US008337.927B2

(12) United States Patent (10) Patent No.: US 8,337,927 B2 Purkayastha et al. (45) Date of Patent: *Dec. 25, 2012

(54) PROCESS FOR MANUFACTURING A 2006, 0083,838 A1 4, 2006 Jackson et al. SWEETENER AND USE THEREOF 2006/0134292 A1 6, 2006 Abelyan et al. 2006/O142555 A1 6, 2006 Jonnala et al. 2007/0082103 A1 4/2007 Magomet et al. (75) Inventors: Siddhartha Purkayastha, Champaign, 2007, 0116800 A1 5, 2007 Prakash IL (US): Avetik Markosyan, Kuala 2007, 0116819 A1 5, 2007 Prakash Lumpur (MY); Magomet Malsagov, 2007, 0116820 A1 5, 2007 Prakash 2007, 0116821 A1 5, 2007 Prakash Kuala Lumpur (MY) 2007, 0116822 A1 5, 2007 Prakash 2007, 0116823 A1 5, 2007 Prakash (73) Assignee: Purecircle SDN BHD, Negeri Sembilan 2007, 0116824 A1 5, 2007 Prakash (MY) 2007, 0116825 A1 5, 2007 Prakash 2007, 0116826 A1 5, 2007 Prakash 2007/01 16827 A1 5, 2007 Prakash (*) Notice: Subject to any disclaimer, the term of this 2007, 0116828 A1 5, 2007 Prakash patent is extended or adjusted under 35 2007, 0116829 A1 5, 2007 Prakash U.S.C. 154(b) by 362 days. 2007, 011683.0 A1 5, 2007 Prakash 2007, 0116831 A1 5, 2007 Prakash This patent is Subject to a terminal dis 2007, 0116832 A1 5, 2007 Prakash claimer. 2007, 0116833 A1 5, 2007 Prakash 2007, 0116834 A1 5, 2007 Prakash (21) Appl. No.: 12/753,470 2007, 0116835 A1 5, 2007 Prakash 2007, 011683.6 A1 5, 2007 Prakash 2007/01 16837 A1 5, 2007 Prakash (22) Filed: Apr. 2, 2010 2007, 0116838 A1 5, 2007 Prakash 2007, 0116839 A1 5, 2007 Prakash (65) Prior Publication Data 2007, 0116840 A1 5, 2007 Prakash 2007, 0116841 A1 5, 2007 Prakash US 2010/O2551.71 A1 Oct. 7, 2010 2007/O128311 A1 6/2007 Prakash 2007/0134390 A1 6/2007 Prakash Related U.S. Application Data 2007/0134391 A1 6/2007 Prakash (63) Continuation-in-part of application No. 1 1/246,066, 2007/0224321 A1 9, 2007 Prakash filed on Oct. 11, 2005, now Pat. No. 7,807,206, and a (Continued) continuation-in-part of application No. 1 1/246,152, filed on Oct. 11, 2005, now Pat. No. 7,862,845, and a FOREIGN PATENT DOCUMENTS continuation-in-part of application No. 12/684,129, BR PIO7O1736 T 2008 filed on Jan. 8, 2010, and a continuation-in-part of (Continued) application No. 12/684,130, filed on Jan. 8, 2010, and OTHER PUBLICATIONS a continuation-in-part of application No. 12/684,981, filed on Jan. 11, 2010, now Pat. No. 8,298,599, and a Chang, S.S. et al., “Stability Studies of Stevioside and Rebaudioside continuation-in-part of application No. 12/720,888, A in Carbonated Beverages'. Materials and Methods, 1-6. Chen, et al., “Enrichment and separation of from filed on Mar. 10, 2010. by a novel adsorbent with pyridyl group. Science in China, vol. 42, No. 3 1999, 277-282. (60) Provisional application No. 61/260,593, filed on Nov. Chen, et al., “Selectivity of polymer adsorbent in adsorptive separa 12, 2009, provisional application No. 61/290,778, tions of stevia diterpene glycisides'. Science in China, vol. 41, No. 4 filed on Dec. 29, 2009. 1998, 436-441. Chen, et al., “Studies on the adsorptive selectivity of the polar resin (51) Int. C. with carbonyl group on rebaudioside A'. Acta Polymeric Schica, No. A23C I/236 (2006.01) 4 1999, 398-403. (52) U.S. Cl...... 426/548; 426/478; 426/489 Fuh, , "Purification of Steviosides by membrane and ion exchange (58) Field of Classification Search ...... 426/478, process”, Journal of Food Science, vol. 55, No. 5 1990, 1454-1457. 426/479, 481,489, 506, 548 Jaitak, et al., “An Efficient Microwave-assisted Extraction Process of See application file for complete search history. Stevioside and Rebaudioside-A from (Bertoni)”. Phytochem. Anal. vol. 20 2009, 240-245. (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner — Leslie Wong 3,723.410 A 3, 1973 Persinos (74) Attorney, Agent, or Firm — Briggs and Morgan, P.A.: 4,082,858 A 4, 1978 Morita et al. Aleya R. Champlin 4,171.430 A 10, 1979 Matsushita et al. 4,219,571 A 8/1980 Miyake (57) ABSTRACT 4,361,697 A 11, 1982 Dobberstein et al. Highly purified Stevioside, Rebaudioside A and a purified 4,599.403 A 7, 1986 Kumar Sweet Steviol mixture were prepared from Sweet 4,892,938 A 1/1990 Giovanetto 5,112,610 A 5, 1992 Kienile glycoside extracts obtained from Stevia rebaudiana Bertoni 5,779,805 A * 7/1998 Morano ...... 127/42 leaves. The resulting Sweeteners are Suitable as non-calorie, 5,962,678 A 10/1999 Payzant et al. non-cariogenic, non-bitter, non-lingering Sweeteners, which 5,972, 120 A 10/1999 Kutowy et al. may be advantageously applied in foods, beverages, and milk 6,031,157 A 2/2000 Morita et al. products. 6,080,561 A 6, 2000 Morita et al. 7,862,845 B2 * 1/2011 Magomet et al...... 426,548 4 Claims, 3 Drawing Sheets US 8,337,927 B2 Page 2

