(51) Int Cl.: A23L 27/30 ( ) C07H 15/256 ( )

Transcription

1 (19) TEPZZ Z ZB_T (11) (12) EUROPEAN PATENT SPECIFICATION (4) Date of publication and mention of the grant of the patent: Bulletin 16/24 (21) Application number: (22) Date of filing: (1) Int Cl.: A23L 27/ (16.01) C07H 1/26 (06.01) (86) International application number: PCT/JP09/0678 (87) International publication number: WO / ( Gazette /14) (4) NEW STEVIOL GLYCOSIDES NEUE STEVIOLGLYKOSIDE NOUVEAUX GLYCOSIDES DE STÉVIOL (84) Designated Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR () Priority: JP (43) Date of publication of application: Bulletin 11/31 (60) Divisional application: (73) Proprietor: Morita Kagaku Kogyo Co., Ltd. Osaka (JP) (72) Inventors: MORITA, Toyoshige Osaka-shi Osaka (JP) FUJITA, Isao Osaka-shi Osaka (JP) MATSUURA, Fumito Fukuyama-shi Hiroshima (JP) OTA, Masaya Fukuyama-shi Hiroshima (JP) (74) Representative: Uexküll & Stolberg Partnerschaft von Patent- und Rechtsanwälten mbb Beselerstraße Hamburg (DE) (6) References cited: EP-A EP-A WO-A2-09/8680 JP-A KOBAYASHI M ET AL: "Dulcosides A and B, new diterpene glycosides from Stevia rebaudiana", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 16, 1 January 1977 ( ), pages 1-18, XP , ISSN: , DOI:.16/S (00) SHINTARO KAMIYA ET AL: "Synthesis and Taste of Some Analogs of Stevioside", AGRICULTURAL AND BIOLOGICAL CHEMISTRY, JAPAN SOC. FOR BIOSCIENCE, BIOTECHNOLOGY AND AGROCHEM, TOKYO, JP, vol. 43, no. 9, 1 January 1979 ( ), pages , XP , ISSN: KASAI,R. ET AL.: Sweet diterpene glycosides of leaves of Stevia rebaudiana Bertoni. SYNTHESIS AND STRUCTURE- SWEETNESS RELATION OF REBAUDIOSIDES A, D, AND E AND THEIR RELATED GLYCOSIDES, NIPPON KAGAKU KAISHI 1981, pages 726-3, XP KAMIYA,S. ET AL.: Synthesis and taste of some analogs of stevioside AGRICULTURAL AND BIOLOGICAL CHEMISTRY vol. 43, no. 9, 1979, pages , XP KUSAMA,S. ET AL.: Transglucosylation into stevioside by the enzyme system from Streptomyces sp AGRICULTURAL AND BIOLOGICAL CHEMISTRY vol. 0, no., 1986, pages 244-1, XP Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). Printed by Jouve, 7001 PARIS (FR) (Cont. next page)

2 Remarks: The file contains technical information submitted after the application was filed and not included in this specification 2

