Stevia-derived molecules, methods of obtaining such molecules, and uses of the same

Abstract

A purified composition of steviol glycoside molecules is described. The composition imparts desirable taste, flavor and flavor modifying properties to food, beverages, and other consumable products.

Description

BACKGROUND OF THE INVENTION

Sugar alternatives are receiving increasing attention due to awareness of many diseases in conjunction with consumption of high-sugar foods and beverages. However, many artificial sweeteners such as dulcin, sodium cyclamate and saccharin were restricted in some countries due to debatable concerns on their safety. Therefore, non-caloric sweeteners of natural origin are becoming increasingly popular. The sweet herb Stevia rebaudiana produces a number of diterpene glycosides which feature high intensity sweetness and sensory properties superior to those of many other high potency sweeteners.

Stevia rebaudiana is a plant species belonging to the Astracea family, and is native to South America and cultivated now in many parts of the world (Gardana et al., 2003; Koyama et al., 2003; Carakostas et al., 2008). Stevia leaves are naturally sweet, and have been used for sweetening food products for hundreds of years in South America (Soejarto et al., 1982). Extracts of Stevia rebaudiana have been used commercially to sweeten foods in Japan and other Southeast Asian countries for a number of years (Koyama et al., 2003). As a product of nature, the stevia plant leaves contain different sweet tasting components, called steviol glycosides. Reportedly, more than 40 steviol glycosides have been identified that are typically present in the stevia leaf extract (Ceunen and Geuns, 2013; Purkayastha et al, 2016). Each of these steviol glycosides has its own unique taste profile and sweetness intensity, which can be up to 350 times sweeter than sugar, but all share a similar molecular structure where different sugar moieties are attached to aglycone steviol (an ent-kaurene-type diterpene).

The leaves of the Stevia plant contain a mixture containing diterpene glycosides in an amount ranging from about 10% to 20% of the total dry weight. These diterpene glycosides are about 30 to 450 times sweeter than sugar. Structurally, many of the diterpene glycosides are characterized by a single base, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19. Typically, on a dry weight basis, the four major steviol glycosides found in the leaves of Stevia are dulcoside A (0.3%), rebaudioside C (0.6-1.0%), rebaudioside A (3.8%) and stevioside (9.1%). Other glycosides identified in Stevia extract include rebaudioside B, D, E, and F, steviolbioside and rubusoside.

Rebaudioside A and stevioside have garnered the most commercial interest and have been extensively studied and characterized in terms of their suitability as commercial high intensity sweeteners. Stability studies in carbonated beverages confirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983) Stability studies of stevioside and rebaudioside A in carbonated beverages. J. Agric. Food Chem. 31: so 409-412.)

Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be 110-270 times sweeter than sucrose and rebaudioside A is between 150 and 320 times sweeter than sucrose. Rebaudioside A has the least astringent, the least bitter, and the least persistent aftertaste thus possessing the most favorable sensory attributes in major steviol glycosides (Tanaka O. (1987) Improvement of taste of natural sweeteners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989) Stevia: steps in developing a new sweetener. In: Grenby T. H. ed. Developments in sweeteners, vol. 3. Elsevier Applied Science, London. 1-43.)

By the early 21st century, only a limited number of the chemical structures of steviol glycosides in Stevia rebaudiana have been characterized including stevioside, rebaudioside A-F, dulcoside A, and steviolbioside (Ceunen and Geuns, 2013). In recent years, many minor steviol glycosides with diverse chemical structures, have been reported from the leaves of Stevia rebaudiana (Chaturvedula et al., 2011a,b,c; Chaturvedula and Prakash, 2011 a,b). These diverse steviol glycosides, which are ent-kaurene-type diterpenes, are connected to various sugars such as glucose, rhamnose, xylose, fructose and deoxy glucose at C-13 and C-19 positions via 1,2-; 1,3-; 1,4- or 1,6-α or β-glycosidic linkages (Purkayastha et al, 2016).