U.S. PATENT DOCUMENTS RU 2198.548 2, 2003 2007,0292582 A1 12, 2007 Prakash et al. W. 386. A. 658 2008/O107775 A1 5, 2008 Prakash 2008/O107776 A1 5, 2008 Prakash OTHER PUBLICATIONS 3888 A. 2. Rish Kitahata, S. et al., “Production of Rubusoside Derivatives by 2008/0292765 A1 11, 2008 PrakashakaS Transgalactosylation of Various b-Galactosidases'. Agric. Biol. 2008/0292.775 A1 11, 2008 Prakash Chem., vol. 53, No.& 8 11 1989, 2923-2928. 2008/0300402 Al 12/2008 Yang et al. Kobayashi, et al., “Dulcoside A and B, New diterpene glycosides 2009.0053378 A1 2/2009 Prakash from Stevia rebaudiana", Phytochemistry, vol. 16 1977, 1405-1408. Kohda, et al., “New Sweet diterpene glucosides from Stevia FOREIGN PATENT DOCUMENTS rebaudiana”, Phytochemistry, vol. 15 1976, 981-983. CN 1049666 3, 1991 Kovylyaeva, et al., “Glycosides from Stevia rebaudiana', Chemistry CN 11OO727 3, 1995 of Natural Compounds, vol.43, No. 1 2007, 81-85. CN 1112565 11, 1995 Liu, et al., “Study of Stevioside preparation by membrane separation CN 1192.447 9, 1998 process”. Desalination, vol. 83 1991, 375-382. CN 1238341 12/1999 Lobov, S.V. et al., “Enzymic Production of Sweet Stevioside Deriva CN 1349.997 5, 2002 tives: Transglucosylation of Glucosidases”. Agric. Biol. Chem... vol. CN 101.2004.80 6, 2008 55, No. 12 1991, 2959-2965. JP 520058OO 1, 1977 Montovaneli, et al., “The effect of temperature and flow rate on the E. 3:63 ET, clarification of the aqueous Stevia-extract in fixed-bed column with JP 521362OO 11, 1977 Zeolites'. Brazilian Journal of Chemical Engineering, vol. 21, No. 3 JP 540301.99 3, 1979 2004, 449-458. JP 54132599 10, 1979 Moraes, et al., “Clarification of Stevia rebaudiana (Bert.) Bertoni JP 55O39731 3, 1980 extract adsorption in modified zeolites'. Acta Scientiarum, vol. 23. JP 55081567 6, 1980 No. 6 2001, 1375-1380. JP 55092400 T 1980 Phillips, K.C., “Stevie: steps in developing a new Sweetener'. In T.H. JP 5512O77O 9, 1980 Grenby, Editor, Developments in Sweeteners-3, Elsevier 1987, 1-43. JP 55138372 10, 1980 Pol, et al., “Comparison of two different solvents employed for JP 55 159770 12, 1980 pressurised fluid extraction of Stevioside from Stevia rebaudiana: E. 3.6% '3. methanol versus water'. Anal Bioanal Chem vol. 388 2007, 1847 1857. E. 5. 88: Shi, et al., “Synthesis of bifunctional polymeric adsorbent and its JP 56121454 9, 1981 application in purification of Stevia glycosides'. Reactive & Func JP 5612145.5 9, 1981 tional Polymers, vol. 50 2002, 107-116. JP 56160962 12/1981 Starratt, et al., “Rebaudioside F, a diterpene glycoside from Stevia JP 57002656 1, 1982 rebaudiana”, Phytochemistry, vol. 59 2002, 367-370. JP 57.005663 1, 1982 Tanaka, O., “Improvement of of natural Sweeteners'. Pure & JP 57046998 3, 1982 Appl. Chem., vol. 69, No. 4 1997, 675-683. JP 57075.992 5, 1982 Teo, et al., “Validation of green-solvent extraction combined with JP 57086264 5, 1982 chromatographic chemical fingerprint to evaluate quality of Stevia JP 58O28246 2, 1983 rebaudiana Bertoni'. J. Sep. Sci., vol. 32 2009, 613-622. E. 585. 33 Yamamoto, K. et al., “Effective Production of Glycosyl-Steviosides JP 58212759 12/1983 by a-1,6 Transglucosylation of Dextrin Dextranase'. Biosci. Biotech. JP 58212760 12/1983 Biochem. vol. 58, No. 9 1994, 1657-1661. JP 59045848 3, 1984 Yoda, et al., “Supercritical fluid extraction from Stevia rebaudiana JP 62166861 7, 1987 Bertoni using CO2 and CO2+ water: extraction kinetics and identi JP 631 73531 T 1988 fication of extracted components”,Journal of Food Engineering, vol. JP 1131.191 5, 1989 57 2003, 125-134. JP 3262458 11, 1991 Zhang, et al., “Membrane-based separation scheme for processing JP 6007108 1, 1994 Sweetener from Stevia leaves'. Food Research International, vol. 33 JP 6192283 T 1994 2000, 617-620. E. 2 4. S. 2. Prakash et al., “Development of rebiana, a natural, non-caloric Sweet JP 8000214 1, 1996 ener,'Jul. Jul. 1,1, 2008., Food and ChemicalUnemical ToxicolIOX1cology, Vol.1.46, 40, IS. 7.f. Sup.S 1, p. S75-S82. 2002, 2.98. Internationalinternational Search Report andan Writtenritten Opinion for PCT/US2010/ RU 2123267 12/1998 055960, mailed Jan. 25, 2011. RU 2156083 9, 2000 RU 2167544 5, 2001 * cited by examiner U.S. Patent Dec. 25, 2012 Sheet 1 of 3 US 8,337,927 B2

FIG.