3 Description TECHNICAL FIELD [0001] The present invention relates to a novel Steviol Glycoside, a sweetener containing Rebaudioside A and the novel Steviol Glycoside, which is included in the variety Stevia Rebaudiana Bertoni containing a high content of Rebaudioside A, a method of manufacturing food, pharmaceuticals, non-pharmaceuticals and cosmetics, confirmation of Stevia varieties and analytical method of the novel Steviol Glycoside. BACKGROUND ART [0002] Stevia is a perennial plant of the Asteraceae family grown in Paraguay, South America. Its scientific name is Stevia Rebaudiana Bertoni. Stevia contains components, whose sweetness is 0 times or more than that of sucrose and is planted to be used as a natural sweetener after extracting the sweet components. [0003] Stevioside (C 38 H 60 O 18 ), Rebaudioside A (C 44 H 70 O 23 ), Rebaudioside C, D and E, Dulcoside A etc. have been known as sweet components of Stevia. In the generally planted Stevia variety, Stevioside (hereinafter ST) is the major component among the aforementioned sweet components, with a contained amount of Rebaudioside A (hereinafter RA) of around weight % content and that of Rebaudioside C being slightly less. But depending on the variety there are various types such as those with Rebaudioside C being the major component. [0004] Because ST has a degree of sweetness of 0 times that of sucrose, it has been widely used in the food industry as a natural sweetener. Its sweetness is relatively similar to that of sugar, but it is known that compared to RA an unpleasant taste of bitterness remains in the mouth. Compared to this, RA has good quality of sweetness, with a degree of sweetness from 1.3 to 1. time that of ST, so in general a sweetener of Stevia with a high RA content ratio is desirable rather than ST. The inventors of the present invention carried out plant breeding through the repetition of selective cross fertilizations of conventional varieties, thereby obtaining only Stevia varieties with very few amounts of ST compared to RA, developing sweeteners from these varieties (see Patent Literature 1 for example). [000] However, among the tastes such as bitterness, astringency and smoothness to the tongue, the smooth taste is quite delicate. This delicate smoothness does not rely upon the ratio between ST and RA alone. When glucose is added to the chemical structure of the various sweet components contained in Stevia, the smooth taste is improved and a method has been developed to improve the smooth and strong tastes by structurally adding glucose to the sweet components of Stevia (Patent Literature 2 and 3). [0006] Thus, even if the content amount is small, it is very important to analyze the unknown components contained in Stevia, in particular to grasp the components in which glucose is structurally added rather than ST, and it is extremely important to execute a careful examination of the glucose structurally added to them from a perspective of taste-quality control. [0007] Simultaneously, as the taste quality is influenced by the sweet components included in the raw material plants themselves, it is important to grasp thoroughly the sweet components in order to develop excellent Stevia varieties and use them. From now on, raw material plant improvement of breed will probably become very popular, but it will be possible to grasp in details the results of the breed improvement by specifying diligently the sweet components contained in the plants developed. [0008] On the other hand, the inventors of the present invention developed a method for establishing a variety for plant varieties newly developed by using genes (Patent Literature 4 and ), but actually there were no means of specifying the raw material plants regarding the sweetener extracted and processed from these raw material plants and the products which make use of them. [0009] 0 [Patent Literature 1] Laid Open Patent Publication JP Gazette [Patent Literature 2] Patent Publication JP Gazette [Patent Literature 3] Laid Open Patent Publication JP Gazette [Patent Literature 4] Laid Open Patent Publication JP , Gazette [Patent Literature ] International Patent Publication of Patent Cooperation Treaty -PCT WO06/ Gazette [00] EP relates to a plant belonging to the Stevia Rebaudiana Bertoni variety which contains 4 parts by weight or more of Rebaudioside A with respect to one part by weight Stevioside and allows a sweetening of a good quality to be produced. [0011] EP relates to a flavouring mixture comprising one or more sweet-tasting substances selected from the group of the naturally occurring sweet-tasting substances and their physiologically acceptable salts without phyllodulcin and its physiologically acceptable salts. Kobayashi et al. Phytochemistry, Pergamon Press, vol. 16, January 1977, 3

