FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention: AN IMPROVED PROCESS FOR MAKING NATURAL
SWEETENER FROM STEVIA LEAVES


2. Applicant(s)
(a) NAME :
(b) NATIONALITY
(c) ADDRESS:

INDIAN INSTITUTE OF TECHNOLOGY
Created by an act of Parliament, Institute of Technologies Act, 1961
Indian Institute of Technology, Bombay, India

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:


FIELD OF INVENTION
The present invention relates to an improved process for the recovery of sweet principles from Stevia leaves by pressurized hot water extraction (PHWE). The final product is devoid of unpleasant smell and bitter after-taste.
BACKGROUND OF THE INVENTION
Stevia (Stevia rebaudiana Bertoni) is popularly known as sweet leaf. It contains compounds responsible for the sweetness of stevia leaves and their extracts belong to the class of diterpene glycosides and among these the most important components are Steviol glycosides. The two primary glycosides present in stevia leaves are stevioside (5-10%) and rebaudioside A (2-4% and sweetest), and have only glucose as glycone. Steviol is the aglycone present in stevia's sweet glycosides. As they are very low in calorie hence they are used as a natural sweetener for diabetics and others on carbohydrate controlled diets. They are used as substitute sweeteners for natural sugar. Steviol glycosides are 100-300 times sweeter than sucrose and used in small amounts. They are thermally stable, pH stable, and do not ferment.
Steviol glycosides are safe, natural non-nutritive sugar substitute and dietary supplement. This natural sweetener does not alter blood glucose and is a source of safe, natural low-caloric sugar substitute It is also used as a hypoglycaemic (lowers blood glucose), hypotensive (lowers blood pressure), cardiotonic (tones, balances, strengthens the heart), and antimicrobial health supplement. Stevioside has melting point of 198°C, is sparingly soluble in water (0.13%), slightly soluble in alcohol, and soluble in chloroform.
Only deterrant to usage of stevia extract is its bitter after-taste and unpleasant smell. The bitter aftertaste, unpleasant odour and colour are due to the non-glycoside fraction (-60%) comprising: essential oils, diterpenes, triterpenes, stigma-sterols, tannins, flavonoids, sesquiterpenes lactones, caryophyllene, spathulenol, etc.
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The prior art discloses various methods for the extraction of stevia where initially leaves are defattened by chloroform and Ca (CO3)2followed by extraction and crystallization which is done by dioxane and methanol respectively as disclosed in US3723410. Such processes had a disadvantage of using hazardous and toxic organic solvents (methanol, dioxane, chloroform) and additional filtration was required to remove Ca (CO3)2
US 4599403 used di or tri-carboxilic acid, calcium oxide and diatomaceous earth for removal of impurities from aqueous extract, followed by liquid-liquid extraction with butanol. But this process involved multiple steps with many solvents and chemicals and the procedure for removal of impurities is long and many hazardous chemicals are used.
In another process disclosed by Adduci et al (1987) extraction of leaves is done at 90-100°C, and subsequently to remove impurities a Direct Current (30 amp) is passed through aqueous extract for 2 hours via aluminium electrodes (with a small amount of HC1 in solution). After filtration the solution is passed through a mixed resin amberlite MB-1, and after evaporation a dry powder is obtained. Such process uses high ampere current and adsorption by resin is not very efficient. In addition, such a process involves high cost because of resin.
Supercritical fluid extraction from Stevia rebaudiana Bertoni uses CO2 and CO2 + cosolvent water. Stevia leaves are pre-treated by SCFE with CO2 (at 200 bar and 30°C) and extraction of the steviol glycosides by SCFE using: CO2 + cosolvent water, CO2 + cosolvent ethanol, and CO2 + cosolvent (water + ethanol) (at 120 and 200 bar at 16, 30, and 45°C) as disclosed by Yoda et al 2002. In another process, Pasquel et al 2000 discloses a similar process, but such type of processes involves very high
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pressure and yield of glycosides is very less. Using SCFE plant is not very economical, as capital investment is very high.
Also hot water was used for extraction as disclosed in GB 2123685 which is mostly used to prepare beverages like tea or coffee. This method of preparation does not mention the pressure employed, except that it is above atmospheric. In other cases where pressure is applied along with hot water for extraction, the applied pressure and. temperature are very high: 118 bar and 300C as disclosed in Analytical Chemistry (2000) pp. 3070-3076.
