environment and labor costs, and product quality is not uniform.
Accordingly, there is a limitation in producing a Rebaudioside A
with high content product through plant breeding, in aspects of
production costs, production amount, quality, etc.
Meanwhile, researchers, in particular, some Japanese
researchers performed various studies on an enzyme transferring
technique for increasing the Rebaudioside A content in steviol
glycoside.
The objective of the studies performed by the researchers is
to artificially increase the content of Rebaudioside A having
good sweetening quality and a high degree of perceived sweetness

in steviol glycoside, thereby increasing the added value of a
product. More recently, researchers carried out a study for
increasing the obtain-ratio of Rebaudioside A by processing a
primary raw material that mainly contains stevioside by using an
enzyme transferring technique to increase the obtain-ratio of
Rebaudioside A, and ultimately, to reduce manufacturing cost.
In particular, Dainppon Ink and Chemicals, Inc., has been
granted a patent right for an invention related to such study
results (see US Patent 4590160). The US patent discloses a
process of producing Rebaudioside A, which comprises reacting
stevioside with a ß-1,3-glycosyl sugar compound in an aqueous
solution or an aqueous suspension in the presence of a
microorganism or enzyme having ß-1,3-glycosyl transferring
activity thereby to form Rebaudioside A.
However, the method also does not provide a satisfactory
purity level. Accordingly, there is a need to develop a method
of manufacturing high purity Rebaudioside A.
SUMMARY OF THE INVENTION
The present invention relates to a method of producing
Rebaudioside A in a high yield and high purity, and in
particular, relates to a Rebaudioside A production method in
which by-products produced from a conventional manufacturing
process for Rebaudioside A, particularly, residual by-products as
a mother liquor produced from a fractional crystallization
process are used as a starting material and subjected to a
series of processes to have a reusable level suitable for the
second step production process (high purity Rebaudioside A
production process), and then the resultant by-products are re-
circulated in the production process for Rebaudioside A. In
particular, residual by-products are subjected to a series of
purification processes, and crystallized to have a reusable
level by using an enzyme transferring method so as to
economically increase the Rebaudioside A content.
A prior art has been developed and applied simply to
increase a Rebaudioside A content in a stevia raw material or a
stevioside product whereas the present invention has a technical
feature developing a re-circulation process in order to increase
the obtain-ratio of the manufacturing process and providing a
product with high purity by processing mother liquor by-product
from cyristallization to a reusable level. According to the
present invention, it has been found that it is more effective
in respects of yield and purity to increase a Rebaudioside A
content by using by-products produced from a first fractional
crystallization process for a stevioside product than to
increase a Rebaudioside A content in the stevioside product.
More particularly, method of the present invention comprises
steps purifying a mother liquor produced from crystallization to
increase a purified solution containing a steviol glycoside
content therein to 90% or higher; adding an insoluble ß-1,3
glucan, such as curdlan, to the purified solution; breaking down
a ß-1,3 bond of the ß-1,3 glucan by ß-1, 3-glucanase to give a
glucose; and connecting the glucose with the stevioside
contained in the purified solution by using a specific glucosyl
transferase to produce a Rebaudioside A of which content is 50%
or higher.
A high content Rebaudioside A according to the prevent
invention is prepared from either the product produced by such
steps alone or a mixture of the product and a conventional raw
material.
The inventors of the present invention have reached the
completion of the invention by producing substantially high
purity Rebaudioside A by setting stevioside contained in
residual by-products produced after the extracted Rebaudioside A
was purified and crystallized as an acceptor, and then by
applying a ß-1,3-glucosyl transferase that transfers glucose and
also is an enzyme suitable for producing Rebaudioside A and a ß-
1,3-glucanase for breaking down ß-1,3 glucose contained in a ß-
1,3-glucosyl oligosaccharide compound.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a flowchart illustrating a process of
manufacturing Rebaudioside A, according to an embodiment of the
present invention.
FIG. 2 shows HPLC (Agilent 1200 Series) component analysis
data of a conventional RA 60 product.
FIG. 3 shows HPLC (Agilent 1200 Series) component analysis
data of a sample of by-products used as a raw material in a
method of producing Rebaudioside A according to an embodiment of
the present invention.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
The invention is described more fully hereinafter with
reference to the accompanying drawings, in which exemplary
embodiments of the invention are shown. This invention may,
however, be embodied in many different forms and should not be
construed as limited to the exemplary embodiments set forth
herein. Rather, these exemplary embodiments are provided so
that this disclosure is thorough, and will fully convey the
scope of the invention to those skilled in the art. In the
drawings, the size and relative sizes of layers and regions may
be exaggerated for clarity. Like reference numerals in the
drawings denote like elements.
A method of producing Rebaudioside A in a high yield and
high purity includes:
i) purifying by-products produced when a high purity
steviol glycoside product is purified by fractional
crystallization;
ii) performing an enzyme transferring reaction on the
purified by-products to adjust a Rebaudioside A content in the
by-products to be in a range of 50 weight (wt)% to 60 wt%; and
iii) fractional-crystallizing either the product produced in
step ii) alone or a mixture of the product produced in step ii)
and a high purity steviol glycoside product to produce
Rebaudioside A.
The by-products in step i) may include 80 wt% or higher of
steviol glycoside, preferably 90 wt% or higher of steviol
glycoside. The by-products in step i) may include 40 to 50 wt%
of stevioside.
According to the present invention, the enzyme transferring
reaction is performed on the purified by-products at a
temperature of 50°C for about 5 hours in the presence of a ß-1,3-
glucosyl oligosaccharide compound, a ß-1,3-glucanase capable of
breaking down (3-1,3 glucose in the compound, and a ß-1,3-glucosyl
transferase.
According to the present invention, the ß-1,3-glucosyl
oligosaccharide compound is a compound that is derived from a
microorganism known in the art and examples thereof are curdlan
and laminarin.
According to the method of the present invention, the
purification of the by-products may be performed by,
desalinization, microfiltration, or purification using an
adsorption resin. However, the purification method is not
limited thereto.
According to the method of the present invention, the ß-1,3-
glucosyl transferase may employ a microorganism or a
corresponding enzyme itself having a ß-1,3-glucosyl transferring
activity, and examples of the microorganism or the enzyme having
ß-1,3-glucosyl transferring activity are those disclosed in US
Patent 4,590,160.
The present invention also relates to a method of using by-
products produced when a high purity steviol glycoside product
is purified by fractional crystallization, in which the method
includes, as a characteristic, re-circulating the by-products in
a manufacturing process for Rebaudioside A after an enzyme
transferring reaction is performed on the by-products.
The term "steviol glycoside product in a high purity" used
herein refers to a product that is produced by extracting a
solution containing steviol glycoside from stevia dry leaves by
using a hydrothermal fluid, ethanol, methanol, or polyalcohols,
and then purifying the solution to give a product containing at
least 70 wt.% of steviol glycoside.
The term "RA 60" or "RA 97" used herein refers to a product
containing 60 wt% or 97 wt% of Rebaudioside A based on the total
weight of the product.
The unit "%" used herein refers to a weight percentage,
unless otherwise defined.
The present invention will now be described in further
detail with reference to the following examples. These examples
are for illustrative purpose only and are not intended to limit
the scope of the invention.
< Examples >
Example 1. Content and ash content analysis of steviol
glycoside in conventional RA 60 product, and in residual by-
products produced from a conventional Rebaudioside A, in
particular, residual by-products as a mother liquor produced
from a fractional crystallization process.
HPLC analysis was performed to identify a content of steviol
glycoside. To do this, 1 g of each of the samples and 1000 ml
of distilled water were loaded into a 1000 ml mass cylinder and
uniformly mixed, and then filtered through a 0.45 urn aqueous
filter. HPLC (Agilent 1200 Series) device was used and 20 µl of
each of the samples was loaded thereto. Analysis conditions
were set to be a flow rate of 0.5 ml/min and a wavelength of 210
nm. As shown in FIGS. 2 and 3, contents of stevioside,
Rebaudioside A, and Rebaudioside C were measured.
As analysis results, the conventional RA60 product included

23.3% of stevioside, 61.4% of Rebaudioside A, and 12.7% of
Rebaudioside C, and the by-products included 42.6% of stevioside,
25% of Rebaudioside A, and 26.9% of Rebaudioside C.
[ Table 1]
Content Difference and Major Content Factor Comparison
An ash content of each of the conventional RA 60 product and
the by-products was analyzed. As a result, it was confirmed
that the conventional RA 60 included 7% of ash and the by-
products included 15% of ash.
Example 2. Purification of By-products
In order to process residual by-products produced following
the conventional RA60 product being manufactured, that is, by-
products as a mother liquor produced from crystallization to
have a reusable level, a content pattern of steviol glycoside in
the by-products that had been decolorized and purified was
analyzed in the same manner as in example 1 to identify contents
of stevioside, Rebaudioside A, and Rebaudioside C.
After the by-products were purified, ash contents of
stevioside, Rebaudioside A, and Rebaudioside C from each of
processes were analyzed.
Example 3. Enzyme Transferring Process
By-products as a mother liquor produced from a fractional
crystallization process were processed to have a reusable level
as in example 2, and stevioside in the by-products was used as
an acceptor and reacted in the presence of a (3-1, 3-glucosyl
transferase, (3-1,3-glucosyl oligosaccharide compound and a (3-1,3-
glucanase for breaking down (3-1,3 glucose contained in the (3-1,3-
glucosyl oligosaccharide compound at a temperature of 50°C for 5
hours, thereby producing Rebaudioside A.
A content pattern of the newly formed steviol glycoside was
analyzed in the same manner as in example 1 to identify contents
of stevioside, Rebaudioside A, and Rebaudioside C.
As apparent from the above description, according to the
method according to the exemplary embodiments, by-products that
are produced when extracted Rebaudioside A (RA) is purified and
crystallized, that is, residual by-products are recycled as a
raw material to increase a production yield, thereby enabling
production of a product having price competitiveness. In
addition, residual by-products as a mother liquor produced from
a fractional crystallization process is used as a starting
material and subjected to a series of processes to have a
reusable level suitable for the second step process (high purity
Rebaudioside A production process), thereby reforming the by-
products into a high value-added product.
It will be apparent to those skilled in the art that various
modifications and variation can be made in the present invention
without departing from the spirit or scope of the invention.
Thus, it is intended that the present invention cover the
modifications and variations of this invention provided they
come within the scope of the appended claims and their
equivalents.
WE CLAIM :
1. A method of manufacturing Rebaudioside A in a high yield,
the method comprising:
i) a step for purifying by-products produced when a high purity
steviol glycoside product is purified by fractional
crystallization;
ii) a step for performing an enzyme transferring reaction on
the purified by-products to adjust a Rebaudioside A content in
the by-products to be in a range of 50 weight (wt)% to 60 wt%;
and
iii) fractional-crystallizing either the product produced in
step ii) alone or a mixture comprising the product produced in
step ii) and a high purity steviol glycoside product.
2. The method of claim 1, wherein the by-products from step
i) comprise 80 wt.% or higher of stevioside glycoside.
3. The method of claim 1, wherein the enzyme transferring
reaction is performed on the purified by-products in the
presence of a ß-1,3-glucosyl oligosaccharide compound, a ß-1,
3-glucanase capable of breaking down ß-1,3 glucose in the
compound, and a ß-1,3-glucosyl transferase.
4. The method of claim 3, wherein the enzyme transferring
reaction is performed at a temperature of 50°C for 5 hours.
5. A method of using by-products produced when a high purity
steviol glycoside product is purified by fractional
crystallization, wherein the method comprising steps of
performing an enzyme transferring reaction on the by-products;
and re-circulating the by-products in a manufacturing process
for Rebaudioside A.
6. The method of claim 5, wherein the enzyme transferring
reaction is performed on the purified by-products in the
presence of a ß-1, 3-glucosyl oligosaccharide compound, a ß-1,3-
glucanase capable of breaking down ß-1,3 glucose in the
compound, and a ß-1, 3-glucosyl transferase.

The present invention relates to a method of producing
Rebaudioside A in a high yield by recycling by-products produced
when Rebaudioside A is produced from leaves of Stevia Rebaudiana
Bertoni containing a sweetening material.