4 pages 1-18 relates to Dulcosides A and B, new diterpene glycosides from Stevia rebaudiana. [0012] Shintaro Kamiya et al, Agricultural and Biological Chemistry, Japan Soc. For Bioscience, Biotechnology and Agrochem, vol. 43, no. 9, 1 January 1979, pages , relates ot Synthesis and Taste of Some Analogues of Stevioside. DISCLOSURE OF INVENTION PROBLEMS TO BE SOLVED 1 2 [0013] Five components of the stevia sweetener (ST, RA, Rebaudioside A, Dulcoside A and Steviol Bioside) have been analyzed and standardized, but there was no knowledge on the other unknown components. In addition, there was no way to confirm their presence even for the sweet components already known, since there was no established analytical method. However, recently the JECFA standard has been set for 7 components of Steviol Glycosides, the sweeteners contained in Stevia have been confirmed and the unknown components clarified, along with the awareness on the importance to know the influence of the delicate taste from these components. [0014] The purpose of the present invention is to define the structure of the small quantity sweeteners contained in the Stevia variety and to confirm their influence on the taste for Stevia sweetener. [001] Also the other purpose is to provide the measures for specifying the Stevia plant, which became a raw material, regarding Stevia sweetener and the products which use it. [0016] The inventors of the present invention have searched new Steviol Glycosides contained in the Stevia variety, main component of which is RA, and discovered components of the novel Steviol Glycosides which may have a subtle influence on taste quality. After then they completed the present invention by discovering that there was a difference in content volume of these components among varieties, and certain components were present only in the specified Stevia varieties which had RA as their main component followed by confirmation that it was possible to use this as a marker of sweetener originated in such plants. EFFECTS OF THE INVENTION [0017] Steviol Glycosides of the present invention have structures in which more glucoses are added than ST or RA and therefore provide a Stevia sweetener having an excellent strong taste. [0018] Besides, it is possible to suppose the origin of the raw material by confirming Steviol Glycoside X in the extract or the crystals, and analysis of the final product enables to judge if the raw material plant is opposed to rights such as patent and others. 3 (The best mode for embodying the invention) 4 [0019] The Stevia variety with RA as main component mentioned in the present invention is a variety which contains more RA content than ST, and described in the applications for patent JP and JP ; the extract obtained from dry leaves has more RA content than ST and also contains Rebaudioside D (R-D), Steviol Glycoside III, V, VI, VII and X, making it possible to obtain an excellent sweetener of strong taste by containing the component in which more glucoses are structurally added than ST and/or RA. [00] In addition, it is possible to efficiently obtain highly pure RA sweetener containing a trace amount of ST and Steviol Glycoside X by recrystalization. [0021] The first embodiment of the present invention is Steviol Glycoside of the formula III to x, where compounds not falling within the claims are considered reference examples useful for understanding the invention: 0 4

5 1 wherein R 1 and R 2 are a sugar chain defined in the next table; No. (Steviol Glyciside) R 1 R 2 III (Rebaudioside I) B-glc-B-glc(3 1) 2 IV (Rebaudioside H) B-glc V (Rebaudioside L) B-glc VI (Rebaudioside K) Bglc-B-glc(2 1) 3 VII (Rebaudioside J) B-glc-α-rha(2 1) VIII (Rebaudioside M) 4 IX (Rebaudioside N) 0 X (Rebaudioside O) [0022] The symbols in the formula are the sugar below: glc: D-glucopyranosyl

