Process for separation, isolation and characterization of steviol glycosides

Abstract

A comprehensive process for the separation, isolation and characterization of a combination of two or more steviol glycosides from extract of Stevia rebaudiana plants and their use in sweetening compositions are disclosed. Combinations of two or more steviol glycosides from Stevia rebaudiana are characterized. The combinations of two or more steviol glycosides can be used as sweetness enhancers, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals. A method for isolating combinations of two or more steviol glycosides is also disclosed.

Claims

1. A process for production a steviol glycoside composition comprising: a) extracting dried Stevia leaves for 2 h at 95° C. to form a crude extract; b) fractionating the crude extract on a reverse-phase column, wherein fractionation is conducted using a gradient of water and ethanol, wherein said gradient is used starting from 1:0, moving to 29.8:70.2, moving to 23.4:76.6, moving to 0:1, and moving to 1:1 water to ethanol, where the ethanol is food grade ethanol; wherein, fractions containing rebaudioside D, N, M, and A are collected and the water and ethanol removed, to provide a steviol glycoside composition comprising 23% rebaudioside D, 21% rebaudiosides N and M, and 2% rebaudioside A; and wherein, rebaudiosides D, N, M, and A are obtained in the claimed percentages without the need for crystallization.

2. The process of claim 1, wherein the Stevia leaves have not been desiccated.

3. The process of claim 1, wherein the Stevia leaves have been desiccated.

4. The process of claim 1, wherein the resulting composition further comprises one or more of rebaudiosides I and O.

5. The process of claim 1, wherein the crude extract is clarified by centrifugation or filtration before fractionation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows steviol glycoside aglycone structure with indicated location of glycosylation (R1 and R2).

(2) FIG. 2 shows the organization of steviol glycosides.

(3) FIG. 3 is a chart that how subjective taste quality and sweetness can be predicted by molecular size of the steviol glycoside.

(4) FIG. 4 shows mass spectra for steviol glycosides of interest.

(5) FIG. 5 shows mass spectra for steviol glycosides of interest.

(6) FIG. 6 shows extraction and purification steps that may be used in accordance with the invention.

(7) FIG. 7 shows the use of simulated moving bed chromatography in accordance with the invention.

(8) The present invention may be more fully understood by reference to the Figures, Detailed Description and Examples which follow.

DETAILED DESCRIPTION OF THE INVENTION

(9) It is believed that one skilled in the art can, based upon the description herein, utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and not as limiting the remainder of the disclosure in any way whatsoever.

(10) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. As used herein, all percentages are by weight unless otherwise specified. In addition, all ranges set forth herein are meant to include any combinations of values between the two endpoints, inclusively.

Definitions

(11) By the term “flavor notes” it is meant subtle sensory aspects typically detected by taste, or smell experienced while exhaling through the nose after ingestion (retronasal olfaction).

(12) By the term “steviol” it is meant the diterpenoic compound hydroxy-ent-kaur-16-en-13-ol-19-oic acid, which is the hydroxylated form of the compound termed “ent-kaurenoic acid”, which is ent-kaur-16-en-19-oic acid.

(13) By the term “steviol glycoside” it is meant any of the glycosides of the aglycone steviol including, but not limited to, stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudisode E, Rebaudisode F, Rebaudioside I, Rebaudiouside M, Rebaudioside N, Rebaudioside O, dulcoside, rubusoside, steviolmonoside, steviolbioside, and 19-O-β-glucopyranosyl-steviol.

(14) Examples of synthetic sweeteners include sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone synthetic derivatives, cyclamate, neotame, dulcin, suosan, N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester (Advantame), N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, salts thereof, and the like.

(15) Examples of natural high intensity sweeteners include Stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside, some mogrosides (for example, Mogroside V), brazzein, neohesperidin dihydrochalcone (NHDC), glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside and others.

(16) Suitable “heat-stable, high-intensity sweeteners” include chemical compounds or mixtures of compounds, which elicit a sweet taste at least five times sweeter than sucrose, as measured in accordance with the test method described in G.B. Patent No. 1,543,167, which is incorporated by reference herein. Typically such sweeteners are substantially free from degradants after being heated for about one hour at about 40° C. Examples of such suitable sweeteners include, but are not limited to, sucralose, neotame, saccharin, acesulfame-K, cyclamate, neohesperdine DC, Stevia, thavmatin, brazzein, aspartame, and mixtures thereof.

