Title of Invention

A PROCESS FOR PRODUCTION OF STEVIOL FROM STEVIA SPECIES

Abstract The present invention is in relation to a process for obtaining steviol from stevia species. In addition, the present invention provides simple process to obtain steviol, more particularly white steviol from stevia species.
Full Text

Field of the Invention
The present invention is in relation to production of white steviol. More particularly the present invention relates to a process for isolation and purification of steviol from stevia plant species.
Background and Prior art of the Invention
The utilization profile of table sugar, the carbohydrate sweetener processed from cane sugar has gradually diminished at global level since the type2 diabetes remains as a health hazard of human population especially in tropical belts. Due to dramatic increase in number of Stevia rebaudiana is a tropical short duration crop with high genetic potential of producing a natural non-carbohydrate sweetener with zero calories. The leaves of the plant have a group of diterpene glycosides collectively named as steviol with a property of natural sweetness (200 to 300 times sweeter than cane sugar). Besides the sweetening property its leaves have been used as herbal teas by diabetic patients in Asian countries for years. It is recommended for diabetes and has been extensively tested on animals and has been used by human with no side effects. In the vegetative parts, mature leaves have the higher deposition than the tender leaves and it indicates the correlation of steviol synthesis and the growth of the plant. The leaves of this splendid plant are 20 to 30 times sweeter than sugar with zero calories whereas purified extract is 200 to 300 times sweeter than sugar. Presently it has been consumed in large quantities in tropical countries such as Japan, Korea, China and Brazil. Presently more than 1300 MT raw material is being cultivated in China, Taiwan and Malaysia for marketing in Japan.
Interest has been rekindled in more recent years especially in the developed world where diet conscious consumers seek a natural low calorie sweetener as an alternative to chemical sweeteners from the time Stevia rebaudiana was first described in the early 1900s, the chemistry of the Stevia sweeteners has attracted interest from chemists and later biochemists. The dry leaves of Stevia rebaudiana have an approximately 10 to 12 percent steviol content, the value varies depending on the

variety. The traditional method of use was to dry the leaves and to use them to sweeten teas and medicines or to chew the leaves as a 'sweet treat'. Crystalline powders and extracts are preferred in commercial situations as they have a fixed known sweetening value. There are a number of patented refining processes registered international. They generally use the four basic steps:
1. Extracting the sweetener in boiling water or other solvent methanol.
2. Ion exchange separation
3. Filtration with precipitation / coagulation
4. Crystallization and drying
The techniques standardized so far demands more advanced devices and hence expensive. As a small duration crop with compatible agricultural practices for small-scale farmers Stevia rebaudiana has more attraction than others. Apart from its traditional and raw use of dried leaves for sweeteners an affordable processing system of the leaves for getting purified and refined steviol is inevitable for exploiting the commercial value of the product at national and international level. In the present study a protocol for the production of white steviol has been developed by processing fresh mature leaves of Stevia rebaudiana. By repeated trials it is evident that the steps of extraction and purification of the crude green extract into fine colorless solution were simple, reliable and economical. Objects of the Invention
The principal object of the present invention is to obtain white steviol from stevia plant species.
Another object of the present invention is to develop a protocol for the production of white steviol from Stevia rebaudiana.
Yet another object of the present invention is to develop a simple, reliable and economical process for the production of white steviol from the leaves of Stevia rebaudiana.
Statement of the Invention
Accordingly, the present invention provides a process for production of steviol from Stevia species, said process comprising steps of: extracting dried plant parts using solvent to obtain crude extract; treating the crude extract with charcoal to remove phenols and pigments; filtering the charcoal treated extract to obtain yellow colored extract; treating the yellow colored extract with celite to remove yellow pigments;

filtering the celite treated extract to obtain light yellow colored solution; adjusting the
pH of light yellow colored solution towards acidic side to obtain colorless solution;
centrifuging the colorless solution to remove soluble impurities; and drying the
centrifuged solution to obtain white crystalline powder of steviol.
Brief Description of the Accompanying drawings
Figure: 1 HPLC chromatogram for crude steviol extracts
Figure: 2 HPLC chromatogram of partially purified steviol
Figure: 3 Flow chart representing the protocol for production of white steviol from
stevia leaves
Detailed Description of the Invention
The present invention is in relation to a process for production of steviol from Stevia
species, said process comprising steps of:
a. extracting dried plant parts using solvent to obtain crude extract;
b. treating the crude extract with charcoal to remove phenols and pigments;
c. filtering the charcoal treated extract to obtain yellow colored extract;
d. treating the yellow colored extract with celite to remove yellow pigments;
e. filtering the celite treated extract to obtain light yellow colored solution;
f. adjusting the pH of light yellow colored solution towards acidic side to obtain
colorless solution;
g. centrifuging the colorless solution to remove soluble impurities; and
h. drying the centrifuged solution to obtain white crystalline powder of steviol. In another embodiment of the present invention, wherein the Stevia species is preferably Stevia rebaudiana Bertoni.
In yet another embodiment of the present invention, wherein the plant parts are selected from a group comprising root, shoot, leaf and seeds or the whole plant and preferably the leaves of the plant.
In still another embodiment of the present invention, wherein the plant parts are dried at a temperature ranging from 70°C to 80°C and for a time period ranging from 8 to 10 hours.
In still another embodiment of the present invention, wherein the plant parts are dried preferably at a temperature of about 70 °C and preferably for a time period of about 8 hours.