6 rha: L-rhamnopyranosyl xyl: xylopyranosyl [0023] extract containing Steviol Glycoside X (Rebaudioside 0) may be obtained by extracting a plant body of Stevia Rebaudiana Bertoni, a plant of the Asteraceae family, or its dry leaves, a main component of which is Rebaudioside A, by water or a solvent containing water. [0024] A method of obtaining highly pure Rebaudioside A containing Steviol Glycoside X (Rebaudioside 0) may be provided through the recrystalization of the aforementioned extract. [002] A manufacturing method for foodstuff may be provided, in which the extract of the above extract is added to food in an amount of equal to or less than 1% of the food. [0026] A manufacturing method for foodstuff may be provided, in which the highly pure Rebaudioside A obtained in the method above is added to food in an amount of equal to or less than 1% of the food. [0027] In the extract obtained from the raw material variety, whose main component is ST, there is no Steviol Glycoside X, but as the extract obtained from raw material varieties, whose main component is RA, has Glycoside X, it is possible to judge the raw material variety by whether the main component is ST or RA. That is, as there is no Glycoside X in the extract containing RA as a main component, which is obtained by eliminating ST by crystalization through the extract obtained from varieties whose main component is ST, or in the high purity products obtained from their recrystalization, it is possible to confirm the raw material varieties. The sixth embodiment of the present invention is the method for confirming Stevia varieties by Steviol Glycoside X. [0028] An analitical method of the Steviol Glycosides III-X may be provided through high performance liquid chromatography (hereinafter HPLC). [0029] In order to accomplish these objectives, the inventors of the present invention extensively studied the sweet components included in the varieties whose main component is RA and the varieties of applications JP and JP , found new sweet components and determined their chemical structure. In addition, they confirmed the usefulness of these components as sweeteners and completed the analytical method and confirmation method of varieties through these varieties. [00] The confirmation of the new components was executed through extraction by water or solvent containing water of the varieties whose main components was RA as in the Example 1 (hereinafter called Variety A) and the dry leaves of applications JP (hereinafter called Variety B) and (hereinafter called Variety C). [0031] After that, the extract solution is directly concentrated, or if necessary, ionic impurities are removed with an ionic exchange resin or a cationic exchange resin, or activated carbon, the sweetening components are allowed to be absorbed into an absorption resin, followed by the elution with a hydrophilic solvent, and if necessary the elute is concentrated and dried and the eluant retreated with an ionic exchange resin or a cationic exchange resin, or activated carbon, and the extract thus obtained or the extract obtained through an appropriate purification means of conventional art such as discoloring can be confirmed. [0032] The Steviol Glycosides of the extract RA-C obtained from the Example 1(1) described hereinafter were separated and analyzed by a device of High performance liquid chromatography mass spectrometry (HPLC-MS) according to Example and each Glycoside structure I-X: 4 0 wherein R 1 and R 2 are respectively a hydrogen atom and the aforementioned sugar chain, was determined. [0033] Steviol Glycoside I (Dulcoside B) is a glycoside with a structure of 788 molecular weight, as confirmed by about 13 minutes of chromatograph Retention Time (hereinafter R.T.) of the HPLC in Figure 1. 6

7 1 2 [0034] Steviol Glycoside II (Rebaudioside G) is a glycoside with a structure of 804 molecular weight, as confirmed by about 1 minutes of R.T. of HPLC in Figure 1. [003] Steviol Glycoside III (Rebaudioside I) is a glycoside with a structure of 1128 molecular weight, as confirmed by about 28 minutes of R.T. of HPLC in Figure 1. [0036] Steviol Glycoside IV (Rebaudioside H) is a glycoside with a structure of 1112 molecular weight, as confirmed by about 29 minutes of R.T. of HPLC in Figure 1. [0037] Steviol Glycoside V (Rebaudioside L) is a glycoside with a structure of 1128 molecular weight, as confirmed by about 34 minutes of R.T. of HPLC in Figure 1. Steviol Glycoside VI (Rebaudioside K) is a glycoside with a structure of 1112 molecular weight, as confirmed by about 34 minutes of R.T. of HPLC in Figure 1. [0038] Steviol Glycoside VII (Rebaudioside J) is a glycoside with a structure of 1112 molecular weight, as confirmed by about 34 minutes of R.T. of HPLC in Figure 1. [0039] Steviol Glycoside VIII (Rebaudioside M) is a glycoside with a structure of 1290 molecular weight, as confirmed by duplicating Rebaudioside D by about 34 minutes of R.T. of HPLC in Figure 1. [00] Steviol Glycoside IX (Rebaudioside N) is a glycoside with a structure of 1274 molecular weight, as confirmed by about 43 minutes of R.T. of HPLC in Figure 1. [0041] Steviol Glycoside X (Rebaudioside O) is a glycoside with a structure of 1436 molecular weight, as confirmed by about 1 minutes of R.T. of HPLC in Figure 1. [0042] However, anyone skilled in the art will understand that the R.T. mentioned above from the analysis using gradient elution is variable. [0043] As mentioned above, it provides with important information for the identification of the raw material variety whether Steviol Glycoside X is present or not in the final product. [0044] Simultaneously, since it is possible to confirm these Steviol Glycosides through HPLC, even in case they are used as sweeteners, quality and taste may be totally controlled with ease by HPLC analysis. [004] The extract or crystals obtained may be used as sweetener in candies, jellies, powder beverages, instant noodles, jams, frozen fruits, chewing gums, Japanese sweets, health foods, chocolate, tabletop sweeteners, fried sweets, delicacies, water boiled foods, fermented lactic-drink, coffee drinks, cocoa drinks, tea drinks, liqueurs, wines, sherbets, cereals, vegetable fiber-containing foods, sauces, soy sauce, soy paste, vinegars, dressings, mayonnaises, catch-up, curry, soups, rice sweets, arare, breads, biscuits, crackers, pancake mix, canning fruits, canning vegetables, meat products, products made with boiled fish paste, salt foods, pickles, combined seasoning, luxury foods, cosmetics, etc, resulting in calorie decrease, sucrose reduction, melting point decrease, improvement of sweetness quality and masking effect, among others, also being possible to be added to other natural and artificial sweeteners and solvents. EXAMPLES 3 Example 1 Manufacturing of RA Extract (1) Extract 4 [0046] 0 g of dry leaves obtained from varieties A, B or C, whose main component is RA, was extracted several times with times amount of water by weight until the sweetness cannot be tasted. The extract was passed through a column filled up with 0 ml of absorption resin (Diaion HP-) and the sweet components of the extract were absorbed to the resin, which was sufficiently washed with water, and the components were eluted with 900ml of methanol. The eluate was passed through a column filled up with 0 ml of ion exchange resin (Diaion WA-), g of activated carbon was added to the eluate and stirred. The mixture was filtered, the filtrate was concentrated and the residue was dried to give 13.0 g of RA-A extract, whose main component is light yellow Rebaudioside A (ST 3.4%, RA 41.7% and RC 9.8%), 11.g of RA-B extract (ST 19.%, RA 8.1% and RC 8.8%) and 12g of RA-C extract (ST.4%, RA 72.3% and RC 8.1%) respectively. 0 (2) RA Recrystallization [0047] Each g of the aforementioned RA-B extract and RA-C extract was dissolved in times the weight of 90% methanol under heating, and it was left stand at 4 C for six days. The resulted crystals were separated, washed with cold methanol and dried under reduced pressure to give 3.9g of white RA-B crystals (ST 0.2%, RA 9.0% and RC 0.2%) and 4.g of RA-C Crystals (ST 0.2%, RA 9.6% and RC 0.1%) respectively. Example 2 Manufacturing of ST Extract [0048] For comparison purposes, the same procedure was carried out as to a variety, the main component of which 7