(17) Stevia is a non-caloric natural sweetener from the plant Stevia rebaudiana. The plant makes a number of sweet compounds collectively referred to as steviol glycosides, which make Stevia up to 300 times sweeter than sucrose. These glycosides can be extracted from the plant with water and other solvents well known to those skilled in the art. They are heat stable, pH stable, do not ferment, and do not induce a glycemic response.

(18) Stevioside, sometimes referred to as 13-[(2-O-β-D-glucopyranosyl-α-D-glucopyranosyl)oxy]-kaur-16-en-18-oic acid β-D-glucopyranosyl ester, and rebaudioside A are exemplary glycosides of the diterpene derivative steviol, extracted and refined from Stevia rebaudiana (also known as Eupatorium rebaudianum) leaves. These glycosides are high intensity sweeteners, about 100 to about 500 times that of sucrose, but have metallic and bitter notes. They can be used in a wide range of low or reduced calorie food products and beverages.

(19) Other sweet glycosides can also be extracted from Stevia rebaudiana. These have varying degrees of sweetness. As used herein “Stevia extract” means a sweet glycoside extracted from a Stevia plant.

(20) Of the glycosides found in Stevia extracts, Rebaudioside A has been generally believed to have the least aftertaste. This aftertaste, which has been described by many as bitter and licorice like, is present in all current Stevia-sweetened products. Such formulations typically require extensive dilution or taste-masking technology.

(21) Like with all high intensity sweetener containing sweetener compositions, Stevia containing sweetener compositions typically have been provided with a bulking agent to aid in measurement and distribution into the users application. Among those disclosed or used include fructooligosaccharide (FOS) and other fibers, maltodextrins, and erythritol. Erythritol is especially popular as it can mitigate some of the bitter taste.

(22) U.S. Patent Applications Nos. 20120201952 and 20120201940 to Catani et al. disclose a method of making a natural sweetening composition comprising steam stripping a crude mixture comprising at least one plant based natural high intensity sweetening compound and filtering the crude mixture.

(23) U.S. Application Serial No. 20100285201 to Catani et al. discloses a synergistic sweetening composition that comprises sucralose and a purified extract of Stevia, wherein the purified extract of Stevia comprises rebaudiosides and dulcosides.

(24) U.S. Patent Application No. 20090017185 to Catani et al. discloses a reduced calorie sweetening composition consisting of a Stevia extract and a simple sugar. The reference discloses that the Stevia extract may have a rebaudioside A level of from about 80 wt % to about 99.5 wt % relative to all steviol glycosides and the simple sugar may be sucrose, fructose or glucose.

(25) U.S. Patent Application No. 20090004355 to Catani discloses a sweetening composition comprising erythritol and a Stevia extract.

(26) The present invention provides a process for the separation, isolation and characterization of a combination of two or more sweet glycosides from Stevia rebaudiana plant extract with molecular weights in the range of about 966 g/mol to about 1436 g/mol and with Rebaudioside A less than 25% of steviol glycosides, more preferably with Rebaudioside A less than 15% of steviol glycosides, more preferably with Rebaudioside A less than 10% of steviol glycosides, and more preferably with Rebaudioside A less than 5% of steviol glycosides.

(27) Advantages of the present invention will become more apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

(28) Among sweet glycosides existing in Stevia, only Stevioside, Rebaudioside A and Rebaudioside C are available at moderate cost at <80% purity and at high cost at >80% purity. The highest purity of commercial product usually is more than 97%. Hereinafter, the term “highly purified” refers to a steviol glycoside composition that includes at least about 90% to about 100% of the steviol glycoside on a dry weight basis. In the market there are no commercial quantities of highly purified Rebaudioside B or Rebaudioside D, two Stevia components with taste quality and intensity comparable to Rebaudioside A. Rebaudiosides E and F, also good-tasting sweeteners, are available in minor quantities as analytical standards. No commercial use has been made of naturally occurring steviol glycosides of molecular mass comparable or larger than Rebaudioside D. The present invention seeks to define a composition of such large molecular mass components, and low-cost method of obtaining that composition, which compensates for the low concentration of the components in Stevia rebaudiana by using them collectively as a sweetening agent.