In still another embodiment of the present invention, wherein the plant parts are
extracted using boiling water for a time period ranging from 2 to 3 hours and preferably
for a time period of about two and half hours.
In still another embodiment of the present invention, wherein the crude extract is green
in color with a pH value of below 7 and preferably a pH value of about 6.8
In still another embodiment of the present invention, wherein said charcoal and celite
are used at a concentration ranging from 5 % to 10 % and preferably at a concentration
of about 5%.
In still another embodiment of the present invention, wherein the pH of yellow colored
solution is ranging from 5 to 6.5 and preferably a pH value of about 6.
In still another embodiment of the present invention, wherein said centrifugation is
carried out at an rpm ranging from 6000 to 10,000.
In still another embodiment of the present invention, wherein said centrifugation is
carried out for a time period ranging from 15 to 20 minutes and preferably for a time
period of about 15 minutes.
In still another embodiment of the present invention, wherein the centrifuged solution is
spray dried.
In still another embodiment of the present invention, wherein the yield of white steviol
powder is ranging from 10 % to 12 % and preferably a concentration of about 12 %.
The present invention is in relation to a white steviol powder obtained from Stevia
species.
In another embodiment of the present invention, wherein the Stevia species is
preferably Stevia rebaudiana Bertoni.
In yet another embodiment of the present invention, wherein said powder particles are
spherical in shape with mean particle size of about 10 |im and moisture content of 3. 9
%.
The technology of the instant Application is further elaborated with the help of
following examples. However, the examples should not be construed to limit the
scope of the invention.
Example: 1
The fresh mature leaves were collected from healthy stevia rebaudiana plants prior to
flowering. The leaves were spread in the chamber of the drier at 70 to 80°C for 8 to 10
hrs. for drying. The drying at low temperature for long duration helped to maintain the

quality of the leaf from over drying as used in conventional drying i.e. 110 C for 2 to 3 hrs. The moisture content was calculated as 20%. Example: 2
The dried leaves (1 kg) were immersed in boiling water (5L) for 2 to 3 hrs and the steviol content was quantified spectrophometrically at 200nm. The extract was filtered through single layered cheesecloth. The pH of the dark green extract was below 7 (pH 6.8). The content of steviol, total phenols and pigments in the crude extract was quantified. The crude green colored extract was treated with 5 to 10% activated charcoal and stirred well for 15 to 20 mts for the first level of purification. Adding boiling water prior to the addition of the extract moisturized the activated charcoal. The extract was filtered through ordinary filter paper. The residual charcoal was again mixed with boiling water for 10 mt. and filtered for getting good recovery of steviol. Both the filtrate was mixed together for the second level of purification. Steviol content of the partially purified extract was determined. The level of total phenols and the pigment content were determined for calculating the level of purity. The yellow colored partially purified extract was treated with celite (5 to 10%) and stirred for 15 to 20 mts. The celite was initially moisturized with boiling water before the treatment of the extract. The mixture was filtered through ordinary filter paper. The remaining celite was again mixed with boiling water for 10 mts and filtered for getting maximum recovery of steviol. Both the filtrates were mixed together and the steviol content was estimated. The color of the extract became very light yellow. The pH of the solution was reduced to slight acidic i.e. pH6 and the final solution was turned into colorless. The colorless extract recovered after celite treatment was centrifuged at 6000 to 10000 rpm for 15 mts for removing all the soluble micro impurities. The clear aqueous solution was decanted and stored. The steviol content of the aqueous preparation was estimated. The final concentration of steviol was 10 to 12%, which is equivalent to international standards.
The steviol solution was spray dried at 180°C inlet temperature and 95°c outlet temperature with 500-psi pump pressure to white crystalline form of steviol. The result clearly demonstrates the feasibility of producing white steviol powder, from fresh mature leaves. Since the percentage of recovery of steviol is equivalent to international refining processes this process technique is more affordable to Indian farmers in both small scale and large-scale conditions. Considering the potential of the

product as an ideal substitute of cane sugar with anti-diabetic property, the market
value of this process is highly promising.
Example: 3
The physical characteristics of white steviol powder are as follows:
Mean particle size = 10 and Moisture content = 3.9 %




