8 is ST, to give 11.3g of ST extract (ST 1.9%, RA 23.7% and RC 7.4%). Example 3 RA-A Mother liquid, ST Mother liquid [0049] Each g of the aforementioned RA-A extract and ST extract was dissolved in times the weight of 90% methanol under heating, and it was left stand at 4 C for six days. The resulted crystals were separated, washed with cold 98% methanol and dried under reduced pressure to give 2.1g of RA-ST crystals, which are white crystals of stevioside, and 3.8g of ST-ST crystals respectively. [000] Each of 8.8g of RA-A mother liquid(st 1.7%, RA 43.8% and RC 6.9%) and 6.1g of ST mother liquor (ST.0%, RA 37.1% and RC 11.2%), whose main component is RA, is concentrated and dried to give a powder of mother liquid, whose main component was a pale yellow RA respectively. Example 4 RA-A Crystal, ST-RA Crystal 1 [001] Each of the mother liquid powder of Example 3 was dissolved in times the weight of 90% methanol under heating and left stand at 4 C for six days. The resulted crystals were separated, washed with cold 98% methanol and dried under reduced pressure to give 2.2g of white RA-A crystals (ST 1.6%, RA 90.4% and RC 1.4%) and 1.2g of ST- RA crystals (ST 1.6%, RA 96.9% and RC 1.4%) respectively. Example Structural Determination of Steviol Glycosides 2 3 [002] As described below, analysis was performed by using HPLC. Separation of the Steviol Glycosides included in each extract was carried out by using Shimazu LC-Advp HPLC using a column of TSKgel Amide-80 (4.6 x mm Tosoh). Acetonitril-water was used as a solvent and a gradient elution in which the ratio of acetonitril:water was changed from 82:18 to 66:34 within 60 minutes was carried out. The flow rate was 0.6ml/min, the column temperature was C and the detection was performed at ultraviolet absorption of 2nm. [003] In the measurement of molecular weight, Waters Alliance HPLC System 269 and Waters Quattro micro (triple quadrupole mass) equipped with electrospray ionization (ESI)-mass spectrometer were used. As to HPLC, a column was TSKgel Amide-80 (2.0 x mm, Tosoh), acetonitril-water was used as a solvent and a gradient elution in which the ratio of acetonitril:water was changed from 82:18 to 66:34 within 60 minutes was carried out. The flow rate was 0.2 ml/min, the column temperature was C. Nitrogen gas was used as a desolvation gas and argon gas was used as a collision gas. As a capillary voltage, 1.0kV were used in the Steviol Glycoside analysis in the negative mode and in the analysis of ABEE-oligosaccharides 13. kv were used in the positive mode. A voltage of V to 80V was used as the cone voltage and the collision voltage at the time of MS/MS analysis. The source temperature and desolvation temperature were 0 C and 0 C respectively and the flow volume of cone gas and desolvation gas were 0 1/hr and 900 1/hr respectively. [004] The results of HPLC Analysis regarding each extract and crystal were illustrated in Figures 1-. [00] The analytical results of each chromatography peak shown in Figures 2- were shown in the following Table [Table 1] - Crystals RA-A CH PKNO T A H MK IDNO CONC N Stev mono Rubuso Rebau B Stev Rebau C Rebau F Rebau A Rebau E+III+IV V+VI+VII V Rebau D+VIII IX 8

9 (continued) CH PKNO T A H MK IDNO CONC N X TOT [Table 2] - Crystals RA-B CH PKNO T A H MK IDNO CONC N V I Rebau B V Stev V Rebau C Rebau F Rebau A III + Rebau E+IV Rebau D+V+VI+VII IX X TOT [Table 3] - Crystals RA-C CH PKNO T A H MK IDNO CONC N Rubuso Rebau B Stev Rebau C Rebau F Rebau A V+VI+Rebau D+VII+VIII IX X TOT

10 1 2 3 [Table 4] - Extract RA-A CH PKNO T A H MK IDNO CONC N V V V V Stev mono V V V V V Stev bio V V V 1.8 Rubuso V V Dulco A V I Rebau B Stev V 9.94 Rebau C V Rebau F Rebau A V III + Rebau E V IV V+VI+VII Rebau D+VIII IX X TOT [Table ] - Extract RA-B CH PKNO T A H MK IDNO CONC N V V Stev mono V V V Stev bio V V V V Rubuso V

11 (continued) 1 2 CH PKNO T A H MK IDNO CONC N V V Dulco A V I II Stev V Rebau C V Rebau F S Rebau A T V V Rebau E+III+IV V V+VI+VII V Rebau D+VIII IX X TOT [Table 6] - Extract RA-C CH PKNO T A H MK IDNO CONC N V V Stev mono V V V V Rubuso V V 0.6 Dulco A Rebau B V Stev Rebau C V Rebau F Rebau A V Rebau E V III+IV 11

12 (continued) CH PKNO T A H MK IDNO CONC N V V V VI+VII V Rebau D+VIII IX X TOT [Table 7] - Extract ST CH PKNO T A H MK IDNO CONC N V V V V V Stev mono V V V V V Stev bio V V Rubuso V V Dulco A V I V V Rebau B V II SV. 916 Stev T V Rebau C V Rebau F SV Rebau A T Rebau E+III+IV V+VI+VII V Rebau D+VIII IX TOT [Table 8] - Crystals ST-ST CH PKNO T A H MK IDNO CONC N