(29) There is a need for an efficient and economical method for comprehensive separation, isolation and/or characterization of combinations of two or more sweet glycosides from Stevia extract with molecular weight greater than about 900 g/mol. Individual steviol glycosides that have been developed for commercial use have limitations in terms of taste quality and sweetness temporal profile. The characteristics and limitations of isolated naturally occurring and man-made sweeteners is described by Grant E. DuBois and Indra Prakash in Annu. Rev. Food Sci. Technol. (2012), 3:353-380 (Non-Caloric Sweeteners, Sweetness Modulators, and Sweetener Enhancers). The present invention eschews the concept of isolation of individual sweet components in favor of separation, isolation and/or characterization of a combination of two or more molecules from Stevia.

EXAMPLES

(30) The known steviol glycosides can be ordered on the basis of the sugar (glycoside) substitution pattern. This allows for the prediction of other missing or as yet unidentified steviol glycosides that may be present, albeit at very low levels in various preparations. The organization of steviol glycosides is illustrated in FIG. 2.

(31) The sweetness quality and intensity of steviol glycosides is found to be correlated with the extent of glycosylation on the steviol aglycone. FIG. 3 below illustrates how subjective taste quality and sweetness can be predicted by molecular size, although exceptions may occur, such as rebaudioside B. This compound may be illustrative of the importance of the specific glycoside structure at R2, even in the absence of glycosylation at R1, in defining sweetness quality and intensity.

(32) Sweetness quality and intensity is also affected by the presence of monosaccharides other than glucose. Thus, the presence of rhamnose (Rh) in the molecule, such as in dulcoside A or rebaudioside C, results in a lesser sweetness intensity and quality relative to steviol glycosides substituted with the same number of glucose-only monosaccharides, such as stevioside or rebaudioside A, respectively.

(33) TABLE-US-00001 TABLE 1 Steviol glycoside glycosylation pattern correlates with sweetness. Steviol #Glucose #Rhamnose Mol. Sweetness Sweetness glycoside R1 R2 (G) (Rh) Wt. Intensity Quality steviol 318 steviolmonoside G- 1 480 40 −3 steviolbioside G-2G- 2 642 40 −3 rubusoside G- G- 2 642 115 −2 dulcoside-A G- Rh2-G- 2 1 788 70 −2 stevioside G- G2-G- 3 804 145 0 rebaudioside B G2-(G3)- 3 804 300 3 G- rebaudioside-C G- Rh2-(G3)- 3 1 950 200 −1 G- rebaudioside-E G2- G2-G- 4 966 200 1 G- rebaudioside-A G- G3-(G2-)G- 4 966 250 2 rebaudioside-D G2- G2-(G3)G- 5 1128 300 3 G- Note: Sweetness quality and intensity data from Osamu Tanaka. “Improvement of taste of natural sweeteners”. Pure & Appl. Chem. 1997, 69(4), 675-683. Sweetness intensity is understood to be relative to an equal weight of sucrose. Sweetness quality is a subjective relative ranking.

(34) Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be about 110 to about 270 times sweeter than sucrose, Rebaudioside A is between about 150 and about 320 times, and Rebaudioside C is between about 40 and about 60 times sweeter than sucrose. Dulcoside A is about 30 times sweeter than sucrose. Sweetness intensity is known to vary somewhat with temperature and viscosity of the carrier medium.

(35) While several steviol glycosides are now known, not all have been evaluated for sweetness quality and intensity. Also, while many processes are employed to isolate and exploit individual steviol glycosides as sweeteners, current knowledge does not permit predicting sweetness characteristics of steviol glycoside blends, particularly in proportions other than that found naturally, or of taste characteristics when combined with other sweeteners, whether caloric or non-caloric, of high intensity or more commensurate with simple mono- and disaccharides like glucose, fructose or sucrose.