We claim:
1) A process for production of steviol from Stevia species, said process
comprising steps of:
a. extracting dried plant parts using solvent to obtain crude extract;
b. treating the crude extract with charcoal to remove phenols and pigments;
c. filtering the charcoal treated extract to obtain yellow colored extract;
d. treating the yellow colored extract with celite to remove yellow pigments;
e. filtering the celite treated extract to obtain light yellow colored solution;
f adjusting the pH of light yellow colored solution towards acidic side to obtain
colorless solution; g. centrifuging the colorless solution to remove soluble impurities; and h. drying the centrifuged solution to obtain white crystalline powder of steviol.
2) The process as claimed in claim 1, wherein the Stevia species is preferably Stevia rebaudiana Bertoni.
3) The process as claimed in claim 1, wherein the plant parts are selected from a group comprising root, shoot, leaf and seeds or the whole plant and preferably the leaves of the plant.
4) The process as claimed in claim 1, wherein the plant parts are dried at a temperature ranging from 70°C to 80°C and for a time period ranging from 8 to 10 hours.
5) The process as claimed in claim 1, wherein the plant parts are dried preferably at a temperature of about 70 °C and preferably for a time period of about 8 hours.
6) The process as claimed in claim 1, wherein the plant parts are extracted using boiling water for a time period ranging from 2 to 3 hours and preferably for a time period of about two and half hours.
7) The process as claimed in claim 1, wherein the crude extract is green in color with a pH value of below 7 and preferably a pH value of about 6.8
8) The process as claimed in claim 1, wherein said charcoal and celite are used at a concentration ranging from 5 % to 10 % and preferably at a concentration of about 5 %.
9) The process as claimed in claim 1, wherein the pH of yellow colored solution is ranging from 5 to 6.5 and preferably a pH value of about 6.

10) The process as claimed in claim 1, wherein said centrifugation is carried out at an rpm ranging from 6000 to 10,000.
11) The process as claimed in claim 1, wherein said centrifugation is carried out for a time period ranging from 15 to 20 minutes and preferably for a time period of about 15 minutes.
12) The process as claimed in claim 1, wherein the centrifuged solution is spray
dried.
13) The process as claimed in claim 1, wherein the yield of white steviol powder is
ranging from 10 % to 12 % and preferably a concentration of about 12 %.
14) A white steviol powder obtained from Stevia species using a process as claimed in claim 1.
15) The white steviol powder as claimed in claim 14, wherein the Stevia species is preferably Stevia rebaudiana Bertoni.

16) The white steviol powder as claimed in claim 14, wherein said powder particles are spherical in shape with mean particle size of about 10 and moisture content of 3. 9%.
17) A process for production of white steviol powder and also white steviol powder


Documents:

1436-CHE-2007 AMANDED CLAIMS 28-05-2010.pdf

1436-che-2007 drawings 28-05-2010.pdf

1436-CHE-2007 EXAMINATION REPORT REPLY RECIEVED 28-05-2010.pdf

1436-CHE-2007 FORM-13 28-05-2010.pdf

1436-che-2007 form-3 28-05-2010.pdf

1436-che-2007 form-5 28-05-2010.pdf

1436-che-2007-abstract.pdf

1436-che-2007-claims.pdf

1436-che-2007-correspondnece-others.pdf

1436-che-2007-description(complete).pdf

1436-che-2007-drawings.pdf

1436-che-2007-form 1.pdf

1436-che-2007-form 26.pdf

1436-che-2007-form 3.pdf

1436-che-2007-form 5.pdf


Patent Number 245903
Indian Patent Application Number 1436/CHE/2007
PG Journal Number 06/2011
Publication Date 11-Feb-2011
Grant Date 04-Feb-2011
Date of Filing 04-Jul-2007
Name of Patentee SCHOOL OF COMMUNICATION AND MANAGEMENT STUDIES INSTITUE OF BIOSCIENCE AND BIOTECHNOLOGY RESEARCH
Applicant Address MANAGEMENT HOUSE SOUTH KALAMASSERY COCHIN 682 033
Inventors:
# Inventor's Name Inventor's Address
1 CHINNAMMA MOHANKUMAR MANAGEMENT HOUSE SOUTH KALAMASSERY COCHIN 682 033
2 JAYASHREE GOPALAKRISHNA PAI MANAGEMENT HOUSE SOUTH KALAMASSERY COCHIN 682 033
3 HARISH MADHAVA WARRIER MANAGEMENT HOUSE SOUTH KALAMASSERY COCHIN 682 033
4 REEJA RAJAB MANAGEMENT HOUSE SOUTH KALAMASSERY COCHIN 682 033
PCT International Classification Number A23F5/40
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA