US 20110091637A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0091637 A1 Abelyan et al. (43) Pub. Date: Apr. 21, 2011

(54) HIGH-PURITY REBAUDIOSIDE D AND Publication Classi?cation LOW-CALORIE CHOCOLATE CONTAINING THE SAME (51) Int. Cl. A23G 1/40 (2006.01) (75) Inventors: Varuzhan Abelyan, Kuala Lumpur (MY); Avetik Markosyan, Kuala Lumpur (MY); Lidia Abelyan, (52) US. Cl...... 426/631 Kuala Lumpur (MY) (73) Assignee: PureCircle Sdn Bhd, Negeri Semb?an (MY) (57) ABSTRACT

21 A 1. N .._ 12/785 507 The invention P rovides methods of P urifyin g RebaudiosideD ( ) pp 0 ’ from the Slevia rebaudiana Bertoni plant extract along With (22) Filed: May 24, 2010 . The methods are useful for producing high purity Rebaudioside D and Rebaudioside A. The invention Related US. Application Data further provides a loW-calorie chocolate containing the puri (63) Continuation-in-part of application No, 12/580,233, ?ed Rebaudioside D and a process for making the low-calorie ?led on Oct 15, 2009 chocolate containing the puri?ed Rebaudioside D.

Stevia Extract I

- Absolute ; - Water; ~ Heating; - Complete dissolving; - Cooling; - Starter of Rebaudioside A; - Aging; - Heating; - Cooling; - Aging.

Suspension of

- centri?lgation; - Washing wth ethanol.

or or

R?baudloslde A Remaining Solution

_- Ethanoi/suspensi‘gz. t e1 ' Soiuilon,' ' - Evaporation' of ethanol; “ Heating. , - Concentration; - Washmg- _ a with_ agitation;. I ~ Spray drying. - Cooling; . “ Washing with agitation. Mixture of Stevio] Glycosides » Cennifugation; - Washing wth ethanol; ~ Drying.

Highly Puri?ed Rebaudioside A

Patent Application Publication Apr. 21, 2011 Sheet 2 0f 11 US 2011/0091637 A1

Stevioside

FIGZ Patent Application Publication Apr. 21, 2011 Sheet 3 0f 11 US 2011/0091637 A1

Rebaudioside B Patent Application Publication Apr. 21, 2011 Sheet 4 0f 11 US 2011/0091637 A1

Rebaudioside D FIG 2 (Cont’d) Patent Application Publication Apr. 21, 2011 Sheet 5 0f 11 US 2011/0091637 A1

Rebaudioside E

m; 2 (Cunt’d) Patent Application Publication Apr. 21, 2011 Sheet 6 0f 11 US 2011/0091637 A1

Dulcoside A FIG 2 (Cont’d) Patent Application Publication Apr. 21, 2011 Sheet 7 0f 11 US 2011/0091637 A1

cHzoH

‘¢=o

0H Steviolbioside HOE-2

Rubugoside 2 (contid) Patent Application Publication Apr. 21, 2011 Sheet 8 of 11 US 2011/0091637 A1

Stevia Extract

- Absolute ethanol; - Water; ~ Heating; - Complete dissolving; - Cooling; - Starter of Rebaudioside A; - Aging; ~ Heating; ' Cooling; - Aging. Suspension of Glycosides

- Centri?igation; - Washing wth ethanol.

or or

Rebaudioside A ‘ Remaining Solution

- Ethanol/water solution; _ Evaporation 9f ethanol; -:I11Spf1mi0n; - Concentmtion; “ eaung; ~ Spraydryin - - Washing with agitation; g

~ Cooling; . . r . m Washing with agitation. Mixture of Steviol G13 cosides - Centrifugation; - Washing wth ethanol; - Drying.

Highly Pun'?ed Rebaudioside A

FIG 3 Patent Application Publication Apr. 21, 2011 Sheet 9 0f 11 US 2011/0091637 A1

(A) (B) RebA / 95.6% RebA / 52.7%

RebD / 43.9% /3.5%

(C)

RebD 95.0%

FIG 4 Patent Application Publication Apr. 21, 2011 Sheet 10 of 11 US 2011/0091637 A1

Stevia Extract i Precipitation of Rebaudioside A i Separation + i’ i Rebaudiioside A (~95-96%) Remaining solution Alcohol‘I tratment Evaporation Separation i Concentration i i High purity High Rebaudioside D Drying Rebaudioside A (>99%) Fraction Stevio! Glycosides Mixture Evaporation i Drying RebD Concentrate

FIG 5 Patent Application Publication Apr. 21, 2011 Sheet 11 0f 11 US 2011/0091637 A1

16

-3. sobo 2000 i000 Wavenumbers (cm-1)