(36) Method of Analysis

(37) Conventional methods of analysis of steviol glycosides require dual solvent gradients to separate steviol glycosides from other non-sweet plant extract components. Consequently, detectors that use refractive index changes in the eluent are not useful. Typically, ultraviolet (UV) detectors are employed to assay steviol glycosides which contain a weak UV chromophore. This method is of limited usefulness to assay crude extracts because of the presence of components with stronger chromophores which obscure the detection of the components of interest because components with a large molar extinction coefficients at the wavelength selected will appear more prominent than components with lower extinction coefficients, which though present at a higher concentration on a mass basis, will produce a much weaker signal. Another alternative to refractive index (RI) for mass-based detection include evaporative light scattering (ELS) and charged aerosol detection (CAD). THERMO Scientific (Determination of Steviol Glycosides by HPLC with UV and ELS Detections. Application Note 241 (2012)) teaches how ELS can present advantages over UV detection in measuring the content of Rebaudioside A and Stevioside in table-top sweetener formulations. However, in our experience, ELS can be of limited use in measuring the concentration of a minor component in the presence of another closely eluting major component. H. Y. Eom et al. (J. Chromatogr. A 1217 (2010) 4347-4354) teach that the detection of saponins derived from the roots of Bupleurum falcatum L. (Umbelliferae) is more sensitive with CAD than with ELS. We have found that using a charged aerosol detector, ESA®, Inc., Chelmsford, Mass., allows for good quantitation of steviol glycosides in crude extracts of Stevia rebaudiana, which preparations are rich in proteins and other plant components that have strong UV absorbance, and also in the presence of larger amounts of polysaccharides or other steviol glycosides which can “blind” the detector to lower levels of the components of interest.

(38) Identification of an effective detector is only part of the system for effective quantitation of the components of interest in a crude preparation. Another part of the system is identification of a solvent system (mobile phase) and solid support (stationary phase) to accomplish the separation. The successful development of a suitable combination of mobile and stationary phases is an empirical process of trial and error which is not predictable a priori. We have experimented with various gradient system mobile phase systems to discover a solvent gradients that useful to separate the steviol glycosides of interest, thus enable their identification by mass spectrometry, and quantitation by CAD. An example is shown below. The gradient can be adjusted to allow greater resolution around a particular peak region such as near Rebaudioside D.

(39) Chromatographic Method of Analysis:

(40) Stationary phase: Phenomenex Kinetex C-18, 150×4.6, 2.6 μm; Column temp.: 55° C.;

(41) Injection Volume is 10 μL.

(42) Mobile phase (MPA and MPB; flow rate: 0.35 mL/min):

(43) MPA: 0.1% formic acid in water

(44) MPB: 0.1% formic acid in acetonitrile

(45) TABLE-US-00002 TABLE 2 Time (minutes) % A % B 0 95 5 5 95 5 30 70 30 45 30 70 55 30 70 55.1 95 5 60 95 5

(46) TABLE-US-00003 TABLE 3 Reference identity RT.sup.1 (min) Mol. Wt. Rebaudioside D 35.920 1128 Rebaudioside A 39.544 966 Stevioside 39.704 804 Rebaudioside F 40.207 936 Rebaudioside C 40.468 950 Rebaudioside A 40.745 788 Rubusoside 41.474 642 Rebaudioside B 42.027 804 Steviolbioside 42.368 642 Steviol 50.012 318 Isosteviol 52.427 318 .sup.1RT—retention time.

(47) See also FIG. 4.

(48) As is known, the gradient can be modified for faster separation (0-2 min 80% MPA, 20% MPB; 2-35 min from 80 to 0% MPA, from 20% to 100% MPB, 35.01-40 min 80% MPA, 20% MPB. Flow rate 0.35 mL/min; injection volume: 5 uL), such as to allow for mass spectrometric identification of steviol glycosides. An example is provided in FIG. 5.

(49) TABLE-US-00004 TABLE 4 Peak identity RT (min) Mol. Wt. Rebaudioside D 14.08 1128 Rebaudioside N 14.33 1274 Rebaudioside M 14.42 1290 Novel steviol glycoside 14.75 Not determined (possible Reb. O) Rebaudioside I 15.97 1128 Rebaudioside A 16.29 966 Stevioside 16.43 804
Integration of Analytical Method to Agricultural Development

(50) We have developed a strategy for optimizing the selection of particular breeding progeny of Stevia plants based on the steviol glycoside content. It is well known historically that native Stevia contained predominantly Stevioside as the main steviol glycoside, but selective breeding has favored the development of progeny in which Rebaudioside A predominates. We have applied our novel analysis to enable identification of breeding progeny that further favor Rebaudioside D or other desirable steviol glycosides with molecular weight (Mol. Wt.) equal or greater than that of Rebaudioside A (Mol. Wt. 966 g/mol), notably Rebaudiosides I, O, M, N, among others that may subsequently be found to occur in new breeding progeny.