U.S. Patent Dec. 25, 2012 Sheet 2 of 3 US 8,337,927 B2

FIG. 2

U.S. Patent Dec. 25, 2012 Sheet 3 of 3 US 8,337,927 B2

FIG. 3

US 8,337,927 B2 1. 2 PROCESS FOR MANUFACTURING A vidual glycosides with standard characteristics and minimal SWEETENER AND USE THEREOF content of accompanying compounds. The invention related to the purification of two main gly RELATED APPLICATIONS cosides—Stevioside and Rebaudioside A and use thereof. A process for the recovery of diterpene glycosides, includ This application is a continuation-in-part application of ing Stevioside from the Stevia rebaudiana plant is described and claims the benefit of priority of U.S. patent application (U.S. Pat. No. 4.361,697). A variety of solvents, having dif Ser. No. 1 1/246,066, filed Oct. 11, 2005, now U.S. Pat. No. ferent polarities, were used in a sequential treatment that 7,807,206, issued on Oct 5, 2010; U.S. patent application Ser. concluded with a high performance liquid chromatographic No. 1 1/246,152, filed Oct. 11, 2005, now U.S. Pat. No. 7,862, 10 (HPLC) separation procedure. 845, issued on Jan. 4, 2011; U.S. patent application Ser. No. A method for the recovery of Rebaudioside A from the 12/684,129, filed Jan. 8, 2010; U.S. patent application Ser. leaves of Stevia rebaudiana plants has been developed (U.S. No. 12/684,130, filed Jan. 8, 2010; U.S. patent application Pat. No. 4,082,858). Again, final purification is achieved by Ser. No. 12/684,981, filed Jan. 11, 2010, now U.S. Pat. No. liquid chromatography Subsequent to an initial extraction 8.298,599; and U.S. patent application Ser. No. 12/720,888, 15 filed on Mar. 10, 2010, the contents of which applications are with water and an alkanol having from 1 to 3 carbon carbons, incorporated by reference herein in their entireties. This preferably methanol. It is also known that water may be used application also claims the benefit of priority of U.S. Provi as the initial solvent; their preferred solvent at this stage is a sional Application Ser. No. 61/260,593, filed Nov. 12, 2009, liquid haloalkane having from 1 to 4 carbon atoms. The and U.S. Provisional Application Ser. No. 61/290,778, filed preferred second solvent is an alkanol having from 1 to 3 Dec. 29, 2009, the contents of which applications are incor carbon atoms, while the preferred third solvent is an alkanol porated by reference herein in their entireties. having from 1 to 4 carbon atoms and optionally minor amounts of water. BACKGROUND OF THE INVENTION Individual sweet glycosides can be obtained from the Ste 25 via rebaudiana plant. A mixture of Sweet glycosides extracted 1. Field of the Invention from the Stevia rebaudiana plant is processed to remove The present invention relates to a process for producing a impurities by using two types of ion-exchangers. After highly purified sweet mixture, Stevioside removing the mixed Sweet glycosides from the second col and Rebaudioside A from the extract of the Stevia rebaudiana umn with methanol, the solution is dried. Upon refluxing the Bertoni plant and use thereof in various food products and 30 dried Solids in a methanol Solution and then cooling the solu beverages. tion, Stevioside precipitates out. The filtrate is further con 2. Description of the Related Art centrated and cooled to precipitate out Rebaudioside A. This In view of food sanitation, the use of artificial sweeteners Rebaudioside A can be further purified as can the previously Such as dulcin, Sodium cyclamate and saccharin has been obtained Stevioside (U.S. Pat. No. 5,962,678). However, a restricted. However natural Sweeteners have been receiving 35 large amount of toxic organic solvent, such as methanol is increasing demand. Stevia rebaudiana Bertoni is a plant that used. produces an alternative Sweetener that has an added advan However, all the above-mentioned methods allow the pro tage of being a natural plant product. In addition, the Sweet duction of Stevioside and Rebaudioside A not in highly puri Steviol glycosides have functional and sensory properties fied grade, which further possess a residual bitterness and Superior to those of many high potency Sweeteners. 40 aftertaste. The extract of Stevia rebaudiana plant contains a mixture On the other hand, the unfavorable taste of the glycosides of different Sweet diterpene glycosides, which have a single can be as a result of contamination of impurities, presented in base—stevioland differ by the presence of carbohydrate resi extract. Highly purified Stevioside and Rebaudioside A pos dues at positions C13 and C19. These glycosides accumulate sessing an improved taste profile and there is a need to provide in Stevia leaves and compose approximately 10%-20% of the 45 an easy and commercially valuable process for the manufac total dry weight. Typically, on a dry weight basis, the four turing the highly purified Stevioside and Rebaudioside A, and major glycosides found in the leaves of Stevia are Dulcoside use thereof in various beverages and food products. A (0.3%), Rebaudioside C (0.6%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other glycosides identified in Stevia SUMMARY OF INVENTION extract include Rebaudioside B, C, D, E, and F. Steviolbioside 50 and Rubusoside. An object of the present invention is to provide a commer The physical and sensory properties are well studied only cially valuable process for producing a highly purified Sweet for Stevioside and Rebaudioside A. They were tested for ener from the extract of Stevia rebaudiana Bertoni plant and stability in carbonated beverages and found to be both heat use thereof in various food products and beverages, which and pH stable (Chang and Cook, 1983). The 55 overcomes the disadvantages of the related art. potency of Stevioside is around 210 times higher than The invention, in part, pertains to the dried and powdered , Rebaudioside A in between 200 and 400 times, and leaves being subjected to water extraction and the resulted Rebaudioside C and Dulcoside Aaround 30 times (Phillips, extracts is purified using treatment with a base Such as cal 1989 and Tanaka, 1997). cium hydroxide and then iron chloride. The filtrate was deion However, apart from its high level of sweetness, they have 60 ized on, e.g., Amberlite FPC23H, Amberlite FPA51, and also intrinsic properties of post-bitter taste and unpleasant Amberlite FPA98C1. The filtrate is concentrated under and undesirable aftertaste. Some undesirable taste character vacuum and spray dried. The isolation and purification of istics of glycosides can be as a result of contamination of Stevioside and Rebaudioside A were developed using alco other Substances, presented in extract. holic precipitation and ultrafiltration. The highly purified One of the main ways to improve the taste quality is the 65 Stevioside and Rebaudioside A were obtained. Any type of enzymatic glycosylation of mixture of semi-purified Steviol existing Stevia extract with various ratios of Sweet steviol glycosides. Another way is to produce highly purified indi glycosides are feasible. US 8,337,927 B2 3 4 The highly purified glycosides were applied in various The plant material was separated from the solution by foods and beverages as Sweetener. filtration, and the pH of the filtrate was adjusted to about 10 The invention, in part, pertains to a purified Sweet glyco with calcium hydroxide and heated between 40-60° C., pref sides extract produced from the Stevia rebaudiana plant, erably from 50° C. to 55° C., for about 0.5-1.0 hours, cooled wherein the main Sweet glycosides are Rebaudioside A and 5 to ambient temperature with slow agitation, and neutralized Stevioside, obtained by a process including drying Stevia by FeCls. After mixing for 10-15 minutes, the precipitate was rebaudiana leaves, treating the leaves to extract an aqueous removed by filtration; the filtrate was passed through the liquid solution containing mixed Sweet Steviol glycosides, Celite, deionized, and decolorized by Amberlite FPC23H, extracting the Stevia rebaudiana leaves, obtaining an extract, Amberlite FPA51, and Amberlite FPA98C1 by conventional filtering the extract, obtaining a filtrate, treating the filtrate 10 manner. The Solution was concentrated and spray dried. with a base Such as calcium hydroxide, treating the extract The resulting Sweet Steviol glycoside mixture was a yellow with trivalent iron chloride, desalting, decolorizing, and powder and had a content of 3.4% Dulcoside, 64.6% Stevio evaporating the filtrate to dryness. side, 6.7% Rebaudioside C and 25.3% Rebaudioside A. In the invention, purified Rebaudioside A and Stevioside An HPLC analysis of the obtained product was carried out can be obtained by dissolving Sweet Steviol glycosides in 15 using an Agilent Technologies 1100 Series (USA) equipped methanol at ambient temperatures to precipitate Stevioside, with Zorbax-NH column using acetonitrile-water gradient filtering the solution to recover a precipitate of Stevioside, from 80:20, v/v (2 minutes) to 50:50, v/v during 70 minutes purifying, recovering a high purity Stevioside, concentrating and UV detector at 210 nm. the remaining solution and evaporating to dryness, Suspend The obtained powder was dissolved in methanol and main ing the powder in ethanol, heating and then cooling the solu tained at a temperatures 20-50° C., preferably at 20-25°C., tion to precipitate Rebaudioside A. Suspending the crystal for 0.5-6.0 hours, preferably 0.5-1.0hours with agitation. The line Rebaudioside A obtained in ethanol-water solution at proportion of extract and methanol