The methods found in the prior art often used extraction that required significant volume of organic solvents and were rather tedious. Hence, methods that are rapid, require low volume of organic solvent and have high extraction efficiency are attractive options.
Conventional methods for the extraction and purification of steviosides are associated almost exclusively with the use of organic solvents, such as methanol, ethanol or chloroform, and many require that the steviosides be adsorbed first on a resin with subsequent elution with an organic solvent. The concentrated, evaporated solutions from these methods usually are treated with methanol or ethanol to bring about the final crystallization of the, end mixture. Other methods make use of iron or aluminium salts to remove impurities. These two materials require a further treatment with sodium hydroxide to remove residues of the iron or aluminium salts. These methods for extraction of glycosides are very elaborate, expensive, and time-consuming, and involve multiple steps. These methods use large amounts of a number of (some toxic) solvents and chemicals .The bitter after taste and unpleasant odour is still not eliminated. Hence there is a need to develop a process which would overcome all the above mentioned drawbacks.
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The present inventors have now found an improved process for the recovery of steviosides by pressurized hot water extraction (PHWE) of Stevia Rebaudiana bertoni leaves by using a protocol of extraction and purification as a result of which the undesirable impurities are removed and the final product is made clear, colourless (in case of a liquid product), and devoid of bitter after-taste and unpleasant smell ( for both solid and liquid products).
OBJECTS OF THE INVENTION
An object of the invention is to provide an improved process for the extraction of steviol glycosides (sweet principles) from the stevia leaves wherein the process is economical, simple, efficient, cost-effective and easy to carry out.
Another object of the invention is to provide an improved process for the extraction of sweet principles, namely steviol glycosides from the stevia leaves wherein the process avoids hazardous, toxic reagents, unacceptable process solvents and conditions.
Another object of the invention is to provide an improved process for the extraction of sweet principles ■ steviol glycosides from the stevia leaves wherein the process results concentrated clear and colourless solution or solid crystals devoid of bitter after-taste and unpleasant smell.
SUMMARY OF THE INVENTION
The present invention is directed to an improved process for the recovery of sweet principles steviosides from Stevia rebaudiana Bertoni; the said process comprises subjecting leaves of Stevia rebaudiana Bertoni to pressurized hot water extraction using CO2 at appropriate range of temperature and pressure followed by removal of impurities.
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BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
The accompanying drawings are included to provide a further understanding of the invention. The drawings illustrate embodiments of the invention and together with the description serve to explain the principles of the embodiment of the invention
Figure 1 shows Pressurized hot water extraction assembly
Figure 2 shows the High Performance Liquid Chromatogram of standard stevia products (A), aqueous extract (B) & aqueous extract after electro coagulation(C).
Figure 3 shows the yield of glycosides from stevia leaves at different temperatures Figure 4 shows the yield of glycosides from stevia leaves at different pressures Figure 5 shows the flow chart for the process of extraction.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention there is provided an improved process for recovering the sweet principles from stevia leaves. The method of the present invention is referred to as pressurized hot water extraction (PHWE) technique which is very useful for recovering bio-active molecules from solid matrices of the natural materials using subcritical (compressed liquid) water and gaseous C02 in the temperature range of 40-140 °C. The process of extraction follows the steps as mentioned in Figure 5
Various parts of plants are used for extraction, leaves are the most preferred. In the present invention leaves are used, preferably dry. These dry leaves are then triturated in a grinder. These powdered leaves can be used as such or optionally may be pre-treated with supercritical carbon dioxide (SCC02) at a moderate condition in a small lab scale SCCO2 extractor. Pre-treated or dried stevia leaf powder is then processed by Pressurised Hot Water Extraction (Fig 1). A known amount of water is taken in two consecutive batches with stirring at a particular range of temperature. The
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temperature ranges from 40-140°C. CO2 is used at a pressure ranging from 1-20 bar. The stirring is maintained continuously during the hour-long of extraction. The extract is then filtered by a fine membrane and residues after the second extraction are discarded. This extract has brown colour and bitter taste.
The aqueous extract obtained is then electrocoagulated to remove color and taste in two steps at the specified condition for 0.5-1 hr. During electrocoagulation the electric current is maintained between 0.9-1.0 amp and a voltage between 20-30 volts in a cell. After the first stage of electro-coagulation is stopped, the solution is filtered. The obtained filtrate is then again electro-coagulated at the same condition. The finally collected filtrate is a clear liquid slightly greenish in colour. This filtrate is then concentrated in a vacuum rotary evaporator (5:1) and the concentrated solution is again electro-coagulated for 15 minutes.
The clear liquid is collected after filtration and is mixed with a known amount of a water-insoluble organic solvent like hexane or petroleum ether for final deodorisation. This immiscible liquid mixture is then charged in a glass flask and is shaken for 1-8 hr then it is allowed to settle for an hour. The aqueous layer is then separated from the upper hexane layer and is further concentrated and deodorised using a vacuum rotary-evaporator. To obtain a colourless and odourless solution this step may be repeated more than once.
Alternatively, the concentrated filtrate after electro-coagulation is mixed with a small amount of methanol in the extractor and treated with SCCO2 at 80-120 bar for an hour for final deodorisation. A dark odorous liquid is collected in the separator after depressurisation of outgoing carbon dioxide stream and to a colourless and odourless solution is obtained as the raffinate from the extractor.
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The deodorised and concentrated solution is then kept in a cool place (at about 5° C) for 24 hrs. After one day crystals are formed and are separated from the mother liquor by filtration. Alternatively the deodorised and concentrated solution is freeze-dried to obtain the solid product. The amount of glycosides in aqueous extract and the solid crystals/ powdered product are estimated based on phenol sulphuric acid method. The process results concentrated solution or solid crystals of glycosides devoid of bitter after-taste and unpleasant smell.
PHWE has been considered in the present process for replacing conventional organic solvents for selective extraction and because of the greater solubility of some organic compounds and insolubility of some other substances in subcritical water. It is noted that at 205 ° C the dielectric constant of water falls to 33 which is equal to that of methanol at 25 C. Hence for purification of the extract an easy and convenient processing protocol has been innovated to obtain steviol glycosides free of bitter taste and unpleasant odour. The present process has much better economic potential or commercial viability in view of its requirement of less expensive equipment and devices, less amounts and number of solvents, lower energy requirement and less time for extraction and purification.
Firstly Pressurized Hot Water Extraction (PHWE) has been used in the present process for the recovery of glycosides from dried and ground stevia leaves. The major advantages of PHWE include a relatively low operating pressure and environmental friendliness of water. Furthermore, the solvating properties of water can be easily altered through a minor change in temperature and pressure. The advantages in yield and quality, as well as considerations on the energy, safety and cleanliness of the product gave the impetus to explore utilization of the PHWE process for extraction. For pressurization of hot water, gaseous CO2 has been used at a moderate (2-20 bar) pressure eliminating the need for steam injection or a pump. The role of CO2 in the innovative process is multi-fold. One of them is lowering of pH enabling selective
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separation of sweet principles. It also removes air from the system and provides inert environment, so that no degradation or oxidative reactions/ conversions take place. Even an "inert" gas (e.g., nitrogen) is used for flushing the system off from oxygen (from air). This step is simultaneously avoided. Presence of slight residual CO2 after extraction helps in slowing down degradation before and during subsequent steps. Thirdly it provides an easy means to pressurise the system without using a steam boiler and for easy controlling of the CO2 solubility for the above two purposes. Fourthly, CO2 is a gas and is removed from the aqueous slurry unlike HC1 or other acids. This eliminates the subsequent steps for neutralization of pH after extraction and other additional steps, if a mineral acid is used. Finally usage of CO2 reduces cost of production, as CO2 is cheaper than nitrogen plus acid and chemicals used for other steps. Accordingly usage of CO2 is not equivalent to addition of acid. This is one innovative aspect of the process, namely, the usage of CO2 in extraction of natural sweetener from stevia leaves along withpressurized hot water, which is distinct from the available prior art.
Another advantage of the present process is that some of the components responsible for bitter after taste are not soluble in pressurized hot water whereas they are soluble in organic solvents. This enables selective separation of steviol glycosides by PHWE in the present process. The presence of CO2 in water lowers the pH value of the system which is also useful for selective separation.
Electrocoagulation at a mild condition (less than 1.0 amp, 20-30V and 15-30 min without adding HC1 or NaCl in the aqueous extract) is used for removal of tannin, pigments and flavonoids towards clarification of the crude extract. Finally, treatment of the aqueous solution with hexane after partial concentration by evaporation under vacuum facilitates complete removal of astringent and bitter after-taste.
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The pre-treatment prior to PHWE of ground stevia leaves and / or post-treatment of the aqueous extract with SC CO2 at moderate condition (at less than 200 bar) is found useful for partial removal of essential oils and bitter principles. However this pre-treatment step is not essential, as the undesirable components are not soluble in water and are not extracted by PHWE. Accordingly this pre-treatment step may be optional, though it slightly improves the yields of steviol glycosides. Processing with SC CO2 at moderate condition is suggested if cost is not a consideration or if SC CO2 facilities are already available.
The process results colorless odorless sweet concentrated solution and/or solid crystals of steviol glycosides.
The HPLC analysis of stevia powder extracts in aqueous medium is done after sufficient dilution. Figure 2 (A) depicts HPLC figure of standard stevia products, third peak at 3.4 min is noted as corresponding to stevioside. After each step of operation, the amount of glycosides is calculated by phenol sulphuric acid method and HPLC analysis is done for the identification. Stevia leaves contain four major sweetening agents called steviol glycosides. After PHWE Extraction HPLC of aqueous extract (B) is done. Three distinct peaks are observed at 1.4, 3.2 and 4.5 minute respectively. Comparing with the standard plot, it is found that the 2ndpeak (at 3.5 min) is stevioside and 3rd peak (at 4.5 min) is rebaudioside
First peak can be considered for impurity (like pigment) in the solution. HPLC of solution after electro coagulation of aqueous extract is shown in Figure 2(C). Similar figure (as Figure 2A) is obtained here with stevioside at 3.2 minute at Rebaudioside A at 4.9 min. It is clear from the Figure 2 C that after electro coagulation relative amount of impurity is reduced.
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High Performance Liquid Chromatography (HPLC)
Effect of temperature: Figure 3B shows the effect of temperature on the yield of
glycosides from stevia leaves. The values of yield are shown in Table Fig 3A. The

Peak No. Retention Time (min) % Area % Height
1 1.3 04.45 08.97
2 2.3 06.28 08.04
3 3.4 89.27 82.98
yield increases very slowly with temperature up to 135 ° C. The change in yield of glycosides is significant when temperature is varied from 30°C to 60 °C but at a temperature above 60°C, it has less effect. Yield of glycosides in the second extract is almost constant with temperature. As seen from Table 3A the yield was 6.7% at 120 and 135 C, so optimum temperature is 120 C. As temperature increases, properties of water like viscosity, density, surface tension, polarity etc decrease that lead to decrease in resistance of mass transfer. So at higher temperatures more glycoside is extracted from leaves. Also at higher temperature, the cell structure of rinds raptures and glycosides from inside cell come out.
Effect of pressure: The effect of pressure on the yield of glycosides is shown in Figure 4B. The yields of total glycosides obtained in two consecutive extracts at different pressures with 40ml/g water are also shown in 4A. It is observed that the yield is almost constant with pressure i.e., the variation of pressure has negligible effect on the yield. However, a higher pressure is maintained in the extractor to keep the water in liquid form and it also reduces the evaporation loss.
SCCO? treatment:
Pre-treatment with SCC02 (at 300 atm, 45°C and at 200 atm, 45°C) has been tried.
2.1% yield of blackish oily materials was collected as extract in the separator with
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SCC02 at 200 atm, 45 °C. In both cases namely for the feed pre-treated with SCC02 and without pre-treatment, aqueous extracts were brown in color. As yield does not change significantly and extract by SCCO2 is insoluble in water, pre-treatment with SCCO2 is found to be not effective. However, a post treatment of the concentrated extract with SCC02 and a small amount of methanol results complete removal of odorous substances. The residual methanol is also removed from the final solution along with the odorous substances carried along with the outgoing SCC02 stream and is collected in a glass separator after depressurization. A similar result is obtained by post treatment of the concentrated, extract with hexane in place of SC C02 post treatment for removal of the final traces of odorous substances, followed by vacuum evaporation.
The following non limiting examples illustrate preferred embodiments of the invention.
EXAMPLE: - Processes for Extraction & Purification of steviol glycoside
> 70 g of fine ground dried leaves of Stevia rebaudiana Bertoni are pretreated with supercritical carbon dioxide (at a moderate condition at 45°C, pressure at 300 atm and flow rate at 0.7-1.0 kg/hr for 3hr.) in a lab scale SCCO2 plant. 5 g pretreated leaf powder is processed by PHWE with 200 ml & 200 ml in two consecutive batches with stirring at a temperature 60 °C, using CO2 at a pressure 1 atm. Continuous stirring is maintained during the hour-long extraction. The extract is filtered by a fine cloth and residues after the second extraction are discarded.
The aqueous extract is electro coagulated to remove brown color and bitter after-taste for 0.5hr.An electric current 0.1-0.8 amp and voltage 20 -30 V are maintained in an electrocoagulating cell. Electrocoagulation is stopped after
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0.5 hr and solution is filtered. This filtrate is again coagulated at same conditions.
Finally clear slightly greenish filtrate is collected. This filtrate is concentrated in a vacuum rotary evaporator (5:1) and this concentrated solution is again electro coagulated for 15 min.
The clear liquid collected after filtration is mixed with a 50 ml of hexane and is charged in a glass flask. The mixture is shaken for 3 to 8 hr and allowed to settle for an hour. After separation of aqueous layer from hexane layer, the solution is further concentrated and deodorized using vacuum rotary evaporator. Solution is then kept in a cool place (at about 5°C) for 24 hrs. Crystals are formed after 24 hours and are separated from mother liquor by filtration.
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We claim:
1. An improved process for the recovery of sweet principles steviosides from Stevia rebaudiana Bertoni, said process comprises subjecting leaves of Stevia rebaudiana Bertoni to pressurized hot water; extraction using CO2 at appropriate range of temperature and pressure followed of removal of impurities.
2. A process as claimed in claim 1 wherein said temperature range is from 40 °C to about 140 °C.
3. A process as claimed in claim 1 wherein said pressure range is from 1-20 bar.
4. A process as claimed in claim 1 wherein CO2 is used to flush the system off air and to pressurize the system
5. A process as claimed in claim 1 wherein CO2 is used to lower the pH for selective recovery of steviosides
6. A process as claimed in claim 1 wherein any degradation in subsequent processing is retarded by residual CO2 in the system
7. A process as claimed in claim 1 wherein CO2 leaves the system after the process obviating the need of additional steps of neutralisation and addition of chemicals
8. A process as claimed in claim 1 wherein dry or wet leaves powder of Stevia is used for extraction.
9. A process as claimed in claim 1 wherein said dry leaf powder is optionally pretreated with supercritical CO2.
10. A process as claimed in claim 1 wherein said impurities are removed by electrocoagulation at a moderate condition without adding HC1 or a salt
11. A process as claimed in claim 1 further comprises treatment of aqueous extract thus obtained with a water-insoluble organic liquid.
12. A process as claimed in claim 1 wherein said organic liquid is selected from food grade hexane, heptane and petroleum ether.
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13. A process as claimed in claim 1 optionally comprises post-treatment of aqueous extract for deodorization with Super Critical C02 and a small amount of methanol for removal of odorous substances
14. A process as claimed in claim 1 wherein the aqueous clear solution is concentrated and deodorized by a rotary evaporator at 60° C and 0.20 atm for removal of traces of impurities.

15. A process as claimed in claim 1 wherein said sweet principles are obtained in clear colourless liquid form
16. A process as claimed in claim 1 wherein said sweet principles are obtained in crystalline form by keeping the concentrated solution at 5° C up to 24 hrs.
17. A process as claimed in claim 1 wherein freeze drying is used to obtain the solid product.
18. A process as claimed in claim 1 wherein said sweet principles are devoid of bitter after-taste, color and unpleasant smell
19. A process for the recovery of the sweet principles steviosides from Stevia rebaudiana Bertoni as substantially described herein with reference to the examples and accompanying drawings.
Dated this 18th day of June 2008
Saloni Rastogi Of S. Majumdar&Co. Applicant's Agent
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