(51) Separation of Desirable Steviol Glycosides without Crystallization

(52) We have further developed the insights obtained from the analytical separation of steviol glycosides into a novel process that avoids the need to isolate and purify a single component (typically Rebaudioside A) by crystallization. We have further developed a solvent system which uses food grade ethanol (grain alcohol) and water to separate the steviol glycosides of interest without the need for crystallization.

Example

(53) A crude extract was prepared by subjecting 208.6 g dried leaves to hot water extraction (3 L) for 2 h at 95° C. A portion of the crude extract (100 mL, containing about 1.7 g solids) was directly fractionated on a preparative scale reverse-phase column (RediSep C18, 360 g) using a water ethanol gradient (100 mL/min) as below:

(54) TABLE-US-00005 TABLE 5 Time % ethanol 0 0 10 0 27.1 70.2 27.5 76.6 28 100 35.6 100 35.6 100 35.6 50 40.3 50 40.7 50

(55) A total of 628 mg of steviol glycosides were recovered. Of that sample 18 mg were recovered comprising about 23% Rebaudioside D, 21% Rebaudiosides N and M, 1.4% uncharacterized steviol glycosides, 2% Rebaudioside A and about 16% other known steviol glycosides. The solid had an off-white to beige appearance and had a clean sweet taste. The product is suitable for use as a sweetening agent without further processing or purification.

(56) TABLE-US-00006 TABLE 6 Taste (by 2 Time Mass Composition Sample indep. (minutes) (mg) (by HPLC/CAD) Appearance assessments) 22.8-23.6 78 Non-steviol Light beige Bitter (not sweet) glycoside solid components 23.6-25.sup.  18 Predominantly Reb. Med. Brown Clean sweet taste D, N, M solid .sup. 26-31.5 610 Reb. A and smaller Light yellow “classic” Reb. A steviol glycosides solid taste, i.e., sweet with bitter notes

Example

(57) The remaining extract was concentrated under vacuum at less than 40° C. clarified by centrifugation, decanted, cooled. An aliquote (53 mL, 9.5 g solids) of the concentrate was withdrawn for fractionation. The major portion was dried by lyophilization to yield 65.3 g of solids. The concentrate was directly fractionated on a preparative scale reverse-phase column (RediSep C18, 360 g) using a water-ethanol gradient. (100 mL/min) as below:

(58) TABLE-US-00007 TABLE 7 Time % Ethanol 0 0 10 0 19.8 39.9 32 100 37.5 100 41 100 41 45.2 44.7 45.2 50 45.2

(59) Approximately 4.3 g of steviol glycosides were recovered. The majority of the rebaudioside D eluting at about 25 minutes (1.6 g; 18% rebaudioside D and 5% rebaudioside A) with some non-steviol glycoside components. The majority of the rebaudioside A eluting at 27 minutes (2.2 g; 78% rebaudioside A and 1% rebaudioside D).

Example

(60) Authentic samples of Rebaudiosides A (0.5 g) and D (0.5 g) were separated on the same system using a more gradual gradient as indicated below:

(61) TABLE-US-00008 TABLE 8 Time (minutes) % Ethanol 0 0 10 0 38.2 100 44.1 100 44.1 50 50 50

(62) An improved separation was obtained with the major portion of rebaudioside D eluting at 28 minutes (2.40 mg; 86% reb. D, 9.3% reb. A), and the major portion of rebaudioside A eluding at 31 minutes (372 mg; 89% reb. A. 3% reb. D).

(63) The steviol glycosides obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc. The examples above show representative proportions which may be employed.

(64) In addition, the steviol glycosides can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed with improved characteristics.

(65) During the manufacturing of foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, chewing gum the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods can be used.

(66) The sweetener obtained in this invention can be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in the combination with other compounds.