The use of steviol glycosides has been limited to date by certain undesirable taste properties, including licorice taste, bitterness, astringency, sweet aftertaste, bitter aftertaste, licorice aftertaste, and become more prominent with increase of concentration. These undesirable taste attributes are particularly prominent in carbonated beverages, where full replacement of sugar requires concentrations of steviol glycosides that exceed 600 mg/L. Use of steviol glycosides in such high concentrations results in significant deterioration in the final product taste.

Accordingly, there remains a need to develop natural reduced or non-caloric sweeteners that provide a temporal and flavor profile similar to the temporal and flavor profile of sucrose.

There remains a further need for methods for purifying glycosides from stevia plants.

SUMMARY OF THE INVENTION

The present invention relates generally to novel diterpene glycosides and compositions and consumables comprising said novel diterpene glycosides, as well as methods for purifying said novel diterpene glycosides, methods for preparing compositions and consumables comprising said novel diterpene glycosides and methods for enhancing or modifying the flavor or sweetness of consumables using the novel diterpene glycosides. The novel diterpene glycosides of the present invention are isolated from Stevia plants.

The present invention is directed to stevia-derived molecules, methods for obtaining such molecules, and uses of such molecules. These stevia-derived molecules may or may not have the steviol backbone structure, but have structures that may be somewhat or substantially similar to steviol glycosides. In some cases, these molecules have structures that are very different from steviol glycosides. These stevia-derived molecules have desirable taste and flavor properties, which may include sweetness imparting properties, flavor modifying properties, a combination of these properties, and other properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative analytical chromatogram of stevia extract A95 using Gradient KM7. The top and middle plots are MS TIC(−) (mass spectrometry total ion current) chromatograms, and the bottom plot is an ELSD (evaporative light scattering detector) chromatogram.

FIG. 2 is a chart of the schematic steps used to isolate different compounds listed in Table 1.

FIG. 3 is a chart of the schematic steps used to isolate different compounds listed in Table 1.

FIG. 4 shows the structure of RSG1 (Related Steviol Glycoside 1).

FIG. 5 shows the structure of RSG2 (Related Steviol Glycoside 2).

FIG. 6 shows the structure of RSG3 (Related Steviol Glycoside 3).

FIG. 7 shows the structure of RSG4 (Related Steviol Glycoside 4).

FIG. 8 shows the structure of RSG5 (Related Steviol Glycoside 5).

FIG. 9 shows the structure of RSG6 (Related Steviol Glycoside 6).

FIG. 10 shows the structure of Rebaudioside T.

FIG. 11 shows the structure of Rebaudioside Y.

FIG. 12 shows the structure of Rebaudioside O2.

FIG. 13 shows the structure of Rebaudioside C2.

FIG. 14 shows the structure of Rebaudioside W.

FIG. 15 shows the structure of Rebaudioside W2.

FIG. 16 shows the structure of Rebaudioside U2.

FIG. 17A shows an RP-HPLC analysis of selected fractions of stevia leaf extract.

FIG. 17B shows ELSD and MS analysis of selected fractions of stevia leaf extract.

FIG. 17C shows ¹H-NMR analysis of selected fractions of stevia leaf extract.

FIG. 17D shows the structure of Rebaudioside W3.

FIG. 18 shows the structure of Rebaudioside V.

FIG. 19 shows the structure of Rebaudioside U.

FIG. 20 shows the structure of Rebaudioside K2.

FIG. 21 shows the structure of Rebaudioside V2.

DETAILED DESCRIPTION

The chemical structures of certain of the stevia-derived molecules of the present invention are shown in the Figures appended hereto. As used herein, “stevia-derived molecules” shall refer to molecules obtained from any part of the plants of any variety of the species Stevia rebaudiana.

These stevia-derived molecules are useful in the preparation of food, beverages, nutraceuticals, pharmaceuticals, tobacco products, cosmetics, oral hygiene products, and the like. Some of the stevia-derived molecules have a steviol backbone, and may be referred to as steviol glycosides. Other stevia-derived molecules of this invention have a different backbone, but may have properties similar to steviol glycosides, or may have other beneficial properties.

These stevia-derived molecules can be used alone or in combination with other ingredients, such as sweeteners, flavors, flavor modifiers, and the like. Such other ingredients may include steviol glycoside ingredients, or ingredients from other natural or synthetic sources.

Methods of obtaining stevia-derived molecules include the methods used to extract steviol glycosides from Stevia plant leaves. Other methods may include extraction from other parts of the plant, or other extraction techniques and solvents.

The following Example demonstrates certain embodiments of the invention, and is not intended to limit the scope of the invention in any way.

Example 1

A stevia extract available from PureCircle USA Inc. of Oak Brook, IL, labeled as “A95”, was used to isolate and characterize major and minor steviol glycoside components using the following analytical methodologies.

1.1 Sample

• • Product Name: Stevia leaf extract A95
  • Batch No.: WIP A95 27A
  • Manufacturing date: 2 Apr. 20161.2 Analytical LCMS (Liquid Crystal Mass Spectrometry)

Analytical LCMS was performed on a Shimadzu single quad UPLC-system (see Table 1). Two different gradient systems were applied (see Tables 2a and 2b) which are identical for the first 40 min. Gradient KM7 was used to resolve all compounds including already identified steviol glycosides #25-#29, while gradient ACD1 was faster and used for the analysis of compounds #1-#24.

Reference samples were prepared by dissolving Stevia leaf extract A95 (20 mg) in a 1:1 mixture of methanol and dimethyl sulfoxide (DMSO). Sonification for 30 min was necessary to achieve a homogenous solution. The solution was stored at 4° C.

The analytical system proved to be very sensitive towards changes in solvent composition and retention time shifts were observed when a new batch of solvents was used. Therefore, reference samples were analyzed before and after every analytical batch and the assignment of retention times was verified.A typical analytical chromatogram using gradient KM7 is shown in FIG. 1.

TABLE 1
LCMS system
HPLC System      Shimadzu LC-30AD, prominence
Interface        Shimadzu CBM-20A
Degasser         Shimadzu DGU-20A5
Autosampler      Shimadzu SIL-30AC, prominence
Column oven      Shimadzu CTO-20AC
MS               Shimadzu 2020 Single quadrupole
DAD              Shimadzu SPD-M20A
ELSD             Sedere ELSD-LT II, Sedex 85
Stationary Phase Agilent Poroshell 120
                 SB-C18 2.7 μm, 4.6 × 150 mm
Flow Rate        0.5 mL/min
Mobile Phase:    A: Water, 25% Acetonitrile,
                 0.2% Acetic acid B: Acetonitrile

TABLE 2
LCMS Gradients
	Gradient	Time	A	B	Flow
	KM7	[min]	[%]	[%]	[ml/min]
		00.00	100	0	0.5
		24.00	100	0	0.5
		50.00	90	10	0.5
		51.00	0	100	0.5
		55.00	0	100	0.5
		56.00	100	0	0.5
		56.01	100	0	1.0
		65.00	100	0	0.5
		70.00	100	0	0
	Gradient	Time	A	B	Flow
	ACD1	[min]	[%]	[%]	[ml/min]
		00.00	100	0	0.5
		24.00	100	0	0.5
		40.00	94.6	5.4	0.5
		41.00	0	100	0.5
		45.00	0	100	0.5
		46.00	100	0	0.5
		46.01	100	0	1.0
		55.00	100	0	0.5
		60.00	100	0	0

1.3 Recrystallisation

Stevia leaf extract A95 (100 g, white powder) were dissolved in ethanol/water 70/30 (750 mL) at a temperature of 65° C.

The milky solution was allowed to cool down to room temperature in a water bath and then filtrated through a suction filter. The collected crystals were washed with ethanol, dried and stored. Mother liquor and wash solution were kept separate and the respective solvent was removed under vacuum.1.4 Reversed Phase MPLC (Medium Pressure Liquid Chromatography)

The respective sample (15 g) is dissolved in methanol, celite (30 g) is added and the solvent removed by a rotary evaporator. The immobilized sample is transferred into a glass column and built into the MPLC system described in Table 3. A time based fractionation leads to 18 fractions (4 min each). Solvents and gradients are described in Table 3.

TABLE 3
MPLC-System and gradients
Pump System
Interface Module	SCPA
Fraction collector	Labomatic Labocol Vario 2000 plus
Stationary Phase	Polygoprep C18, 50-60 μm, glas column 50 × 250 mm
Mobile	A: Water	C: Methanol
Phase:	B: Aceton	D: 2-Propanol
	Time [min]	A [%]	B [%]	C [%]	D [%]	Flow [ml/min]
Gradient	00.00	85	15	0	0	90
A	51.00	65	35	0	0	90
	56.00	0	0	100	0	90
	61.00	0	0	0	100	90
Mobile	A: Water	C: Methanol
Phase:	B: Methanol	D: 2-Propanol
	Time [min]	A [%]	B [%]	C [%]	D [%]	Flow [ml/min]
Gradient	00.00	75	25	0	0	90
B	51.00	50	50	0	0	90
	56.00	0	0	100	0	90
	61.00	0	0	0	100	90

1.5 Normal Phase Chromatography

The respective sample (20 g) is dissolved in methanol, silica (40 g) is added and the solvent removed by a rotary evaporator. The immobilized sample is transferred into a glass column and built into the high pressure liquid chromatography (HPLC) system described in Table 4. Air is removed from the transfer column by washing with Ethyl acetate/methanol 1:1. A time based fractionation leads to 90 fractions (0.5 min each) which are combined based on the UV and ELSD data generated during fractionation. Resulting fractions are analyzed by LCMS. Solvents and gradients are described in Table 4.

TABLE 4
Preparative HPLC System 2 (HTP-II, NP-Fractionation)
HPLC System	Knauer K-1800
Autosampler	Merck L-7250
UV-detector	Knauer
ELSD	Biotage ELSD-A120
Fraction collector	Merck L-7650
Stationary Phase	Silica, 50-60 μm
Mobile Phase	A: Aceton/Ethyl acetate/Water (50/40/10);
	B: Aceton/Ethyl acetate (85/15)
	Time	A	B	Flow
Gradient A	[min]	[%]	[%]	[ml/min]
	00.00	100	0	35
	372.00	0	100	35

1.6 Reversed Phase HPLC

The respective sample (up to 3.5 g) is dissolved in methanol, C-18 RP material is added and the solvent removed by a rotary evaporator. The immobilized sample is transferred into a column and built into the HPLC system described in Table 5. A time based fractionation leads to 120 fractions (27 sec each) which are combined based on the UV and ELSD data generated during fractionation. Resulting fractions are analyzed by LCMS. Solvents and gradients are described in Table 5.

TABLE 5
Preparative HPLC System 3 (SEPbox)
HPLC System	Sepiatec SEPbox lite
UV-detector	Merck L-7400
ELSD	Sedere Sedex 75
Fraction collector	Merck L-7650
Stationary Phase	Lichrospher Select B, 10 μm 50 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol/Acetonitril (1/1),
	ammonium formate (5 mmol),
	formic acid, pH 3
Delay before fractionation	215 sec	Fraction	29 sec
	Time	A	B	Flow
Gradient A	[min]	[%]	[%]	[ml/min]
	00.00	72	28	80
	57.7	46	54	80
	58	0	100	80
	105	0	100	80
Stationary Phase	Kromasil C18, 10 μm 25 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol, ammonium formate
	(5 mmol), formic acid, pH 3
Delay before fractionation	215 sec	Fraction	29 sec
	Time	A	B	Flow
Gradient B	[min]	[%]	[%]	[ml/min]
	00.00	61	39	30
	57.7	43	57	30
	58	0	100	30
	105	0	100	30
Stationary Phase	Lichrospher Select B, 10 μm 50 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol/Acetonitril (1/1),
	ammonium formate (5 mmol),
	formic acid, pH 3
Delay before fractionation	215 sec	Fraction	29 sec
	Time	A	B	Flow
Gradient C	[min]	[%]	[%]	[ml/min]
	00.00	70	30	80
	57.7	62	38	80
	58	0	100	80
	105	0	100	80
Stationary Phase	Lichrospher Select B, 10 μm 50 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol/Acetonitril (1/1), ammonium
	formate (5 mmol), formic acid, pH 3
	215 sec
Delay before fractionation	215 sec	Fraction	29 sec
	Time	A	B	Flow
Gradient D	[min]	[%]	[%]	[ml/min]
	00.00	68	32	80
	57.7	53	47	80
	58	0	100	80
	105	0	100	80
Stationary Phase	Kromasil C18, 10 μm 50 × 250 mm
Mobile Phase	A: Water, formic acid 0.1%, pH 3
	B: Acetonitril, formic acid 0.1%, pH 3
Delay before fractionation	180 sec	Fraction	22 sec
	Time	A	B	Flow
Gradient E	[min.sec]	[%]	[%]	[ml/min]
	00.00	76	24	109
	40.50	70	30	109
	41.00	0	100	109
	45.00	0	100	109
Stationary Phase	Kromasil C18, 10 μm 25 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol, ammonium formate
	(5 mmol), formic acid, pH 3
Delay before fractionation	180 sec	Fraction	22 sec
	Time	A	B	Flow
Gradient F	[min]	[%]	[%]	[ml/min]
	00.00	46	54	47
	40.50	38	62	47
	41.00	0	100	47
	45.00	0	100	47
Stationary Phase	Lichrospher Select B, 10 μm 50 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol/Acetonitril (1/1),
	ammonium formate (5 mmol),
	formic acid, pH 3
Delay before fractionation	215 sec	Fraction	29 sec
	Time	A	B	Flow
Gradient G	[min]	[%]	[%]	[ml/min]
	00.00	70	30	80
	57.7	55	45	80
	58	0	100	80
	105	0	100	80
Stationary Phase	Kromasil C18, 10 μm 25 × 250 mm
Mobile Phase	A: Water, ammonium formate (5 mmol),
	formic acid, pH 3
	B: Methanol, ammonium formate
	(5 mmol), formic acid, pH 3
Delay before fractionation	180 sec	Fraction	22 sec
	Time	A	B	Flow
Gradient H	[min]	[%]	[%]	[ml/min]
	00.00	50	50	47
	40.50	49	61	47
	41.00	0	100	47
	45.00	0	100	47
Stationary Phase	Kromasil C18, 10 μm 50 × 250 mm
Mobile Phase	A: Water, formic acid 0.1%, pH 3
	B: Acetonitril, formic acid 0.1%, pH 3
Delay before fractionation	180 sec	Fraction	22 sec
	Time	A	B	Flow
Gradient K	[min.sec]	[%]	[%]	[ml/min]
	00.00	78	22	109
	40.50	68	32	109
	41.00	0	100	109
	45.00	0	100	109
Stationary Phase	Kromasil C18, 10 μm 50 × 250 mm
Mobile Phase	A: Water, formic acid 0.1%, pH 3
	B: Acetonitril, formic acid 0.1%, pH 3
Delay before fractionation	180 sec	Fraction	22 sec
	Time	A	B	Flow
Gradient L	[min.sec]	[%]	[%]	[ml/min]
	00.00	75	25	109
	40.50	68	32	109
	41.00	0	100	109
	45.00	0	100	109

1.7 HILIC (Hydrophilic Interaction Liquid Chromatography)

The respective sample is dissolved in 2 mL of a 3:1 mixture of solvents A and B (see Table 6). Sample Injection takes place after 9.95 min. A time based fractionation leads to 96 fractions (43 sec each, starting after 18 min) which are combined based on the UV and ELSD data generated during fractionation. Resulting fractions are analyzed by LCMS. Solvents and gradients are described in Table 6.

TABLE 6
Preparative HPLC System 1 (HTP-I, HILIC-Fractionation)
HPLC System	Knauer K-1800
Autosampler	Merck L-7250
UV-detector	Knauer
ELSD	ELSD Sedex 75
Fraction collector	Merck L-7650
Stationary Phase	Kromasil 60-10-HILIC-D 50 × 250 mm
Flow Rate	80 mL/min
Mobile Phase:	A: Acetonitril; 0.1% Acetic acid;
	B: Methanol/Water/Acetic acid (95/4.9/0.1)
				Flow
Gradient	Time [min]	% A	% B	[ml/min]
	00.00	75	25	80
	11.50	75	25	80
	65.00	65	35	80
	70.00	0	100	80
	75.00	0	100	80

1.8 NMR (Nuclear Magnetic Resonance)

Isolated compounds were identified by NMR spectroscopy using a Bruker 500 Mhz NMR spectrometer. Identification of the aglycon was based on reference ¹H-NMR spectra using C17, C18 and C20 proton signals as primary indicators. Especially C20 proton shifts indicated alterations as seen in compounds #4 and #18. Glycosides were elucidated using H-H-Cosy, HSQC and HMBC and experiments using spectra of literature known steviosides as reference.

1.9 Results

FIG. 1 shows the HPLC chart containing the major peaks identified in Table 7 by using analytical methodology as described above. The schematic steps to isolate different compounds in Table 7 are shown in FIG. 2 and FIG. 3.

TABLE 7
	Formula		KM7
Peak	(based on	Trivial	tr	Base Peak	Fraction
Identifier	structure)	Formula	(min)	Mass	ID	MW
#1	C21H30O11		6.97	517.3	C-2314-B-07	458
#2	C44H70O24		6.99	981.4	C-2293-E-02_NF2	982
#3	C32H52O15		7.32	735.4	C-2283-C-07_NF2	676
#4	C50H80O28		7.64	1127.4	C-2374-I-05	1128
#5	C44H70O24		8.56	981.4	C-2314-B-12	982
#6	C50H80O28	SvGal1G4	8.95	1127.5	C-2376-E-09	1128
ACD1				965.1	C-2387-K	966
ACD2	C55H88O32	SvA1G5		1259.5	C-2376-E-12	1260
ACD14	C62H100O37	SvR1G6			C-2376-E-15	1436
#7 REB E	C44H70O23	SvG4	10.37	965.1	C-2321-E-E09	966
#8 REB O	C52H100O37	SvR1G6	11.45	1435.0	C-2348-G-04	1436
#9 REB D	C50H80O28	SvG5	12.16	1127.1	C-2340-N-A01	1128
#10 REB K	C50H80O27	SvR1G4	12.69	1111.1	C-2293-E-07_NF2	1112
#11 REB N	C56H90O32	SvR1G5	13.19	1273.1	C-2321-I-04	1274
#12 REB M	C56H90O33	SvG6	15.22	1289.5	C-2340-N-12	1291
#13	C44H70O22	SvR1G3	15.79	949.2	C-2353-K-03	950
#14 REB J	C50H80O27	SvR1G4	16.46	1111.1	C-2340-N-03	1112
#15	C49H78O27	SvA1G4	17.93	1097.1	C-2353-K-05	1098
#16			18.31	1289.4
#17b	C49H78O27	SvA1G4		1097.5	C-2376-D-09	1098
#17a	C49H78O27	SvX1G4	18.80	1097.5	C-2376-B-02	1098
#18	C44H70O23		19.49	965.2	C-2376-D-03	966
#19	C49H78O27	SvA1G4	20.26	1097.4	C-2348-F-11	1098
ACD6	C55H88O32	SvX1G5	20.95	1259.6	C-2374-D-10	1260
#20	C49H78O27	SvXG4	21.14	1097.4	C-2283-F-11_NF2	1098
#21	C50H80O27	SvR1G4	23.31	1111.4	C-2374-D-07	1112
#22	C55H88O32	SvX1G5	25.51	1259.6	C-2283-F-14_NF2	1260
#23 REB H	C50H80O27	SvR1G4	30.71	1111.5	C-2321-F-08_NF2	1112
#24			32.14	1111.5
#25 REB I		SvG5	37.49	1127.5
#26 REB A	C44H70O23	SvG4	40.32	965.1		966
#27 Stevioside		SvG3	40.53	641.3
#28 REB C	C44H70O22	SvR1G3	50.17	949.5		950
#29 REB B	C38H60O18	SvG3	53.76	803.5	C-2321-B-22	804

A list of novel stevia-leaf-derived molecules isolated by using the method of Example 1 is shown in Table 8 and Table 9.

TABLE 8
Related Steviol Glycoside Components
Related Steviol
Glycoside				Retention
Components	Molecular	Trivial		time
(Peak ID)	Weight	Formula	Formula	(min)
RSG1 (#1)	458	NA	C21H30O11	6.97
RSG2 (#2)	982	NA	C44H70O24	6.99
RSG3 (#3)	676	NA	C32H52O15	7.32
RSG4 (#4)	1128	NA	C50H80O28	7.64
RSG5 (#5)	982	NA	C44H70O24	8.56

TABLE 9
Novel Steviol Glycoside Components
Steviol				Retention
Glycoside	Molecular	Trivial		Time
(Peak ID)	Weight	Formula	Formula	(min)
Rebaudioside	1128	SvGal1G4	C50H80O28	8.95
T (#6)
Rebaudioside	1260	SvA1G5	C55H88O32	—
Y (#ACD 2)
Rebaudioside	1436	SvR1G6	C52H100O37	—
O2 (#ACD 14)
Rebaudioside	950	SvR1G3	C44H70O22	15.79
C2 (#13)
Rebaudioside	1098	SvA1G4	C49H78O27	17.93
W (#15)
Rebaudioside	1098	SvA1G4	C49H78O27	NA
W2 (#17b)
Rebaudioside	1098	SvX1G4	C49H78O27	18.8
U2 (#17a)
Rebaudioside	1098	SvA1G4	C49H78O27	20.26
W3 (#19)
Rebaudioside	1260	SvX1G5	C55H88O32	20.95
V (#ACD6)
Rebaudioside	1098	SvX1G4	C49H78O27	21.14
U (#20)
Rebaudioside	1112	SvR1G4	C50H80O27	23.31
K2 (#21)
Rebaudioside	1260	SvX1G5	C55H88O32	25.51
V2 (#22)

Example 2: Identification and Characterization of a Novel Compound

This Example outlines the isolation, identification and characterization of Rebaudioside W3 (#19) as an example. Similar analysis was carried out for all novel steviol glycoside molecules.

Isolation

100 g stevia leaf extract A95 were recrystallized according to the method described in section 1.3 (Example 1) yielding 33.2 g of enriched minor compounds from mother liquor. The enriched minor compounds were fractionated using normal phase chromatography as described in section 1.5 using gradient A (see Table 4). Fractions 49-60 yielded 1.32 g of enriched minor compounds which were further fractionated using reversed phase HPLC according to section 1.4 using gradient L.

RP (Reversed Phase)—HPLC & LCMS

Fractions 51+52 are marked (FIG. 17A) by a red rectangle, ELSD trace (blue) and UV trace (orange) yielded 37.5 mg of #19. Fractions 66+67 (FIG. 17B) with preparative RP-HPLC chromatogram yielded 3.85 g of enriched minor compounds, Fractions 66+67 were analyzed by LCMS according to section 3.2 (see FIG. 17B). 37.5 mg of compound #19 were obtained with 89% purity (ELSD).

NMR

The structure of compound #19 was determined by NMR on a 500 MHz Bruker-NMR in d₄-Methanol (δ_C=48.5 ppm; δ_H=3.3 ppm). The data are shown in Table 10 and the NMR analysis is shown in FIG. 17C. The structure of compound #19 is shown in FIG. 17D.

TABLE 10
Assignment of the 1H-and 13C-NMR-Signals (based on HH-COSY,
HSQC, HMBC and HSQC-TOCSY experiments)
Position	δC [ppm]	δH [ppm]	J [Hz]/(INT)	HMBC (H -> C)
Aglycon moiety
1	40.6 t	0.87 m
		1.90 m
2	19.1 t	1.46 m
		1.96 m
3	37.4 t	1.56 m
		1.98
4	43.5 s	—
5	57.5 d	1.15 m
6	21.9 t	1.88 m
		2.03
7	41.7 t	1.48 m
		1.60 m
8	54.0 s	—
9	54.2 d	1.00 m
10	39.0 s	—
11	19.6 t	1.67 m
		1.80 m
12	38.0 t	1.08 m
		2.17 m
13	87.6 s	—
14	44.2 t	1.59 d	11.6
		2.25 d	11.6
15	47.5 t	2.07 d	15.9	7, 8, 9, 14
		2.16 d	15.9
16	152.5 s	—
17	104.7 t	4.90 br s		13, 15, 16
		5.26 br s
18	27.7 q	1.24 s	(3H)	3, 4, 5, 19
19	177.3 s	—
20	15.4 d	0.99 s	(3H)	1, 5, 9, 10
Sugar moiety
β-D-Glucopyranoside
1i	96.5 d	4.64 d	8.4	13
2i	79.0 d	3.67 t	8.4
3i	86.7 d	3.78 t	8.4
4i	69.6 d	3.38 t	8.4
5i	77.5 d	3.41 m
6i	61.7 t	3.68 m
		3.93 m
β-D-Glucopyranoside
1ii	103.0 d	4.87 d	8.4	2i
2ii	74.8 d	3.23 t	8.4
3ii	77.1 d	3.29 t	8.4
4ii	71.2 d	3.19 t	8.4
5ii	77.1 d	3.26 m
6ii	61.8 t	3.66 m
		3.89 m
β-D-Glucopyranoside
1iii	103.4 d	4.70 d	8.4	3i
2iii	74.5 d	3.30 t	8.4
3iii	77.3 d	3.33 t	8.4
4iii	70.2 d	3.28 t	8.4
5iii	76.7 d	3.35 m
6iii	61.3 t	3.67 m
		3.88 m
β-D-Glucopyranoside
1iv	94.5 d	5.40 d	8.4	19
2iv	73.2 d	3.37 t	8.4
3iv	77.5 d	3.47 t	8.4
4iv	69.7 d	3.45 t	8.4
5iv	76.6 d	3.58 m
6iv	67.9 t	3.86 m
		4.09 m
β-D-Arabinopyranoside
1v	103.7 d	4.33 d	8.4	6iv
2v	71.5 d	3.60 t
3v	73.0 d	3.58 t
4v	68.5 d	3.83 br s
5v	65.5 d	3.53 m
		3.89 m

Each of these minor molecules identified above, preferably at purity levels ranging from 80-99%, including 90-95% purity, 99% purity, and 89% purity and higher, either as isolated or in combination with other stevia-derived molecules, are believed to have numerous desirable effects on the sweetness, taste and flavor profiles of products containing stevia-based ingredients. These molecules can be useful in imparting specific tastes or modifying flavors, or both, in food, beverage, nutraceutical, pharmaceutical, and other comestible or consumable products.

It is to be understood that the foregoing description and specific embodiments shown herein are merely illustrative of the best mode of the invention and the principles thereof, and that modifications and additions may be easily made by those skilled in the art without departing for the spirit and scope of the invention, which is therefore understood to be limited only by the scope of the appended claims.

Claims

1. A stevia-derived composition having taste imparting properties, flavor modifying properties, or a combination thereof, at a purity level of greater than 80%, comprising one or more molecules selected from the group consisting of:

2. A food, beverage, nutraceutical, pharmaceutical or other consumable product comprising the stevia-derived composition of claim 1.