13 (continued) 1 CH PKNO T A H MK IDNO CONC N Rubuso I S Stev T C Rebau F V A Rebau E+III+IV TOT [Table 9] - Crystals ST-RA CH PKNO T A H MK IDNO CONC N Rebau B Stev Rebau C Rebau F Rebau A Rebau E+III+IV V+VI+VII Rebau D+VIII IX TOT [006] The abbreviations used in the tables above are as follows: 4 0 PKNO: Peak Number T: Time (minutes) A: Peak area H: Peak Height CONC: Concentration (%) N: Glycoside s Name TOT: Total Stev mono: Steviol monoside Stev bio: Steviol bioside Rebuso: Rubososide Rebau: Rebaudioside Stev: Stevioside Dulco: Dulcoside [007] The concentration is calculated from the total area of the ultraviolet absorption spectrum at 2 nm, and it is necessary to correct the molecular weight in order to measure the volume contained. I to X in the chromatograph chart indicate the novel Steviol Glycoside I to X. 13

14 Example 6 Evaluation of taste quality [008] 0.0% aqueous solution of each extract and 0.03% aqueous solution of the crystals were evaluated by people familiar with a sensory test of Stevia, and the results of averaged evaluation are shown in Table below: Evaluation : Excellent, 4: Good, 3: Ordinary, 2: Bad, 1: Worst 1 2 [Table ] Subject matter of the sensory test 1) 2) 3) 4) ) 6) RA-A Extract RA-A Crystal RA-B Extract RA-B Crystal RA-C Extract RA-C Crystal ST Extract ST-RA Crystal ) Sweetness Quality 2) Taste remaining in the mouth 3) Astringency 4) Delicate taste ) Refreshing feeling 6) Sweetness running out [009] The three types of RA Extract containing the Steviol Glycosides II to X (Rebaudioside G to O) are excellent in delicate taste compared to the three types of RA Crystals, but the RA Crystals are excellent in the other evaluations. Except for the delicate taste evaluation, the ST extract is inferior to the ST-RA Crystals. From this result, it is verified that the Steviol Glycosides II-X influence the delicate taste. 3 4 Example 7 Determination of Variety [0060] According to the HPLC analysis of each extract and/or crystal, the extract obtained from the varieties containing RA as a main component(hereinafter Variety RA) includes more Rebaudioside D, and also include the Steviol Glycoside X (Rebaudioside O) compared with the extract obtained from the varieties containing ST as a main component (hereinafter Variety ST). Further it is found that the Steviol Glycoside X is also included in the RA crystals obtained by the purification of the extract of RA varieties, although trace amount. [0061] On the other hand, there is no Steviol Glycoside X in the extract obtained from the ST varieties. Naturally the Steviol Glycoside X is not found out in the ST-RA crystals obtained from the ST varieties and it is therefore possible to confirm that the extract or the crystal is obtained from the RA variety, if the presence of Steviol Glycoside X is confirmed. Example 8 Analytical method of Steviol Glycoside 0 [0062] According to the HPLC condition described in Example, it is possible to confirm each Steviol Glycoside I-X. In principle, it is possible to confirm the presence of Steviol Glycoside from R.T. in the HPLC analytical chart, but each Steviol Glycoside I-X can be confirmed by measuring the molecular weight after preparative isolation of each Glycoside. Example 9 Tabletop sugar [0063] 1) Tabletop sugar was prepared by mixing 1g of RA-A Crystals and 99g of powder sugar. 2) Tabletop sugar was prepared by mixing 1g of RA-B Crystals and 99g of erythritol. 3) Tabletop sugar was prepared by mixing 1g of RA-C Crystals and 99g of high-fructose corn syrup. 14