FIG6 US 2011/0091637 A1 Apr. 21, 2011

HIGH-PURITY REBAUDIOSIDE D AND hydroxyphenyl)propyl]-L-0t-aspartyl]-phenylalanine LOW-CALORIE CHOCOLATE CONTAINING 1-methyl ester, salts thereof, and the like, and combination THE SAME thereof. [0009] Non-limiting examples of natural high intensity RELATED APPLICATIONS sweeteners include Stevioside, Rebaudioside A, Rebaudio side B, Rebaudioside C, Rebaudioside E, Rebaudioside F, [0001] This application is a continuation-in-part applica Steviolbioside, Dulcoside A, Rubusoside, mogrosides, tion of Us. patent application Ser. No. 12/580,233, ?led Oct. braZZein, neohesperidin dihydrochalcone (NHDC), glycyr 15, 2009. rhiZic acid and its salts, thaumatin, perillartine, pemandulcin, mukuroZiosides, baiyunoside, phlomisoside-I, dimethyl FIELD OF THE INVENTION hexahydro?uorene-dicarboxylic acid, abrusosides, perian [0002] The invention relates to a process for isolation and drin, camosi?osides, cyclocarioside, pterocaryosides, poly puri?cation of individual sweet glycosides from Slevia podoside A, braZilin, hemandulcin, phillodulcin, rebaudiana Bertoni plant extract, and more particularly to glycyphyllin, phloriZin, trilobatin, dihydro?avonol, dihydro isolation and puri?cation of Rebaudioside D from Slevia quercetin-3-acetate, neoastilibin, trans-, rebaudiana Bertoni plant extract and further to low-calorie monatin and its salts, selligueain A, hematoxylin, monellin, chocolate sweetened with high intensity sweetener. osladin, pterocaryoside A, pterocaryoside B, mabinlin, pen tadin, miraculin, curculin, neoculin, chlorogenic acid, DESCRIPTION OF THE RELATED ART cynarin, siamenoside and others. [0010] High intensity sweeteners can be derived from the [0003] Sweeteners are critical ingredients in food supply. The demand of healthy low calorie beverages and food prod modi?cation of natural high intensity sweeteners, for ucts results in the increasing consumption of sweeteners; thus example, by fermentation, enZymatic treatment, or derivati Zation. there is a need to reduce the calories contributed by sweeten [0011] At present about eleven high intensity sweeteners ers. This goal can be achieved by using high intensity sweet are used worldwide. These are acesulfame-K, alitame, aspar eners. [0004] High intensity sweeteners possess level tame, cyclamate, , NHDC, saccharin, Stevioside, many times exceeding that of . They are essentially sucralose, thaumatin, neotame, and Rebaudioside A. non-caloric and used widely in manufacturing of diet and [0012] The high intensity sweeteners can be grouped into reduced calorie food. Although natural caloric sweetener three generations. The ?rst generation represented by cycla mate, glycyrrhiZin and saccharin has a long history of use in such as sucrose, , and provide the most desir able to consumers, they are caloric. High intensity food. The second generation includes acesulfame-K, aspar sweeteners do not affect the blood glucose level and provide tame, NHDC and thaumatin. Alitame, neotame, sucralose, Stevioside, and Rebaudioside A belong to the third genera little or no nutritive value. [0005] However, high intensity sweeteners that generally tion. are used as sucrose substitutes possess taste characteristics [0013] The standard sweetening power associated with different than that of sugar, such as sweet taste with different each high intensity sweetener is given in TABLE 1. However, temporal pro?le, maximal response, ?avor pro?le, mouth when they are used in blends, the sweetening power can feel, and/or adaptation behavior than that of sugar. For change signi?cantly. example, the sweet taste of some high-potency sweeteners is slower in onset and longer in duration than that of sugar and TABLE 1 thus changes the taste balance of a food composition. Because Sweetener Sweetness power of these differences, usage of high-potency sweetener in replacing such a bulk sweetener as sugar in a food or beverage Saccharose 1 causes an unbalanced temporal and/or ?avor pro?le. If the AcesulfaIne-K 200 Alitalne 2000 taste pro?le of high-potency sweeteners could be modi?ed to Aspartame 200 impart desired taste characteristics, it can provide low calorie Cyclamate 30 beverages and food products with taste characteristics more Glycyrrhizin 50 desirable for consumers. NHDC 1000 saccharine 300 [0006] On the other hand, high-potency sweeteners may Stevioside 200 have some cost and functional advantages compared to sugar. Rebaudioside A 450 The competition among sugar and non-sugar high-potency Thaurnatin 3000 sweeteners is tough in soft drinks industry, in countries where Sucralose 600 their use and production is permitted and also in countries with overvalued sugar prices. [0014] On the other hand, ‘natural’ and ‘organic’ foods and [0007] At present high intensity sweeteners are used world beverages have become the “hottest area” in the food indus wide. They can be of both synthetic and natural origin. try. The combination of consumers’ desire, advances in food [0008] Non-limiting examples of synthetic sweeteners technology, new studies linking diet to disease and disease include sucralose, potassium acesulfame, aspartame, alitame, prevention has created an unprecedented opportunity to saccharin, neohesperidin dihydrochalcone synthetic deriva address public health through diet and lifestyle. tives, cyclamate, neotame, dulcin, suosan, N-[N-[3-(3 -hy [0015] A growing number of consumers perceive the abil droxy-4-methoxyphenyl)propyl]-L-(x-aspartyl]-L-phenyla ity to control their health by enhancing their current health lanine 1-methyl ester, N-[N- [3 -(3 -hydroxy-4 and/or hedging against future diseases. This creates a demand methoxyphenyl) -3 -methylbutyl] -L-0t-aspartyl] -L for food products with enhanced characteristics and associ phenylalanine 1-methyl ester, N-[N-[3-(3-methoxy-4 ated health bene?ts, speci?cally a food and consumer market US 2011/0091637 A1 Apr. 21, 2011

trend towards “Whole health solutions” lifestyle. The term times sWeeter than sugar. The leaves have been traditionally “natural” is highly emotive in the World of sWeeteners and has used for hundreds of years in Paraguay and BraZil to sWeeten been identi?ed as one of key trust, along With “Whole grains”, local teas and medicines. “heart-healthy” and “loW-sodium”. ‘Natural’ term is closely [0021] At present there are more than 230 Slevia species related to ‘healthier’. With signi?cant sWeetening properties. The plant has been [0016] In this respect, natural high intensity sWeeteners can successfully groWn under a Wide range of conditions from its have better commercial potential. native subtropics to the cold northern latitudes. [0017] NeW formulations of food products having [0022] Steviol glycosides have Zero calories and can be improved nutritional characteristics, including, for example, used Wherever sugar is used. They are ideal for diabetic and loWer calorie content, are desirable. Also, there is perceived loW calorie diets. In addition, the sWeet steviol glycosides market demand for foods having improved ?avor pro?les, possess functional and sensory properties superior to those of including good taste, mouthfeel, etc. In addition, today there many high potency sWeeteners. is an increasing interest in loW-calorie foods and beverages. [0023] The extract of Slevia rebaudiana plant contains a There is consumer interest in food products, Whose formula mixture of different sWeet diterpene glycosides, Which have a tions make greater use of natural ingredients, that is, ingredi single base-steviol and differ by the presence of carbohy ents distilled, extracted, concentrated or similarly obtained drate residues at positions C13 and C19. These glycosides from harvested plants and other naturally occurring sources, accumulate in Slevia leaves and compose approximately With limited or no further processing. Alternatives to sucrose 10%-20% of the total dry Weight. Typically, on a dry Weight serve a number of purposes. It is important that such alterna basis, the four major glycosides found in the leaves of Slevia tives don’t cause signi?cant changes in the sensory charac are Dulcoside A (0.3%), Rebaudioside C (0.6%), Rebaudio teristics of the product. It is also very important that diabetic side A (3.8%) and Stevioside (9.1%). Other glycosides iden (With no sugar) or reduced calorie foods have as feW differ ti?ed in Slevia extract include Rebaudioside B, C, D, E, and F, ences as possible from conventional foods. Steviolbioside and Rubusoside (FIG. 1). Among steviol gly [0018] The development of neW food formulations, for cosides only Stevioside and Rebaudioside A are available in example, employing sWeeteners, ?avorants, ?avor enhancing commercial scale. agents and the like, presents challenges in addressing associ [0024] The chemical structures of the diterpene glycosides ated bitterness and/ or other off-. In addition, such chal of Slevia rebaudiana Bertoni are presented in FIG. 2. lenges typically are presented in formulations developed for [0025] The physical and sensory properties are Well studied improved nutritional characteristics and/ or ?avor pro?les. only for Stevioside and Rebaudioside A. The sWeetness Also, there is need for neW beverage formulations Which can potency of Stevioside is around 210 times higher than satisfactorily meet the combination of objectives including sucrose, Rebaudioside A in betWeen 200 and 400 times, and nutritional, ?avor, shelf life, and other objectives. Rebaudioside C and Dulcoside A around 30 times. Rebau [0019] An object of the invention is to provide a food prod dioside A is considered to have most favorable sensory uct, particularly a reduced calorie chocolate, having excellent attributes of the four major steviol glycosides (TABLE 2). taste pro?le and mouthfeel. The product comprises at least [0026] The glycosides from leaves can be extracted using one non-nutritive sWeetener in an amount suf?cient to pro either Water or organic solvent extraction. Supercritical ?uid vide perceptible sWeetening. extraction and steam distillation Were described as Well. [0020] Slevia rebaudiana Bertoni is a perennial shrub of the Methods for recovery of diterpene sWeet glycosides from Asteraceae (Compositae) family native to certain regions of Slevia rebaudiana using membrane technology, and Water or South America. The leaves of the plant contain from 10 to organic solvents, such as methanol and ethanol also are 20% of diterpene glycosides, Which are around 150 to 450 described.

TABLE 2 Optical rotation [(11250 TMeh, Mol. (H2O, Solubility Relative Quality of Name Formula ° C. Weight 1%, W/v) in Water, % sweetness taste

Steviol C2OH3OO3 212-213 318.45 ND ND ND Very bitter Steviolmonoside C26H4OO8 ND 480.58 ND ND ND ND Stevioside C38H6OO18 196-198 804.88 —39.3 0.13 210 Bitter Rebaudioside A 70 23 242-244 967.01 —20.8 0.80 200-400 Less Bitter Rebaudioside B C38H6OO18 193-195 804.88 —45 .4 0.10 150 Bitter Rebaudioside C C44H7OO22 215-217 951.01 —29.9 0.21 30 Bitter Rebaudioside D C5OH8OO28 248-249 1129.15 —29.5 1.00 220 Like sucrose (ethanol) Rebaudioside E C44H7OO23 205-207 967.01 —34.2 1.70 170 Like sucrose Rebaudioside F C43H68O22 ND 93 6.99 —25 .5 ND (methanol) Dulcoside A C38H60Ol7 193-195 788.87 —50.2 0.58 30 Very bitter Steviolbioside C32H5OO13 188-192 642.73 —34.5 0.03 90 Unpleasant Rubusoside C32H5OO13 ND 642.73 642.73 ND 110 Very bitter US 2011/0091637 A1 Apr. 21, 2011

[0027] There are several publications on puri?cation of [0033] An improved method for the recovery of steviol some individual steviol glycosides. glycosides from Slevia rehaudiana Bertoni plant, Which does [0028] Generally production of extract includes extraction not require the use of special separation equipment such as ion exchange and/or chromatographic columns Was of plant material With Water or Water-organic solvent mixture, described in Us. Pat. No. 4,599,403. The extraction Was precipitation of high molecular Weight substances, deioniZa carried out With Water. The resulting aqueous extract is treated tion, and decoloriZation, puri?cation on speci?c With citric acid to remove metallic and other impurities as macroporous polymeric adsorbents, concentration and dry Well as to loWer the pH to about 3.0. The mixture Was ?ltered ing. through Celite and pH of the ?ltrate adjusted to 10.5 by [0029] Us. Pat. No. 3,723,410 discloses an extraction of calcium oxide. The formed precipitate Was remover by ?ltra Steviosides from Slevia rebaudiana Bertoni. The method tion. The ?ltrate Was concentrated and extracted With n-bu tanol. Puri?ed Stevioside crystals Were then recovered by included defatting of Slevi a leaves by treatment With chloro cooling the Water layer obtained from the solvent extraction form for more than 150 hours at boiling temperatures and step. The major draWbacks of the method are the losses of three times treatment With dioxane in the presence of calcium glycosides during extraction by n-butanol and also loW yield carbonate for tWo hours at boiling temperatures. After ?ltra of Stevioside crystals from aqueous solution. The salt content tion the dioxane ?ltrates Were combined and concentrated to in the ?nal product can be high. There are no data about the syrup state under reduced pressure at 50° C. An equal volume ?nal purity of Stevioside. The process is dif?cult to apply on of methanol Was then added to the syrup and the resulting commercial scale. solution set aside over night to alloW crystalliZation to occur. [0034] Us. Pat. No. 4,892,938 and JP No. 01-131191 dis The crystals Were collected by ?ltration and Washed thor close a puri?cation process in Which the extract of the plant oughly With ice cold methanol. The residual solution Was Was obtained through treatment in Water at a temperature concentrated, an equal volume of methanol Was added, and from room to about 650 C. With stirring and subsequent ?l the mixture set aside overnight to crystalliZe. The crystals tration and centrifugation. This extract Was treated With cal Were removed by ?ltration and dried in vacuum at 100° C. The cium hydroxide and the precipitate Was removed by ?ltration yield of Stevioside Was 6.5% from air-dried leaves. The or centrifugation. This ?ltrate Was treated With a strong acidic method is very complicated With the usage of toxic organic ion exchange resin and subsequently With a Weak basic ion solvents. There is no information about purity of Stevioside, exchange resin. The sWeet glycosides remained in the Water hoWever in described conditions Rebaudiosides Will precipi and Were recovered by evaporation of the Water. The disad tate along With Stevioside. The process is dif?cult to apply on vantage is that the ?nal product has quite loW purity. The sWeet glycosides content in the ?nal product Was only about commercial scale. 70%. [0030] A method for the production of Slevia extract With [0035] Us. Pat. No. 5,112,610 discloses a natural sWeet further isolation of Rebaudioside A is developed in Us. Pat. ener preparation process based on Slevia rebaudiana. The No. 4,082,858. The air-dried Slevia leaves Were extracted method included extracting the plant material of Slevia With hot Water, and the extract Was dried under vacuum. The rebaudiana With an organic solvent and subjecting the solu resulted mixture Was extracted With methanol and from com tion to supercritical gas (CO2) extraction to obtain a residue, bined extracts methanol Was removed by distillation under Which Was free from undesired and taste-impairing constitu reduced pressure. The obtained syrup Was subjected to chro ents. Generally speaking, the method concerned to removal of matographic separation on a silica gel column using mixture curticle Waxes, chlorophyll, other pigments and especially of n-propanol, Water and ethyl acetate as mobile phase. The taste-impairing components from Slevia leaves or extract. method is useful in laboratory scale only and has various HoWever direct treatment of the leaves required a great quan disadvantages on the commercial scale. tity of starting material so that the use of leaves Was non economical even When increasing the bulk density of the [0031] Us. Pat. No. 4,171,430 discloses a puri?cation of dried or comminuted leaves by pressing into pellets prior to Stevioside from Slevia extract. The method included extract the extraction. The treatment of poWdered extract, Which Was ing Slevia leaves With Water, concentrating the solution and obtained from leaves by conventional method, alloWed the extracting With methanol. Stevioside Was crystalliZed from removal of taste-impairing components only to a lesser methanol solution and puri?ed on styrene type gel With tet degree, and Without employing entrainers (loW molecular rahydrofuran as mobile phase. The method is useful in labo Weight alcohols, suitable hydrocarbon or mixture of the sol ratory scale only. The process is dif?cult to apply on com vents) achieves not entirely satisfactory results. Moreover, mercial scale. there are no quanti?ed data on the actual purity of extract. The [0032] Us. Pat. No. 4,361,697 discloses an extraction, process is dif?cult to apply on commercial scale. separation and recovery of diterpene glycosides from Slevia [0036] Us. Pat. No. 5,962,678 describes a multi-step rebaudiana. The process included the steps of sequential extraction and puri?cation process of Rebaudioside A from extracting of plant material ?rst With a solvent of intermediate Slevia rebaudiana plant. The extract of the plant Was obtained polarity (such as chloroform), and then With a second solvent through treatment in Water at a temperature ranging from of high polarity (such as methanol). The resulting extract Was ambient to about 650 C. With stirring and sub sequent ?ltration subjected to a liquid chromatography separation. The steviol and centrifugation. This extract Was treated With calcium glycosides Were in the methanol fraction. The major draW hydroxide and the precipitate Was removed by ?ltration or backs Were the use of various toxic solvents to extract and centrifugation. This ?ltrate Was treated With a strong acidic process sWeet glycosides. Final puri?cation of glycosides ion exchange resin and subsequently With a Weakly basic ion Was achieved by column chromatography using sorbents like exchange resin. The sWeet glycosides remained in the Water silica gel as a stationary phase and eluting the column With and Were recovered by evaporation of the Water. The content tWo solvents sequentially running through the column. Pro of steviol glycosides in the extract in this stage Was 70% only. cess is not environmentally-friendly and dif?cult to carry out For further puri?cation the product Was passed through the in the large scale. column With Amberlite XAD-7, Which Was able to adsorb US 2011/0091637 A1 Apr. 21, 2011

steviol glycosides. After Washing With Water the glycosides re?uxed mixture or a stirred Wash mixture, (3) optionally, one Were desorbed With methanol. The purity of the extract Was or more stirred Wash stages, and (4) an ethanol purge and around 95% With content of signi?cant amount of so called drying stage. In embodiments that used loWer quality Slevia yellow oil. To isolate individual Stevioside and Rebaudioside starting material, a second re?ux stage Was typically added A the dried solid Was re?uxed in anhydrous methanol solution before the stirred Wash stage to maximiZe purity of the Rebau and then cooled to precipitate Stevioside With 91.6% of dioside A ?nal product. In the reported method, an EtOH purity. However, the yield of Stevioside Was only 15% from formulation stage Was conducted in order to formulate a the Slevia extract containing 60% Stevioside. Stevioside can desired re?ux solvent for use in the re?ux step(s). Typically, be further puri?ed by re?uxing it in methanol-Water solution. the re?ux solvent Was a mixture of ethanol and Water With Purity of the product Was about 99%. about 5% to 15% by volume Water. The process further [0037] A more puri?ed product can be produced by the included one or more energy-intensive re?uxing steps that combined use of micro?ltration, ultra?ltration, and nano?l Were typically conducted at a temperature of about 890 C. to tration as it is described in Us. Pat. No 5,972,120. The 900 C. for about 1 hour. The method reportedly produced extraction Was uninterruptedly carried out in continuous ?oW 100% pure, Water-soluble Rebaudioside A. columns. The optimum mean particle siZe of leaves had to be [0041] Us. PatentApplication No. 2006/0134292 reports a about 20 mm. With smaller particles, the ?ltration rate sub process for recovering sWeet glycosides from Slevia rebau stantially decreased as the column Was blocked. Initial Water diana plant material. The dried and poWdered leaves Were Was added in a quantity of 0.05 parts per one part of dry leaves treated With Water in the presence of a pectinase, cellulase, (by Weight). The column temperature Was set to not more than and alpha-amylase. The use of such enZymes Was reported to 40 C., and extraction Was carried out With Water at pH Within considerably increase the extraction rate and facilitates the the range 2.0-4.0 (adjusted With phosphoric acid). At loW next stages of puri?cation. The resulting extract Was puri?ed temperatures and pH, a more selective extraction occurred using treatment With calcium hydroxide and ultra?ltration. and nearly colorless solution Was obtained. The extract Was Permeate Was passed through the column packed With ben then ?ltered through tubular ceramic membranes and, then, tonite and concentrated to syrup state under vacuum. The through ultra?ltration membranes. The produced ?ltrate Was treatment With ethanol alloWed separating the practically separated from loW-molecular impurities on nanomembranes pure Rebaudioside A from the mixture. The Rebaudioside A at elevated temperatures. With high purity Was obtained after Washing the crystals With [0038] Method of preparation of Slevia extract is described 88-95% of ethanol. in US. Pat. No. 6,031,157 and 6,080,561. The dry leaves [0042] US. PatentApplication No. 2007/0082103 reports a Were extracted With 10 to 20 parts of Water several times. The process for preparing of Slevia extract and highly puri?ed resulting extracts Were combined and passed sloWly through Stevioside and Rebaudioside A. The dried and poWdered a column ?lled With cation-exchange resin and then a column leaves Were subjected to Water extraction and the resulted ?lled With anion-exchange resin. The treated solution then extract Was puri?ed using treatment With a base such as Was passed through a column packed With a resin (Amberlite calcium hydroxide and then iron chloride. The ?ltrate Was XAD-2) to adsorb the sWeetening components, and then deioniZed using ion-exchange resins, concentrated under Washed With Water. After the Water Was drained from the vacuum and spray dried. Highly puri?ed Rebaudioside A and column, it Was eluted With three volumes of methanol to Stevioside Were obtained by dissolving the extract in metha isolate the sWeetening components. The ef?uent Was concen nol to precipitate Stevioside. The remaining solution after trated and further dried under a reduced pressure to obtain a isolation of Stevioside Was dried and Rebaudioside A Was pale yelloW poWder. The major draWback of the method is the isolated by treatment With ethanol. The ?nal puri?cation of loW quality of extract. Treatment With ion-exchangers and Rebaudioside A Was developed by treatment With ethanol speci?c adsorbents only, cannot result in high quality Slevia Water solution. The purity Was at least 98%. extract With White color and high content of steviol glyco [0043] Us. Published Patent Application No sides. 20070292582 discloses puri?cation of Rebaudioside A. The [0039] Us. Published Patent Application No 2006/ method comprised the steps of combining crude Rebaudio 0142555 discloses a process for the production of Steviosides sideA and an aqueous organic solvent to form a Rebaudioside from Slevia rebaudiana plant. The method included extrac A solution, the aqueous organic solution comprising Water in tion of plant poWder by direct steam injection into the extrac an amount from about 10% to about 25% by Weight, and tor folloWed by ?ltration to get aqueous extract and calcium crystalliZing from the crude Rebaudioside A solution, in a hydroxide treatment to remove impurities in the form of pre single step, substantially pure Rebaudioside A in purity cipitate. The ?ltrate Was treated With strong cation-exchange greater than 95%. In the case of ethanol-methanol-Water mix resin and then Weak base anion-exchange resin. The aqueous ture the yield of Rebaudioside A With purity more than 97% eluate containing Steviosides Was concentrated to obtain Was 32.5% from starting material containing 77.4% Rebau puri?ed Steviosides With 45.47-65.5% Stevioside content in dioside A. The yield from starting material containing the ?nal product. The provided method is suitable for produc 80.37% Rebaudioside A Was in the range of 54.6-72.0%. tion of Slevia extract With various content of Stevioside but Other co-solvents used along With ethanol such as ethyl not for highly puri?ed steviol glycosides. acetate, 1-butanol, 2-butanol, tent-butanol, sec-butanol, [0040] Us. Patent Application Publication No. 2006/ acetonitrile, isopropanol, and 1-propanol Were not suitable 0083838 reports a method of isolating and purifying Rebau for the production of Rebaudioside A With greater than 97% dioside A from commercially available Slevia rebaudiana purity. In the case of use ethanol With various amounts of starting material. The method comprised: (1) an EtOH for Water as crystallization solvent the yield of Rebaudioside A mulation stage to formulate a selected EtOH solvent, (2) a Was in the range 39.6%-76.4% from starting material con ?rst re?ux stage using the Slevia starting material and option taining 80.37% Rebaudioside A. The process used the mix ally additional re?ux stages using retentate isolated from a ture of tWo organic solvents, Which recovery and puri?cation US 2011/0091637 A1 Apr. 21, 2011

in large scale Was very complicated. Moreover, in commer philic organic solvent or hydrous hydrophilic organic solvent. cial scale When centrifugation may take relatively long time, Treatment With only non-polar synthetic adsorbent is unable the co-precipitation of Stevioside, Rebaudioside C, and to result in high quality extract; no measures are taken for Rebaudioside D may occur. residual salts and the color of the product. [0044] Us. PatentApplication No. 2008/0300402 and Chi [0051] JP No. 55-159770 concerns the extraction and puri nese Patent No 101200480 report a method for producing ?cation of Stevioside by extracting Slevia leaves With Water puri?ed Rebaudioside A comprising the folloWing steps: or hydrous alcohol. The extract Was concentrated to solid separation of Rebaudioside A on chromatographic column content from 10 to 50%, added 01-50% of calcium chloride packed With silica gel using the mixture of ethyl acetate, to coagulate and precipitate the colloidal impurities existing ethanol and Water as mobile phase. Rebaudioside A fractions in the extract. From concentrated solution using CaCl2 most Were combined and dried. The solid Was treated With ethanol of impurities cannot be removed. There are no desalting and containing from 2 to 10% of Water and Rebaudioside A Was decoloriZing stages. crystallized by cooling the mixture at —200 C. The purity of [0052] JP No. 55-162953 concerns the preparation of Rebaudioside A can reach to more than 99%. For the puri? Stevioside by extracting Slevia leaves With 10-15 volumes of cation of Rebaudioside A the ?ltrate after separation of Water at 60-800 C. The extract Was treated With slaked lime Stevioside Was concentrated and cooled to 00 C. overnight for With aeration, and the pH of suspension Was adjusted to about 16 hours. The resulting precipitate of Rebaudioside A around 8.0 by adding sulfuric or citric acid. The resulting Was ?ltered, Washed With a small volume of cold methanol, slightly soluble salt Was ?ltered off and the ?ltrate Was then and dried to obtain Rebaudioside A With 79.0% purity and contacted With a polyamide resin to remove impurities. The 3.3% yield from initial extract. This crude Rebaudioside A ?ltrate Was further extracted With n-butanol and the organic Was further puri?ed by re?uxing in anhydrous methanol or phase Was distilled under the vacuum to recover the Stevio methanol-Water mixture. From starting material containing side as White crystals. Content of salts in such product Will be 90.2% of Rebaudioside A the output of the product Was high. Puri?cation process using the n-butanol extraction is around 67% With 98.6% of purity. HoWever the method of dif?cult to apply on commercial scale. improving the purity of Rebaudioside A from 79% to 90.2% [0053] JP No. 55-081567 describes the extraction and puri is not available. The major draWback of the process is loW ?cation of Stevioside. The extract of Slevia leaves prepared yields of the ?nal highly products, Which makes the process by Water or hydrous alcohol extraction Was concentrated, and not suitable for commercial production of highly puri?ed one or more types of Water-soluble salts of Ca, Fe, and Al and Stevioside and Rebaudioside A. a Water soluble organic solvent, e.g. ethanol or acetone, Were [0045] Various Japanese patents also concern about the added to the concentrate to precipitate and remove the colloi preparation of extract from Slevia rebaudiana Bertoni. dal impurities. The resulting liquid With pH 3-7 Was passed [0046] JP No. 52-100500 describes the puri?cation and through a strong cation-exchange resin and a Weak anion concentration of aqueous Stevioside extract by treating the exchange resin. The obtained solution Was passed through the extract With speci?c ion-exchange resin of high decoloriZing speci?c adsorbent. The fractions of Stevioside Were com capacity, folloWed by treatment With Amberlite XAD type bined. The process is similar to the traditional Chinese tech speci?c adsorbent. Treatment With only ion-exchangers and nology, Which can result in yelloW poWder With only 85-86% adsorption/desorption is unable to result in high quality steviol glycosides content. extract. [0054] JP No. 55-120770 concerns the puri?cation of [0047] JP No. 52-136200 discloses a preparation ofStevio Stevioside solution. The leaves and stalks of Slevia rebaudi side solution by extraction With hot Water or hydrous alcohol ana Bertoni Were extracted With Water or an alcoholic solu folloWed by membrane separation. The molecular Weights of tion, to Which a Water-soluble tin salt, e. g., stannous chloride, sWeet glycosides and sterebins are very close and membrane stannous sulfate, stannic sulfate, etc, Was added and dis systems cannot result satisfactory resolution of these com solved. An alkali substance, e.g., sodium hydroxide or lime pounds, Which Will affect to the purity of extract. Content of Was added to the resulting solution to adjust the pH value salts in the ?nal product Will be high. around 5-10. The formed precipitate Was separated. This pro [0048] JP No. 52-005800 discloses a method of preparation cess is unable to result in extract free from salts and other of puri?ed Stevioside from leaves of Slevia rebaudiana by loW-molecular Weight impurities. extraction and treatment With cation-exchange resin. Such [0055] JP No. 55-138372 describes the puri?cation of treatment Will result in yelloW poWder With apparently loW Stevioside solution. Stevioside Was extracted from the leaves content of sWeet glycosides. and stalks of Slevia rebaudiana With Water, hot Water, or a [0049] Japanese Patent JP54030199 discloses the process hydrous alcohol, and the extract or its concentrate Was mixed for preparation of Slevia sWeetening agent free from charac With slaked lime or lime milk. The mixture Was then ?ltered teristic smell and bitter taste, by extracting leaves of Slevia and mixed With an equimolar amount of Water-soluble iron rebaudiana Bertoni With Water, treating the extract With a compound, eg ferrous sulfate, and stirred to precipitate the non-polar synthetic adsorbent resin folloWed by desorption, iron ions as a sparingly soluble hydroxide, Which Was and further treating With an ion-exchange resin. The process removed With the coloring substances adsorbed on it. The is very similar to traditional Chinese technology, Which process is unable to result in extract free from salts and other alloWs producing Slevia extract With steviol glycosides con loW-molecular Weight impurities. tent not more than 85-86%. [0056] JP No. 55-039731 concerns the extraction of Stevio [0050] JP No. 54-132599 discloses a separation and puri side. 1 kg of dried leaves of Slevia rehaudiana Was extracted ?cation of Stevioside by extracting Slevia leaves With hot With 3-10 volumes of Water or hydrous alcohol. The extract Water, treating the extract With a non-polar synthetic adsor Was concentrated to solid content of 10-50% and 0.1-5% of a bent, Washing the resin With an aqueous solution of slaked metallic chloride, e.g. calcium, aluminum, or iron chloride, lime, and eluting the Stevioside from the resin With a hydro Was added. The precipitate of impurities Was removed by US 2011/0091637 A1 Apr. 21, 2011

?ltration. The subsequent puri?cation procedures With ion passed through a polyamide column to absorb glycosides and exchange resins, adsorbent, and ultra?ltration membranes remove impurities. The puri?ed extract Was then concen can be carried out further. Most of impurities cannot be trated under reduced pressure, pH adjusted to 8-9 With aque removed from concentrated solution using salts. The content ous ammonia and extracted With n-butanol to afford crude of loW-molecular Weight impurities can be high. Stevioside, Which Was then recrystallized from methanol. [0057] JP No. 56-160962 discloses a puri?cation ofStevio HoWever, the content of residual salts can be high; there is no side containing solution by extracting Slevia leaves With decoloriZing stage; extraction With n-butanol and recrystalli Water, concentrating the extract obtained to 25-50% solids Zation from methanol is not viable commercially. content, mixing the concentrate With a loW molecular Weight [0063] JP No. 57-075992 concerns the puri?cation of aliphatic alcohol, and removing the precipitated impurities Stevioside. The Water extract of Slevia rebaudiana Bertoni from the mixture. The amount of the alcohol Was at least 5 Was mixed With a ?occulant (e.g. aluminum or polyaluminum times volume of the aqueous extract, or 3-6 times volume of chloride) to ?occulate and remove the colloidal impurities, the concentrate. The treatment is not suitable to remove loW and then treated With a non-polar resin (e.g. Duolite ES-861) molecular Weight impurities. There are no decoloriZing to adsorb the sWeetening substance. The adsorbed substances stages. Process is dif?cult to apply on commercial scale. Were eluted With an organic solvent (e.g. methanol, acetone, [0058] JP No. 56-109568 discloses a puri?cation ofSZevia etc.), and the solution Was discolored and puri?ed With acti sWeetening sub stances by extracting Slevia leaves With Water vated charcoal and activated clay. Activated charcoal can or hydrophilic organic solvent. The extract Was treated With absorb the Stevioside ?rmly from aqueous solution and the an organic solvent selected from the group consisting of 4-8C decoloriZing and puri?cation effects of activated charcoal can ether, 4-7C ester, and 1-4C organic chlorine compound, and be promoted by the combined use With activated clay. HoW the ingredient soluble in the solvent Was separated. Diethyl ever, haZardous solvents are used, Which can present in the ether, diisopropyl ether, ethyl acetate, methyl chloride, car ?nal product. Process is dif?cult to apply on commercial bon tetrachloride, etc. may be cited as the purifying solvent. scale. The bitter taste can be removed effectively With simultaneous [0064] JP No. 57-086264 concerns the isolation of princi decoloriZing. HoWever, used haZardous solvents, can remain pal sWeetening component of Slevia. Dried stalks and leaves in the ?nal product. Process is dif?cult to apply on commer of Slevia Were extracted With cold Water, hot Water, hydrous cial scale. alcohol, etc. The extract Was coagulated or precipitated With [0059] JP No. 56-099768 concerns the preparation of ste an adsorbent, and the precipitate Was removed by ?ltration or viol glycosides. A solution containing steviol glycosides, eg centrifugation to obtain a clear liquid containing the sWeet an aqueous extract of Slevia rehaudiana Bertoni, Was treated ening components. The components Were adsorbed to a syn With magnesium silicate aluminate to adsorb impurities, e.g. thetic polymer adsorbent, puri?ed to 80-90% purity, concen pigments or proteins. HoWever, salts content in the ?nal prod trated, dried, and dissolved in 3-8 volumes of hot methanol or uct can be high. There are no decoloriZing and additional hot ethanol. Stevioside and RebaudiosideA Were crystallized puri?cation stages. Steviol glycosides content in the ?nal from the solution simultaneously. After complete removing product can be loW. of the solvent, the mixed crystals Were heated together With a [0060] JP No. 57-002656 concerns the discoloration and 3-6 volumes of alcohol and separated into the solution part puri?cation of Slevia extract. Slevia extract Was treated With and the solid part by hot ?ltration. Stevioside can be obtained an aqueous solution of a barium compound that is readily from the solution and the Rebaudioside A can be prepared by soluble in Water and then neutraliZed With sulfuric acid. Washing and drying the solid part. Method can result to the Barium hydroxide Was added until pH Was 7-9 and the sus puri?ed Stevioside and Rebaudioside A; hoWever the quality pension again Was treated With sulfuric acid to pH 3-4. The of extract can be loW because of the absence of deioniZation precipitate Was separated. The main drawbacks are that salt and decoloriZing stages. The content of loW-molecular content in the ?nal product can be high, there are no decol Weight impurities can be high. oriZing and additional puri?cation stages, and, as a result, [0065] JP No. 58-212759 andNo. 58-212760 describedthe steviol glycosides content in the ?nal product can be loW. puri?cation of Slevia sWeetening substance. The leaves of [0061] JP No. 57-005663 concerns the puri?cation of Slevia rebaudiana Bertoni Were extracted With Water or an Stevioside through extraction. An extracted solution of Slevia alcohol at pH 4. The extract Was treated With calcium hydrox leaves With Water or Water-containing alcohol Was concen ide and formed precipitate Was ?ltered off. A Water-soluble trated to 10-50% of solids content. A salt or a base of calcium, organic solvent such as methanol Was added to the ?ltrate, and iron, or aluminum Was added and the precipitate Was removed precipitate Was removed. The amount of the Water-soluble by ?ltration. The ?ltrate pH Was adjusted betWeen 5-7, and organic solvent Was from 5% to 50% based on the ?ltrate. The the formed precipitate Was removed. The ?ltrate is treated ?ltrate obtained Was puri?ed by ion-exchange resins or With a cation exchange and an anion exchange resins and adsorption resin. The main draWback is that haZardous sol evaporated to dryness. The major draWback of the method is vent is used, Which can present in the ?nal product. Process is the loW quality of extract. The treatment With alkali and dif?cult to apply on commercial scale. ion-exchangers only is not enough to produce the Slevia [0066] JP No. 58-028246 described the preparation of extract With White color and high content of steviol glyco Stevioside. The raW leaf of Slevia Was extracted With Water, sides. hot Water or a Water-alcohol mixture, and if necessary the [0062] JP No. 57-046998 concerns the preparation of extract Was then concentrated. A mixture of calcium hydrox Stevioside. RaW leaves of Slevia rebaudiana Were extracted ide With calcium chloride in an amount of 0.5-2.0 times that of With 10-20 volumes of Water and the ?ltrate Was treated With the solid content in the extract Were added to the extract or calcium hydroxide in an amount of 10-30% of the raW leaves concentrated extract preferably While bloWing gaseous car Weight. The pH of the suspension Was then adjusted to 4-6 bon dioxide. The impurities Were precipitated in the form of With sulfuric acid or citric acid. After ?ltration the extract Was a colloidal material, Which Was separated by ?ltration. HoW US 2011/0091637 A1 Apr. 21, 2011

ever, the extract quality can be loW because of high content of [0073] JP No. 56-121453, JP No. 56-121454, and JP No. salts and loW-molecular Weight compounds. 56-121455 concern the separation of Stevioside and Rebau [0067] JP No. 58-028247 concerns the puri?cation method dioside A. A mixture of Stevioside and Rebaudioside A of Stevioside solution. The raW leaves of Slevia Were extracted from the leaves of Slevia rebaudiana Bertoni Was extracted With Water, hot Water or a Water-alcohol mixture, mixed With 75% aqueous solution of methanol and main and the extract Was concentrated. Calcium hydroxide and a tained at ambient temperature for about 3 hours. The resulted Water-soluble high polymeric ?occulant, e. g. polyacrylamide crystals With 65% Stevioside and 25.2% Rebaudioside A high polymer, in an amount of 1-2.5 times that of the solid content Were separated by ?ltration and dried. In the case of content in the extract Were added to the extract or concen application of 90% aqueous solution of ethanol the ?nal mix trated extract to precipitate impurities, Which Were then ?l ture contains 57.4% Stevioside and 31.9% Rebaudioside A. tered off. A transparent and almost colorless Stevioside solu Further re-crystallization from 90% aqueous solution of etha tion Was obtained. HoWever, the extract quality can be loW nol resulted in product With higher content of Rebaudioside because of high content of salts and loW-molecular Weight A. The purity of the product Was around 80%. Stevioside compounds. further can be puri?ed up to 86.1% by additional Washing [0068] JP No. 59-045848 concerns the preparation of Sle With Water. [3-Type crystals of Stevioside and ot-type crystals via sWeetener With high content of Rebaudioside A. Dried of Rebaudioside A Were obtained. leaves of Slevia variety containing 1.57 times more Rebau [0074] JP No. 57-046998 concerns the preparation of dioside A than Stevioside Were extracted With Water or a Stevioside. RaW leaves of Slevia rebaudiana Were extracted Water-containing solvent. The prepared extracted solution With 10-20 volumes of Water and the ?ltrate Was treated With Was treated With a cation-exchange resin and an anion-ex calcium hydroxide in an amount of 10-30% of the raW leaves change resin. The solution Was adsorbed on an absorption Weight. The pH of the suspension Was then adjusted to 4-6 resin, eluted With a hydrophilic solvent, and the solution Was With sulfuric acid or citric acid. After ?ltration the extract Was concentrated to yield a natural sWeetener. The process is passed through a polyamide column to adsorb glycosides and similar to the traditional Chinese technology, Which can result remove impurities. The puri?ed extract Was then concen to the yelloW poWder With steviol glycosides content up to trated under reduced pressure, pH adjusted to 8-9 With aque 85-86% only. ous ammonia and extracted With n-butanol to afford crude [0069] JP No. 62-166861 concerns the extraction and puri Stevioside, Which Was then recrystallized from methanol. ?cation of sWeetener component from dry leaves of Slevia. [0075] JP No. 57-086264 concerns the isolation of princi Dried leaves of Slevia rebaudiana Bertoni Were extracted pal sWeetening component of Slevia. Dried stalks and leaves With 7-14 volumes of Water at 50-700 C. for 3-6 hr With of Slevia Were extracted With cold Water, hot Water, hydrous agitation to obtain a broWn liquid extract With total solids alcohol, etc. The extract Was coagulated or precipitated With content of 2-3% and containing 0.7-0.8% of Stevioside. The an adsorbent, and the precipitate Was removed by ?ltration or extract Was concentrated 7-8 times at about 600 C. under centrifugation to obtain a clear liquid containing the sWeet reduced pressure. The concentrated liquid Was treated With ening components. The components Were adsorbed on a syn 0.5-2% CaCl2 to separate impurities as ?occulent precipitate. thetic polymer adsorbent, puri?ed to 80-90% purity, concen The solution Was treated With an Al, Mg oxide of an amount trated, dried, and dissolved in 3-8 volumes of hot methanol or corresponding to 15-20% of the solid content at 40-550 C. hot ethanol. Stevioside and RebaudiosideA Were crystallized under vigorous agitation. Then the precipitate Was removed from the solution simultaneously. After complete removal of by ?ltration. The Stevioside can be further puri?ed on speci?c the solvent, the mixed crystals Were heated together With a 3 -6 adsorbents. HoWever, the process is dif?cult to commercial volumes of alcohol and the solids Were separated from solu ize; salts quantity used for the puri?cation of extract is high tion by hot ?ltration. Stevioside can be obtained from the and there are no deionization and decolorizing stages. The solution and the Rebaudioside A can be prepared by Washing content of loW-molecular Weight compounds can be high. and drying the solid part. [0070] JP No. 06-007108 concerns the method for extract [0076] JP No. 06-192283 and JP No. 08-000214 discloses ing and separating sWeet substances of Slevia rebaudiana puri?cation of Rebaudioside A by gel-?ltration on Toyo Peri Bertoni. Leaves of Slevia rebaudiana Bertoni Were extracted HW-40. Rebaudioside C and Dulcoside Were obtained by With a Water-miscible alcohol such as methanol. The HPLC. Method is useful only in laboratory scale. extracted solution Was mixed With Water and passed through [0077] JP 63173531 describes a method of extracting sWeet an ultra?ltration membranes having 20-150 kDa cutting glycosides from the Slevia rebaudiana plant. The ?rst step of capacity and then through the ultra?ltration membranes With the process Was to extract a liquid solution of sWeet glyco 1-10 kDa cutting capacity. HoWever, hazardous solvents are sides from the Slevia rebaudiana plant. Secondly, the liquid used, Which can present in the ?nal product. Process is di?i solution of sWeet glycosides Was passed through a non-polar cult to apply on commercial scale. porous resin and eluted With a Water-soluble organic solvent, [0071] JP No. 52083731 deals on isolation and puri?cation preferably methanol. Thirdly, the eluted solution Was concen of Rebaudioside A and Rebaudioside B by column chroma trated and dried to give a poWdery material. This procedure tography on silica gel. Further puri?cation is developed by isolates a mixture of sWeet glycosides, but does not isolate a crystallization from organic solvents such as methanol or single pure sWeet such as Rebaudioside A. ethanol. [0078] JP No. 07-143860 discloses puri?cation of Rebau [0072] JP No. 55-092400 concerns the preparation of dioside A through crystallization and re-crystallization from Stevioside. An aqueous solution containing Stevioside Was 10-20% of aqueous methanol solution. The purity of Rebau extracted With 1H,1H,5H-octa?uoro-1-pentanol. After sepa dioside A Was around 90%. rating the solvent Was distilled off, and the residue Was dried. [0079] JP No. 07-177862 discloses puri?cation of Rebau The precipitate Was recrystallized from methanol. The purity dioside A and Stevioside. Puri?ed Slevia extract Was treated of Stevioside Was more than 95%. With loW concentrations of alcohol to obtain crystals With US 2011/0091637 A1 Apr. 21, 2011