(67) TABLE-US-00009 TABLE 9 shows the chemical structure of steviol and the steviol glycosides present in the Stevia rebaudiana Bertoni leaves. U.S. Patent Oct. 30, 2012 Sheet 1 of 11 U.S. Pat. No. 8,299,224 B2 FIG. 1 Compound name R.sub.1(C-19) R.sub.2(C-13) 1. Steviol H H 2. Steviolmonoside H β-Glc 3. Ruboside β-Glc β-Glc 4. Steviolbioside H β-Glc-β-Glc(2 1) 5. Stevioside β-Glc β-Glc-β-Glc(2 1) 6. Rebaudioside B β-Glc 7. Rebaudioside B H 8. Rebaudioside C   (Dulcoside B) β-Glc 9. Rebaudioside D β-Glc-β-Glc(2 1) 10. Rebaudioside E β-Glc-β-Glc(2 1) β-Glc-β-Glc(2 1) 11. Rebaudioside F β-Glc 12. Dulcoside A β-Glc β-Glc-α-Rha(2 1)

(68) TABLE-US-00010 TABLE 10 Proposed structures and their relative percentage of the steviol glycosides from the leaves of S. rebaudiana Morita and S. rebaudiana Bertoni. Steviol Glycoside R.sub.1 R.sub.2 Morita (%).sup.1 Bertoni (%).sup.1,2 SG1 (steviolmonoside) H— Glcβ1- 1.7 1.7 SG2 (steviolbioside) H— Glcβ1-2Glcβ1- 1.0 5.0 SG3 (rubusoside) Glcβ1- Glcβ1- 0.8 ND.sup.3 SG4 (dulcoside B).sup.4 H— Rhaα1-2(GlcB1-3)Glcβ1- 0.6 0.8 SG5 (dulcoside A) Glcβ1- Rhaα1-2Glcβ1- 0.3 2.6 SG6 (rebaudioside B) H— Glcβ1-2(Glcβ1-3)Glcβ1- 2.5 2.0 SG7 (rebaudioside G).sup.4 Glcβ1- Glcβ1-3Glcβ1- 1.1 0.8 SG8 (stevioside) Glcβ1- Glcβ1-2Glcβ1- 9.2 49.8 SG9 (rebaudioside C) Glcβ1- Rhaα1-2(Glcβ1-3)Glcβ1- 7.5 6.8 SG10 (rebaudioside F) Glcβ1- Xy1β1-2(Glcβ1-3)Glcβ1- 1.9 1.4 SG11 (rebaudioside A) Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 61.6 21.5 SG12 (rebaudioside I).sup.4 Glcβ1-3Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 0.1 ND.sup.3 SG13 (rebaudioside E) Glcβ1-2Glcβ1- Glcβ1-2Glcβ1- 0.3 0.9 SG14 (rebaudiodide H).sup.4 Glcβ1- Glcβ1-3Rhaα1-2(Glcβ1-3)Glcβ1- 0.5 ND.sup.3 SG15 (rebaudioside L).sup.4 Glcβ1- Glcβ1-6G1cβ1-2(Glcβ1-3)Glcβ1- 0.3 ND.sup.2 SG16-I (rebaudioside K).sup.4 Glcβ1-2Glcβ1- Rhaα1-2(Glcβ1-3)Glcβ1- 0.3 ND.sup.3 SG16-II (rebaudioside J).sup.4 Rhaα1-2Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 0.5 0.1 SG17 (rebaudioside M).sup.4 Glcβ1-2(Glcβ1-3)Glcβ1 Glcβ1-2(Glcβ1-3)Glcβ1- 1.0 ND.sup.3 SG18 (rebaudioside D) Glcβ1-2Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 2.1 0.4 SG19 (rebaudioside N).sup.4 Rhaα1-2(Glcβ1-3)Glcβ Glcβ1-2(Glcβ1-3)Glcβ1- 1.4 <0.1 SG20 (rebaudioside O).sup.4 Glcβ1-3Rhaα1-2(Glcβ1-3)Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 0.6 ND.sup.3 *From Ohta et al. (2010). .sup.1Relative amounts are expressed as percentage of total peak areas detected on the basis of their UV absorbance at 210 nm by Amide-SO/HPLC. The ratio of SG16-I and SG16-II was obtained by the relative intensities of the product ions at m/z 787 and 803, respectively, by the CID voltage of 60 V in ESI-MS/MS analysis as precursor ion [M − H].sup.− at m/z 1111. .sup.2The structures were proposed on the basis of the results on HPLC mobility and ESI-MS and MS/MS analyses. .sup.3Not detected. .sup.4Names were proposed in this study.