15 Example Candies [0064] Candy was prepared from 0.3g of RA-C extract, 0g of palatinit and an appropriate volume of spices. Example 11 Milk jelly [006] Milk jelly was prepared form 1g of sugar, 0.08g of RA-B extract, g of milk, g of gelatin and an appropriate volume of milk flavor. Example 12 Sports drinks [0066] Sports drink was prepared from 0.07% of RA-B Crystals, 0.11% of calcium lactate, 0.04% of citric acid, 0.03% of trisodium citrate, 0.01% of magnesium chloride, 0.00% of glutamic acid and 99.72% of water. 1 Example 13 Carbonate drinks [0067] Carbonate drink was prepared by adding 0.012% of RA-B Crystals, 8.4% of fructose, 0.6% of citric acid, 0.12% of arginine, 0.1% of inositol, 0.002% of caffeine, % of calcium pantothenate, 0.003% of niacin amide, 0.002% of vitamin B6, % of vitamin B2, % of vitamin B12, and appropriate volumes of spices and water to adjust 0% of whole ingredients, and then introducing carbon dioxide gas. INDUSTRIAL APPLICABILITY 2 [0068] By HPLC analysis of the novel Steviol Glycosides provided by the present invention, it is possible to manufacture sweeteners and other foodstuff with a certain sweetness degree and quality, and delicate taste. Also it enables to presume the raw material varieties, and the invention is helpful to judge the correctness of indication of origin, cultivation area of the Stevia varieties, or infringement of right. BRIEF DESCRIPTION OF DRAWINGS [0069] [Fig. 1] It shows the HPLC Analysis Chart for the extract RA-C. 3 [Fig. 2] It shows the HPLC Analysis Chart for Crystals RA-A. [Fig. 3] It shows the HPLC Analysis Chart for Crystals RA-B. [Fig. 4] It shows the HPLC Analysis Chart for Crystals RA-C. [Fig. ] It shows the HPLC Analysis Chart for the extract RA-A. [Fig. 6] It shows the HPLC Analysis Chart for the extract RA-B. 4 [Fig. 7] It shows the HPLC Analysis Chart for the extract RA-C. [Fig. 8] It shows the HPLC Analysis Chart for the extract ST. 0 [Fig. 9] It shows the HPLC Analysis Chart for Crystals ST-ST. [Fig. ] It shows the HPLC Analysis Chart for Crystals ST-RA. Claims 1. A Steviol Glycoside of the formula III, VII, VIII, IX and X: 1

16 wherein R 1 and R 2 are a sugar chain defined in the next table; 1 No. R 1 R 2 III B-glc-B-glc(3 1) VII B-glc-α-rha(2 1) 2 VIII IX 3 X 2. Steviol Glycoside of the formula III according to claim Steviol Glycoside of the formula VII according to claim Steviol Glycoside of the formula VIII according to claim 1.. Steviol Glycoside of the formula IX according to claim Steviol Glycoside of the formula X according to claim 1. Patentansprüche 1. Steviolglycosid mit der Formel III, VII, VIII, IX und X: 16

17 wobei R 1 und R 2 eine in der nachfolgenden Tabelle definierte Zuckerkette sind: 1 No. R 1 R 2 III B-glc-B-glc(3 1) VII B-glc-α-rha(2 ) 2 VIII IX X 3 2. Steviolglycosid mit der Formel III nach Anspruch Steviolglycosid mit der Formel VII nach Anspruch Steviolglycosid mit der Formel VIII nach Anspruch Steviolglycosid mit der Formel IX nach Anspruch Steviolglycosid mit der Formel X nach Anspruch 1. 0 Revendications 1. Glycoside de stéviol de formule III, VII, VIII, IX et X : 17

18 dans laquelle R 1 et R 2 sont une chaîne de sucre définie dans le tableau suivant ; 1 No. R 1 R 2 III B-glc-B-glc(3 1) VII B-glc-α-rha(2 1) 2 VIII IX 3 X 2. Glycoside de stéviol de formule III selon la revendication Glycoside de stéviol de formule VII selon la revendication Glycoside de stéviol de formule VIII selon la revendication Glycoside de stéviol de formule IX selon la revendication Glycoside de stéviol de formule X selon la revendication

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29 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description JP A [0009] JP B [0009] JP A [0009] JP A [0009] WO A [0009] EP A [00] EP A [0011] JP A [0019] [0029] [00] JP A [0019] [0029] Non-patent literature cited in the description KOBAYASHI et al. Phytochemistry. Pergamon Press, January 1977, vol. 16, 1-18 [0011] SHINTARO KAMIYA et al. Agricultural and Biological Chemistry, Japan Soc. For Bioscience, Biotechnology and Agrochem, 01 January 1979, vol. 43 (9), [0012] 29