about 75% content of Stevioside and Rebaudioside A. The structure of the sorbent on the selectivity Was investigated in crystals Were further recrystallized from Water to provide the detail. TWo separation methods Were applied in the enrich slightly Water-soluble sWeetener With ratio of Stevioside and ment of Rebaudioside A. They Were selective elution using Rebaudioside A around 1:2, W/W. methanol or ethanol solution as solvent, and dynamic chro [0080] JP No. 2002,262,822 discloses a sWeetener matographic separation using pyridyl resin With high selec extracted from dried leaves of Slevia plant and its extraction tivity. Results shoW that the chromatographic separation method. This process used Water or aqueous solvent to extract method can effectively enrich Rebaudioside A from Slevia Slevia glycosides from the dried leaves. In the obtained prod extract With high content of Stevioside. The ratio of Rebau uct, the content of RebaudiosideA is 2.56 times the amount of dioside to Stevioside can increase from 0.75 to 3.94. Further Stevioside. puri?cation of Rebaudioside A Was possible by crystalliza [0081] Isolation of Steviolbioside, Rebaudioside A, tion from methanol. Rebaidioside B Was carried out by Kohda et al., 1976. Dried [0087] A method for clari?cation of Slevia extract using leaves Were extracted With hot methanol and ?ltrate Was modi?ed zeolites is described by Moraes et al. (2001) and concentrated to dryness. The residue Was extracted With n-bu Montovaneli et al. (2004). Synthetic or natural zeolites Were tanol and, after drying the residue Was re-crystallized from modi?ed by treatment With calcium or barium ions and Slevia methanol. The mother liquor Was subjected to chromato extract Without any pretreatment Was contacted With modi?ed graphic separation on silica gel using chloroform-methanol zeolite. lt resulted in 70-80% of clari?cation in batch and only Water mixture as mobile phase. Further puri?cation Was 55-60% in continuous conditions. The clari?cation process developed by thin-layer chromatography. The method can be Was meant to adsorb the pigments that make the extract applied only in laboratory scale for the production of small broWnish, and not the glycosides, Which are responsible for amounts of abovementioned sWeet glycosides. the sWeet taste. HoWever, there are no data on the steviol [0082] Dulcoside A and B Were isolated and identi?ed glycosides content in the ?nal product. Obviously only this using crystallization from methanol-ethanol mixture and fur type of treatment cannot result in highly puri?ed extract, ther puri?ed by chromatography on silica gel (Kobayashi et especially because of polysaccharides, heavy metals and al., 1977). sterebins, Which remain in clari?ed extract. Moreover, no [0083] The combination of ultra?ltration, dia?ltration, data about half-life and adsorption capacity of the carrier reverse osmosis and ion exchange treatment Was used for the Which is very important When process is carried out in con puri?cation of Slevia extract (Fuh and Chiang, 1990). The tinuous conditions. cutting capacity of ultra?ltration membranes Was 25,000 and [0088] Polymeric adsorbents With iN+(CH3)3 groups 100,000 Daltons. The mixtures of strong and Weak cation Were designed and applied for the puri?cation of steviol gly and anion-exchange resins Were used as ion-exchangers. The cosides and enrichment of RebaudiosideA (Shi et al., 2002). recovery of total steviol glycosides Was around 90%; hoWever In the series of ?ve columns the content of Rebaudioside A the ?nal product purity Was 46% only. increased from the ?rst column product to the ?fth column [0084] A method for the puri?cation of steviol glycosides product. At the same time the adsorbent displayed decolori by membrane technology is described by Liu et al. (1991) and zation ability. Zhang et al. (2000). Dried leaves Were placed in a standard [0089] Rebaudioside F Was isolated by liquid chromatog glass column and extraction Was carried out With reverse raphy on 3-aminopropyl-functionalized silica gel by Starratt osmosis Water. The extract Was pretreated With a ceramic et al., 2002. The fractions Which Were rich in Rebaudioside C tubular membrane and then With an ultra?ltration membrane and Rebaudioside F Were combined and separated by HPLC in dia?ltration mode. Permeate Was Washed from loWer on a Waters carbohydrate column With linear gradient of molecular Weight impurities by a nano?ltration membrane in acetonitrile and Water. a dia?ltration mode at elevated temperatures. Addition of [0090] Preparation of Slevia extract by supercritical ?uid lime and/ or other ?occulating agent to ultra?ltration feed extraction is described by Yoda et al. 2003. It is a tWo-step improved the ?ux signi?cantly. The process could provide a process: (i) CO2 extraction at 200 bar and 300 C., and (ii) relatively high purity sWeetener concentrate. HoWever there CO2+Water extraction. Approximately 72% of the CO2 are no data about the purity of the extract and the recovery of soluble compounds Were recovered and the major compound steviol glycosides. The loW pH values used for the extraction Was austroinulin. The system SZevia+CO2+Water Was able to required special acid resistant reactors. LoW temperatures remove approximately 50% of the original Stevioside and during extraction increased the operational cost of the pro about 72% of Rebaudioside A. The main drawbacks of the duction. These both (loW temperatures and pH) resulted in method are the requirement of high pres sure and loW extrac large amount of diluted initial extract. Dilution of extract tion rate of sWeet compounds. Besides, there is no informa occurred also during micro?ltration and ultra?ltration. For tion about content of minor compounds and total steviol the ?nal puri?cation ion-exchange treatment is necessary. glycosides content in the ?nal extract. The process is dif?cult These factors are substantially increasing the production cost to apply on commercial scale. and decreasing the yield of ?nal product in unit of time. Initial [0091] Pressurized ?uid extraction using Water or methanol investment is high as Well. Was employed for the extraction of Stevioside from Slevia [0085] A series of polar resins based on polystyrene With rebaudiana Bertoni (Pol et al. (2007). A temperature of 1100 carbonyl groups Were used for the adsorption of steviol gly C. Was determined to be optimal for extraction of Stevioside cosides and partial separation of Stevioside and Rebaudioside from Slevia rebaudiana leaves using either Water or metha A (Chen et al., 1998; 1999). The ratio of Rebaudioside A to nol. An increased temperature resulted in signi?cant degra Stevioside can increase from 0.72 to 2.24. dation of Stevioside in the media of both solvents or in a [0086] The adsorptive capacity and selectivity of a novel decline in the extraction yield in Water. Both solvents dem adsorbent With pyridyl group toWard steviol glycosides Were onstrated Stevioside extraction With very similar reproduc studied (Chen et al., 1999). The effect of polarity and physical ibility and the proposed extraction parameters are the same