was between 1:2-1:7, w/v. cool conditions (10-12°C.) prepares a high purity of Rebau preferably 1:5. During this time the precipitate was formed, dioside A. Stevioside or Rebaudioside A has a purity of at which was filtered and dried. According to the HPLC analy least 98%. Applications are found in various foods such as 25 sis, the powder contents were around 90-91% of Stevioside. A chocolate, ice cream, beverage, dairy products, as a Sweetener second treatment by methanol was not efficient to prepare in a tablet form. high purity Stevioside. It is to be understood that both the foregoing general For the further purification, the powder was mixed with description and the following detailed description are exem two volumes of 90% of ethanol and at 10-12°C. and main plary and explanatory and are intended to provide further 30 tained for about 30 minutes with slow agitation. The precipi explanation of the invention as claimed. tate was separated by filtration and dried under vacuum. The Stevioside with about 98.5-99.4% purity was obtained. BRIEF DESCRIPTION OF THE DRAWINGS The filtrates were combined and used for recovery of Rebaudioside A. For this purpose the remaining solution was The accompanying drawings are included to provide a 35 evaporated to remove the methanol, the syrup obtained further understanding of the invention. The drawings illus diluted with water and passed through polysulfone based trate embodiments of the invention and together with the ultrafiltration membranes (with a filtering discrimination of description serve to explain the principles of the embodi 2.5 kDa) (Liumar Technologies, Ottawa, Canada) with dia ments of the invention. filtration. The filtrate was concentrated and spray dried. The FIG. 1 shows a sensory evaluation of raw Stevia extract, 40 obtained powder was mixed with 96.2% ethanol and main Stevioside, and Rebaudioside A: tained at 45-50° C. for about 30 minutes with agitation. The FIG. 2 shows a sensory evaluation of Stevioside with a proportion of syrup and ethanol was between 1:2-1:7, w/v. different grade of purity; and preferably 1:5. During this time the precipitate was formed, FIG.3 shows a sensory evaluation of Rebaudioside A with which was filtered and dried. Rebaudioside A with 88-90% a different grade of purity. 45 purity was obtained. For the further purification the powder was mixed with two volumes of 92% ethanol and maintained DETAILED DESCRIPTION at 10-12°C. for about 60 minutes with slow agitation. The crystals were filtered and dried. Rebaudioside A with 98.9% Advantages of the present invention will become more purity was obtained. apparent from the detailed description given hereinafter. 50 Based on the results of preliminary test on the Sweetening However, it should be understood that the detailed description power of the Sweeteners, aqueous Solutions of commercial and specific examples, while indicating preferred embodi Stevia extract (0.05%) commercialized by Ganzhou Julong ments of the invention, are given by way of illustration only, High-Tech Food Industry Co., Ltd (China), Stevioside since various changes and modifications within the spirit and (0.07%), and Rebaudioside A (0.028%) were prepared. scope of the invention will become apparent to those skilled in 55 The organoleptic test was carried out with 30 previously the art from this detailed description. trained panel members. It was observed that Rebaudioside A The dried leaves of Stevia rebaudiana Bertoni were has the highest sweetness level (5.96), followed by Stevioside extracted by 10 volumes of water. The proportion of extrac with a mean score of 4.62, and commercial Stevia extract had tion water preferably was about 5 liters to about 15 liters (pH the lowest mean score of 2.96. Rebaudioside Ahad the lowest 6.0 to 7.0) to one kilogram of leaves. Greater volumes of 60 score forbitterness (1.76), and commercial Stevia extract was solvent can be used, however, it was not preferable from the the most bitter compared to the other samples. For overall practical standpoint. The duration of extraction may be from acceptability, Rebaudioside A had the highest score of 4.05 0.5 hours to 24 hours, with a period of from about 1 hours to followed by Stevioside (3.81) and raw extract (3.16) (FIG. 1). about 6 hours preferred. The taste profile of Stevioside with 99.3% of purity was The extraction temperature can be in the limits of 25-90° 65 more preferable as compared with 90.2 and 95.4% (FIG. 2). C., however the temperatures between 45-75° C. are more The similar feature was obtained for Rebaudioside A with preferable. various grades of purity (FIG. 3). US 8,337,927 B2 5 6 The highly purified sweeteners can be favorably used for EXAMPLE 2 seasoning various food products (for instance, soy sauce, Soy sauce powder, soy paste, soy paste powder, dressings, may Preparation of Stevioside onnaise, vinegar, powdered vinegar, bakery products and 100 grams (on the base of dry material) of the sweet steviol confectioneries, frozen-desserts, meat products, fish-meat 5 glycoside powder obtained by the process of EXAMPLE 1 products, potato salad, bottled and canned foods, fruit and vegetables) in intact or mixed forms with other sweeteners, was mixed with 0.5 liters of methanol and maintained at 25° Such as corn syrup, , maltose, Sucrose, lactose, aspar C. for 45 minutes with slow agitation. The precipitate Stevio tame, Saccharin, Sugar alcohols, organic and amino acids, side was filtered and dried. 61.2 grams of Stevioside with flavors and/or coloring agents. 90.6% purity was obtained. The products are favorably usable as a low-cariogenic and 10 For the further purification the powder was mixed with two low-calorie sweetener because it is less fermentable by oral parts of 90% of ethanol, and maintained at 10-12°C. for about dental-caries causative microorganisms. Exemplary applica 30 minutes with slow agitation. The precipitate was separated tions include low-cariogenic food products Such as confec by filtration and dried under vacuum. The product weighed tioneries including chewing gum, chocolate, biscuits, cook 58.8 grams and contained 99.3% Stevioside. In another ies, toffee and candy. Additionally applications include soft 15 embodiment, Stevioside with a purity of at least about 98% drinks such as coffee, cocoa, juice, carbonated drinks, Sour was obtained. milk beverage, yogurt drinks and alcoholic drinks, such as brandy, whisky, vodka and wine. In addition to the above EXAMPLE 3 described uses, the Sweeteners are usable for Sweetening drugs and cosmetics. Preparation of Rebaudioside A The following examples illustrate preferred embodiments The remaining solutions after separation of Stevioside (EXAMPLE 2) were combined, and methanol was removed of the invention. by evaporation. The syrup was diluted with water and passed EXAMPLE1 through polysulfone based ultrafiltration membranes (with a 25 filtering discrimination of 2.5 kDa) (Liumar Technologies, Extraction of Sweet Steviol Glycosides Ottawa, Canada) with diafiltration. The filtrate was concen The leaves of Stevia rebaudiana are dried at 55°C. for three trated and spray dried. 40.8 grams of powder with content of hours in a vacuum oven and powdered (30 mesh). One kg of Rebaudioside A of around 60% were obtained. The powder the obtained material was mixed with 10 liters of water (pH was mixed with five volumes (w/v) of 96.2% ethanol and 6.5) and heated to 55°C. with slow agitation for 10 hours. The 30 maintained at 50° C. for 30 minutes with slow agitation. The plant material was separated from the solution by filtration precipitate was filtered and dried. Rebaudioside A with and the pH of the filtrate was adjusted to 10 with about 24 89.8% purity was obtained. The powder was mixed with two grams of calcium hydroxide and heated to 50° C. for 0.5 Volumes of 92% of ethanol and maintained at 12° C. for 60 hours. The obtained mixture was cooled to ambient tempera minutes with slow agitation. The crystals were filtered and ture and the pH was adjusted to about 7.0 by about 53 grams 35 dried. 23.6 grams of Rebaudioside A of 98.9% purity was of FeCls. After mixing for 15 minutes the precipitate was obtained. In another embodiment, Rebaudioside A with a removed by filtration. purity of at least about 98% was obtained. The slightly yellow filtrate was passed through the Celite, deionized, and decolorized by conventional manner on EXAMPLE 4 Amberlite FPC23H, Amberlite FPA51, and Amberlite FPA98C1 commercialized by ROHM & HAAS Co., Ger 40 Low-Calorie Orange Juice Drink many. The Solution was concentrated and spray dried. The Orange concentrate (35%), citric acid (0.38%), ascorbic yield was 122 grams of powder with a content of sweet acid (0.05%), sodium benzoate (0.02%), orange red color glycosides of about 91%. The sweet Steviol glycoside mixture (0.01%), orange flavor (0.20%), and sweetener (0.06%) con contains 3.4% Dulcoside, 64.6% Stevioside, 6.7% Rebaudio taining 90.2, 95.4 or 99.3% of Stevioside, or 80,90, or 98.9% side C and 25.3% Rebaudioside A. In another embodiment, a 45 of Rebaudioside A were blended and dissolved completely in sweet steviol glycoside mixture with a purity of at least about the water (up to 100%) and pasteurized. The sensory evalua 95% was obtained. In a further embodiment, a Sweet Steviol tion of the samples is summarized in the TABLE 1. The data glycoside mixture with a purity of at least about 98% was shows that best results were obtained for highly purified obtained. Rebaudioside A and Stevioside. TABLE 1.

Comments

Sample Flavor Aftertaste Mouth feel Stevioside Sweet and balanced flavor Slight bitterness in Acceptable 90.2% aftertaste Stevioside Sweet and balanced flavor Slight bitterness in Acceptable 95.4% aftertaste Stevioside Sweet, pleasant, balanced flavor Clean, no bitterness Quite full 99.3% Rebaudioside Sweet, rounded and balanced Almost no any Acceptable A - 80.0% Flavor bitterness Rebaudioside Sweet, rounded and balanced Almost no any Full A-90.0% Flavor bitterness Rebaudioside High quality of Sweetness, Clean, no unpleasant Quite full A - 98.9% pleasant, taste similar to aftertaste Sucrose, balanced flavor US 8,337,927 B2 7 8 By the same way can be prepared juices from other fruits, -continued Such as apple, lemon, apricot, cherry, pineapple, etc. Ingredients Quantity, kg EXAMPLE 5 Green tea extract 2SO 5 Salt O.3 Low-Calorie Carbonated Lemon-Flavored Beverage Lemon tincture 1.O.O. L. The formula for the beverage was as below: Juniper tincture 8.0 L Sodium benzoate O.17 Carbonated water up to 1000 L

10 Ingredients Quantity, kg Sensory and physicochemical characteristics of the drink Sugar 3O.O are presented in the TABLE 2. Sweetener 0.4 Citric acid 2.5 The drinks with highly purified Rebaudioside A and Stevioside were superior with an excellent flavor and taste. TABLE 2

Characteristics

Stevioside - Rebaudioside Rebaudioside Item Stevioside -90.2% 99.3% A-90.0% A - 98.9% Appearance Transparent liquid, Transparent Transparent Transparent ree of sediment liquid, free of liquid, free of liquid, free of and strange sediment and sediment and sediment and impurities. A light strange strange Strange opalescence, caused impurities. A impurities. A impurities. A by features of used light ight ight raw materials is opalescence, opalescence, opalescence, possible. caused by caused by caused by features of eatures of eatures of used raw used raw used raw materials is materials is materials is possible. possible. possible. Color From light yellow up to From light From light From light Yellow yellow up to yellow up to yellow up to yellow yellow yellow Taste Sour-sweet, some Sour-sweet, Sour-sweet, Sour-sweet, bitterness in expression of almost no any expression of aftertaste Sweetness is bitterness, Sweetness is rapid. The expression of rapid. taste is Sweetness is satisfactory. rapid.

40 EXAMPLE 6 Low-Calorie Carbonated Drink The formula for the beverage was as below: 45 Ingredients Quantity,% Cola flavor O.340 Phosphoric acid (85%) O.100 Sodium citrate O.310 50 Sodium benzoate O.O18 Citric acid O.O18 Sweetener O.O3O Carbonated water to 100 The beverages prepared with different sweeteners were 55 given to 10 judges for comparison. TABLE 3 shows the results. TABLE 3 Number of panelists

Comparison Stevioside- Stevioside- Rebaudioside A Rebaudioside A Point 90.2% 99.3% 90.0% 98.9%

Bitter taste 6 2 3 O Astringent taste 6 2 3 O Aftertaste 6 2 3 O US 8,337,927 B2 9 10 TABLE 3-continued Number of panelists Comparison Stevioside- Stevioside- Rebaudioside A Rebaudioside A Point 90.2% 99.3% 90.0% 98.9% Quality of Sweet, Clean (7 of the Sweet, some Clean (10 of the Sweet taste bitterness in 10 judges) bitterness in 10 judges) aftertaste (6 of aftertaste (5 of the 10 judges) the 10 judges) Overall Satisfactory (5 Satisfactory (8 Satisfactory (8 Satisfactory (10 evaluation of the 10 of the 10 of the 10 of the 10 judges) judges) judges) judges)

The above results show that the beverages prepared using 15 EXAMPLE 9 highly purified Stevioside and Rebaudioside A possessing good organoleptic characteristics. Yogurt In 5 kg of defatted milk, 4.0 grams of Sweetener, prepared EXAMPLE 7 according to EXAMPLES 2 and 3, were dissolved. After 20 pasteurizing at 82°C. for 20 minutes, the milk was cooled to Chocolat 40° C. A starter in amount of 150 grams was added and the OCOa mixture was incubated at 37° C. for 6 hours. Then, the fer A composition containing 30kg of cacao liquor, 11.5 kg of mented mass was maintained at 10-15°C. for 12 hours. cacao butter, 14 kg of milk powder, 44 kg of Sorbitol, 0.1 kg The product is a low-calorie and low-cariogenic yoghurt of salt, and 0.1 kg of Sweetener prepared according to the 25 without. foreign taste and odor. EXAMPLES 2 or 3, was kneaded sufficiently, and the mix ture was then placed in a refiner to reduce its particle size for 24 hours. Thereafter, the content was transferred into a EXAMPLE 10 conche, 300grams of lecithin was added, and the composition 30 was kneaded at 50° C. for 48 hours. Then, the content was Table Top Tablet placed in a shaping apparatus, and solidified. A mixture, consisting of 58.5% lactose, 10% calcium sili Th cate, 5% cross-carmellose, 5% L-leucine, 1% aerosol 200, eproducts are low-cariogenic and low-calorie chocolate 0.5% magnesium stearate, and 20% of a sweetener, obtained with excellent texture. Also, the organoleptic test carried out according to the EXAMPLE 2 or 3, was kneaded sufficiently. with 20 panelists revealed no lingering after-taste. The most 35 Then the mi haped with th f bletti desirableCS18. ones were the: prproducts withO Rebaudioside-98.9% machine,en the equippedmixture was with shaped punchers with of the6.2 usemm ofdiameter, a tabletting into (19 members) and Stevioside 99.3% (16 members). tablets of 70 mg each, 3.0 mm thick, and 10+1 kg hardness The tablets can be easily administrated due to their appro EXAMPLE 8 40 priate Sweetness. However, the formulations using low grade of Stevioside and Rebaudioside A were somewhat sticky with Ice-Cream solubility about 3-4 minutes in water at 25 C. The tablets, O prepared with highly purified Rebaudioside A show the best 1.5 0kg of whole milk were heated to 45°C., and 300 grams characteristics with the solubility around 20-30 seconds. of milk cream, 100 grams of tagatose, 90 grams of sorbitol. 6 45 grams of carrageenan as a stabilizer, 3 grams of polysorbate 80 as an emulsifier, and 1.0 gram of sweetener prepared EXAMPLE 11 according to the EXAMPLES 2 or 3, were added into the milk and was stirred until the ingredients completely dissolved. Tooth Paste The mixture then was pasteurized at a temperature of 80°C. so A tooth paste was prepared by kneading a composition for 25 seconds. The homogenization of the obtained mixture comprising of calcium phosphate, 45.0%; carboxymethylcel was carried out at a pressure of 800 bars and the samples were lulose, 1.5%: carrageenan, 0.5%; glycerol. 18.0%; polyoxy kept at a temperature of 4° C. for 24 hourStO complete the ethylene sorbitan mono-ester, 2.0%; beta-cyclodextrin, 1.5%: aging process. flavor (1.0% of the m1Xture weight) and sodium laurylsarcosinate, 0.2%; flavoring, 1.0%; preserva coloring (0.025% of the mixture Weight) are added into the ss tive, 0.1%; Rebaudioside A or Stevioside, obtained according mixture after aging. The mixture was then transferred to ice to the EXAMPLE 2 or 3, 0.2%; and water to 100%, by usual cream maker to produce ice cream automatically. Samples of way. The product possesses good foaming and cleaning abili ice creams produced were transferred to seal containers and ties with appropriate Sweetness. were kept in the freezer at a temperature of -18°C. The application of Sweeteners does not affect the physico- 60 EXAMPLE 12 chemical properties of ice cream, as well as the overall attributes of color, Smoothness, Surface texture, air cell, Vanilla aroma intensity, Vanilla taste, chalkiness, iciness and Soy Sauce melting rate. Organoleptic test carried out with 20 panelists. 0.8 g of Rebaudioside A/Stevioside mixture (1:1, ww) The most desirable ones were the products with 98.9% 65 obtained according to the invention was added to 1000 mL of Rebaudioside A (18 members) and 99.3% Stevioside (14 soy sauce and mixed homogenously. The products had an members). excellent taste and texture. US 8,337,927 B2 11 12 EXAMPLE 13 1 1/246,066, 12/684,129 and 12/684,130, the entire contents of which are incorporated by reference herein. The processes Bread described in these co-pending applications involve an enzy 1 kg of wheat flour, 37.38 grams of fructooligosaccharide matic transglycosylation process using CGTases (cyclodex syrup, 80 grams of margarine, 20 grams of salt, 20 grams of 5 trin glycosyltransferase) produced by cultures of Bacillus yeasts, and 0.25 grams of high purity Rebaudioside A or Stearothermophilus to produce Stevia-based Sweeteners, Stevioside, obtained according to the EXAMPLE 2 or 3 were including, but not limited to, Steviosides, Rebaudioside A and placed into the blender and mixed well. 600 ml of water was purified Sweet Steviol glycoside mixtures. poured into the mixture and kneaded sufficiently. At the EXAMPLE 16 completion of the kneading process, the dough was shaped 10 and raised for 30 to 45 minutes. The ready dough was placed Sugar (Sucrose) Based Liquid Sweetener with Rebaudio in oven and baked for 45 minutes. Bread samples had creamy side A 100 g of Rebaudioside A obtained according to white color, and Smooth texture. EXAMPLE 3 and 26 kg of sucrose were dissolved in 26.2 L EXAMPLE 1.4 15 of water to make a solution about 50% w/w total solids content. The obtained solution was evaporated under vacuum Diet Cookies at 60° C. to prepare a syrup with total solids content of 68% wfw. Flour (50.0%), margarine (30.0%), (10.0%), The produced liquid Sweetener is clear colorless syrup maltitol (8.0%), whole milk (1.0%), salt (0.2%), baking pow having a taste profile identical to Sucrose, with Sweetness der (0.15%), (0.1%), Rebaudioside A or Stevioside power about or at least about 2 times higher than Sugar. (0.55%), obtained according to this invention were kneaded In one embodiment similar Sweetener was prepared using well in dough-mixing machine. After molding of the dough Stevioside obtained according to EXAMPLE 2 and yet in the cookies were baked at 200° C. for 15 minutes. another embodiment Sweetener was prepared using Steviol The product is a low-calorie diet cookie with excellent taste glycosides obtained according to EXAMPLE 1. 25 and appropriate Sweetness. EXAMPLE 17 EXAMPLE 1.5 Invert Sugar Based Liquid Sweetener with Rebaudioside A 100 g of Rebaudioside A obtained according to Cake EXAMPLE 3 and 44.6 kg of granulated sugar and 5.7 kg 123 golfhen eggs, 45g of Sugar, 345 g of sorbitol liquid, 2.0 30 invert sugar syrup with 65% total solids content were dis g of sucrose fatty acid ester, 0.35 g of Rebaudioside A or solved in 46.3 L of water to make a solution with about 50% Stevioside was mixed with 100g of wheat flour and 200 g of w/w total solids content. The obtained solution was evapo water in order to prepare a cake according to a conventional rated under vacuum at 60° C. to prepare a syrup with total method. The product had an excellent taste with an optimal solids content of 70% w/w. sweet flavor. 35 The produced liquid Sweetener is clear colorless syrup The following examples describe a process to make a having taste profile identical to Sucrose, with Sweetness sucrose-based sweetener with Stevia-based Sweeteners hav power about or at least about 2 times higher than Sugar. ing improved taste, mouthfeel and flavor properties. Because In one embodiment similar Sweetener was prepared using sucrose and stevia-based sweeteners have very different melt Stevioside obtained according to EXAMPLE 2 and in yet in ing characteristics and solubility, conventional co-crystalli 40 another embodiment Sweetener was prepared using Steviol Zation techniques would not result in a Suitable product. For glycosides obtained according to EXAMPLE 1. example, while sugar is about 66% soluble in water, Rebau EXAMPLE 1.8 dioside A is only about 1% soluble, and Stevia 95 (containing Steviol glycosides at 95% purity) is only about 34% soluble in Corn Syrup Based Liquid Sweetener with Rebaudioside A water. Sugar has a melting point of about 186° C., while 45 100 g of Rebaudioside A obtained according to Rebaudioside A has a melting point of about 240° C., and EXAMPLE 3 and 65 kg of corn syrup with 77% total solids Stevia 95 is mostly amorphous. Because of these and other content were dissolved in 35 L of water to make a solution disparities, conventional processes would not lead to a Suit with 50% w/w total solids content. The obtained solution was able product. evaporated under vacuum at 60° C. to prepare a syrup with Instead, the following process was developed to overcome 50 total solids content of 80% w/w. the difficulties associated with working with sweetening The produced liquid Sweetener is clear colorless syrup components having such different properties. The resulting having taste profile identical to Sucrose, with Sweetness low-calorie Sweetener can be used in any food, beverage or power about or at least about 2 times higher than Sugar. consumer healthcare product. Other ingredients can be incor In one embodiment similar Sweetener was prepared using porated into the low-calorie Sweetener using this process, 55 Stevioside obtained according to EXAMPLE 2 and in yet in including but not limited to Sugar, salt, vitamins, minerals, another embodiment Sweetener was prepared using Steviol Supplements, homeopathic agents, preservatives, citric acid, glycosides obtained according to EXAMPLE 1. juniper tincture, ascorbic acid, Sodium benzoate, colorings, In one embodiment, the dry weight ratio of sucrose to the flavorings, and a combination thereof. sweet steviol glycoside mixture, Stevioside or rebaudioside A Although the processes described in the following 60 is in the range of about 50:1 to about 300:1 (w/w). Examples Examples make references to the Sweeteners made in of other ratios of sucrose to steviol glycosides (“Stevia95” Examples 1-3, it is to be understood that these processes can which contains 95% total steviol glycosides), Stevioside or be used with any Stevia-based Sweeteners, including but not rebaudioside A (Reb A) suitable for the present invention are limited to Rebaudioside A, Stevioside, and purified sweet summarized in Table 4. As used in the Table 4, "Calorie Steviol glycoside mixtures, made by using any process, 65 Reduction” refers to the reduced calories of the sweetener as including the enzymatic transglycosylation processes compared to 100 kg of pure sugar. Each of the following described in co-pending U.S. patent application Ser. Nos. blends provides the equivalent sweetness of 100 kg of sugar. US 8,337 927 B2 14 TABLE 4 It is to be understood that the foregoing descriptions and specific embodiments shown herein are merely illustrative of “Stevia the best mode of the invention and the principles thereof, and RebA 95° Stevio that modifications and additions may be easily made by those Sugar Stevioside glycoside mixture 5 skilled in the art without departing for the spirit and scope of the invention, which is therefore understood to be limited Calorie to Sugar to Sugar to only by the scope of the appended claims. Re- Sugar Weight RebA Weight Stevioside Weight Stevia duction (kg) (kg) ratio (kg) ratio (kg) 95 ratio What is claimed is: 25% 75 O.096 780 0.132 570 O. 119 630 10 1. A process for producing a liquid low-calorie Sweetener 35% 65 O.13S 483 0.188 345 O.167 390 composition, comprising the steps of SO% 50 O.192 26O O.278 18O O.238 210 a) drying Stevia rebaudiana leaves; 75% 25 0.357 70 0.480 52 O.429 58 b) treating the dried Stevia rebaudiana leaves with water to extract Sweet glycosides, wherein a leaves-water mix In one embodiment, the dry weight ratio of the combined is ture is formed; granulated and invert Sugar syrup to the Sweet Steviol glyco c) filtering the leaves-water mixture to obtain an aqueous side mixture, Stevioside or rebaudioside A is in the range of filtrate; about 50:1 to about 300:1 (w/w). Examples of other dry d) directly treating the aqueous filtrate with calcium weight ratios of invert Sugar syrup to Steviol glycosides (“Ste hydroxide or calcium oxide to raise the pH of the aque via95” which contains 95% total steviol glycosides), stevio ous filtrate to about 10; side or rebaudioside A (Reb A) suitable for the present inven e) directly neutralizing the treated aqueous filtrate with tion are summarized in Table 5. As used in the Table 5, trivalent iron chloride; and “Calorie Reduction” refers to the reduced calories of the f) filtrating the neutralized aqueous filtrate to obtain a fil Sweetener as compared to 100 kg of dried invert Sugar syrup. trated aqueous filtrate, and evaporating the filtrated Each of the following blends provides the equivalent Sweet aqueous filtrate to dryness to obtain purified Sweet gly ness of 100 kg of dried invert Sugar syrup. cosides to provide a high intensity Sweetener; TABLE 5

“Stevia 95' Stevio RebA Stevioside glycoside mixture

Invert Total Total Invert Sugar Sugar to Sugar to Sugar to Calorie Sugar Syrup Weight RebA Weight Stevioside Weight Stevia 95 Reduction (Kg) (kg) (kg) ratio (kg) ratio (kg) ratio 25% 70 7.7 0.096 780 O.132 570 O. 119 630 35% 60 7.7 0.135 483 O.188 345 O.167 390 SO% 44 9.2 0.192 260 O.278 18O O.238 210 75% 23 3 0.357 70 O480 52 O.429 58

40 In one embodiment, the weight ratio of corn syrup, con g) providing Sugar granules as a low intensity Sweetener, taining 77% dry-solid, to the sweet steviol glycoside mixture, h) dissolving the high intensity Sweetenerand low intensity Stevioside or rebaudioside A is in the range of about 50:1 to Sweetenerin water to obtaina solution with 45-55% total about 300:1 (w/w). Examples of other ratios of corn syrup to Solids; and either steviol glycosides (“Stevia95” which contains 95% 45 i) evaporating the Solution of high intensity Sweetener and total steviol glycosides), Stevioside or rebaudioside A (RebA) low intensity Sweetener under vacuum to obtain the liq suitable for the present invention are summarized in Table 6. uid low-calorie Sweetener composition with a total Sol As used in the Table 6, "Calorie Reduction” refers to the ids content of about 60% to about 90%. reduced calories of the Sweetener as compared to 100 kg of 2. The process claim 1, wherein a dry weight ratio of the dried corn syrup. Each of the following blends provides the 50 low intensity Sweetener to high intensity Sweetener is in the equivalent sweetness of 100 kg of dried corn syrup. range of about 50:1 to about 300:1 (w/w). TABLE 6

“Stevia 95' Stevio RebA Stevioside glycoside mixture

Corn Corn Corn Corn Syrup to Syrup to Syrup to Calorie Syrup RebA Weight Stevioside Weight Stevia 95 Reduction (kg) Weight (kg) ratio (kg) ratio (kg) ratio

25% 98 O.096 102O O.132 742 O. 119 824 35% 84 O.13S 622 O.188 447 O.167 503 SO% 65 O.192 338 O.278 234 O.238 273 75% 33 0.357 92 O480 69 O.429 77 US 8,337,927 B2 15 16 3. The process of claim 1, wherein the purified sweet gly- 4. The process of claim 3, further comprising the steps of cosides obtained in Step (f) are dissolved in methanol at combining remaining alcohol Solutions, removing the alco temperatures ranging frO about 20-50° C. in an amount of hol, ultrafiltering to obtain a filtrate, recovering Rebaudioside 1:2-1:7 (W/v) to precipitate Stevioside from Solution, filtering A, mixing the Rebaudioside A with dilute alcohol, and filter the solution to recover Stevioside precipitate, suspending the 5 ing and drying to result in Rebaudioside A with a purity of at precipitate in an alcohol-water solution to further purify the least 98% to provide the high intensity sweetener. Stevioside, and recovering the higher purity of Stevioside with a purity of at least 98% to provide the high intensity SWeetener. k . . . .