STEVIOL GLYCOSIDE

TECHNICAL FIELD

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

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 300 times or more than that of sucrose and is planted to be used as a natural sweetener after extracting the sweet components.

Stevioside (C₃₈H₆₀O₁₈), Rebaudioside A (C₄₄H₇₀O₂₃). 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 40 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.

Because ST has a degree of sweetness of 300 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 1.5 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).

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).

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.

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.

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 5), 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.

[Patent Literature 1] Laid Open Patent Publication JP2002-262822 Gazette
[Patent Literature 2] Patent Publication JP1957-18779 Gazette
[Patent Literature 3] Laid Open Patent Publication JP1997-107913 Gazette
[Patent Literature 4] Laid Open Patent Publication JP2003-009878, Gazette
[Patent Literature 5] International Patent Publication of Patent Cooperation Treaty -PCT WO06/093229 Gazette

DISCLOSURE OF INVENTION
Problems to be Solved

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 Glycoside, 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.

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.

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.

The inventors of the present invention have searched new Steviol Glycosides contained in the Stevia variety, main component of which is RA, and discovered 10 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

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.

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.

THE BEST MODE FOR EMBODYING THE INVENTION

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 JP2001-200944 and JP2007-506004; 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.

In addition, it is possible to efficiently obtain highly pure RA sweetener containing a trace amount of ST and Steviol Glycoside X by recrystallization.

The first embodiment of the present invention is Steviol Glycoside of the formula I to X:

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wherein R₁and R₂are a hydrogen atom or a sugar chain defined in the next table;

No.

(Steviol Glyciside)
R₁
R₂

I (Dulcoside B)

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H

II
β-glc-β-glc(3→1)
β-glc

(Rebaudioside G)

III (Rebaudioside I)

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β-glc-β-glc(3→1)

IV (Rebaudioside H)

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β-glc

V (Rebaudioside L)

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β-glc

VI (Rebaudioside K)

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β-glc-β-glc(2→1)

VII (Rebaudioside J)

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β-glc-α-rha(2→1)

VIII (Rebaudioside M)

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IX (Rebaudioside N)

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X (Rebaudioside O)

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The symbols in the formula are the sugar below:

glc: D-glucopyranosyl

rha: L-rhamnopyranosyl

xyl: xylopyranosyl

The second embodiment of the present invention is an extract containing Steviol Glycoside X (Rebaudioside O), 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.

The third embodiment of the present invention is a method of obtaining highly pure Rebaudioside A containing Steviol Glycoside X (Rebaudioside O) through the recrystallization of the extract of the aforementioned embodiment.

The fourth embodiment of the present invention is a manufacturing method for foodstuff, in which the extract of the second embodiment above is added to food in an amount of equal to or less than 1% of the food.

The fifth embodiment of the present invention is a manufacturing method for foodstuff, in which the highly pure Rebaudioside A obtained in the third embodiment above is added to food in an amount of equal to or less than 1% of the food.

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 crystallization through the extract obtained from varieties whose main component is ST, or in the high purity products obtained from their recrystallization, 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.

The seventh embodiment of the present invention is an analytical method of the Steviol Glycosides I-X through high performance liquid chromatography (hereinafter HPLC).

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 JP2001-200944 and JP2007-506004, 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.

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 JP2001-200944 (hereinafter called Variety B) and 2007-506004 (hereinafter called Variety C).

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.

The novel 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 5 and each Glycoside structure I-X:

embedded image

wherein R¹and R²are respectively a hydrogen atom and the aforementioned sugar chain, was determined.

Steviol Glycoside I (Dulcoside B) is a glycoside with a structure of 788 molecular weight, as confirmed by about minutes of chromatograph Retention Time (hereinafter R.T.) of the HPLC in FIG. 1.

Steviol Glycoside II (Rebaudioside G) is a glycoside with a structure of 804 molecular weight, as confirmed by about 15 minutes of R.T. of HPLC in FIG. 1.

Steviol Glycoside III (Rebaudioside I) is a glycoside with a structure of 1112 molecular weight, as confirmed by about 28 minutes of R.T. of HPLC in FIG. 1.

Steviol Glycoside IV (Rebaudioside H) is a glycoside with a structure of 1128 molecular weight, as confirmed by about 29 minutes of R.T. of HPLC in FIG. 1.

Steviol Glycoside V (Rebaudioside L) is a glycoside with a structure of 1112 molecular weight, as confirmed by about 34 minutes of R.T. of HPLC in FIG. 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 FIG. 1.

Steviol Glycoside VII (Rebaudioside J) is a glycoside with a structure of 1128 molecular weight, as confirmed by about 34 minutes of R.T. of HPLC in FIG. 1.

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 FIG. 1.

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 FIG. 1.

Steviol Glycoside X (Rebaudioside O) is a glycoside with a structure of 1436 molecular weight, as confirmed by about 51 minutes of R.T. of HPLC in FIG. 1.

However, anyone skilled in the art will understand that the R.T. mentioned above from the analysis using gradient elution is variable.

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.

Simultaneously, since it is possible to confirm these novel Steviol Glycosides through HPLC, even in case they are used as sweeteners, quality and taste may be totally controlled with ease by HPLC analysis.

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
Example 1
Manufacturing of RA Extract
(1) Extract

100 g of dry leaves obtained from varieties A, B or C, whose main component is RA, was extracted several times with 20 times amount of water by weight until the sweetness cannot be tasted. The extract was passed through a column filled up with 300 ml of absorption resin (Diaion HP-20) and the sweet components of the extract were absorbed to the resin, which was sufficiently washed with water, and the components were eluted with 900m1 of methanol. The eluate was passed through a column filled up with 200 ml of ion exchange resin (Diaion WA-30), 10 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 35.4%, RA 41.7% and RC 9.8%), 11.5 g of RA-B extract (ST 19.5%, RA 58.1% and RC 8.8%) and 12 g of RA-C extract (ST5.4%, RA 72.3% and RC 8.1%) respectively.

(2) RA Recrystallization

Each 5 g of the aforementioned RA-B extract and RA-C extract was dissolved in 10 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 3.9 g of white RA-B crystals (ST 0.2%, RA 95.0% and RC 0.2%) and 4.5 g of RA-C Crystals (ST 0.2%, RA 95.6% and RC 0.1%) respectively.

Example 2
Manufacturing of ST Extract

For comparison purposes, the same procedure was carried out as to a variety, the main component of which is ST, to give 11.3 g of ST extract (ST 51.9%, RA 23.7% and RC 7.4%).

Example 3
RA-A Mother Liquid, ST Mother Liquid

Each 10 g of the aforementioned RA-A extract and ST extract was dissolved in 10 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 2.1 g of RA-ST crystals, which are white crystals of stevioside, and 3.8 g of ST-ST crystals respectively. Each of 8.8 g of RA-A mother liquid(ST 15.7%, RA 43.8% and RC 6.9%) and 6.1 g of ST mother liquor (ST 20.0%, 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

Each of the mother liquid powder of Example 3 was dissolved in 10 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 2.2 g of white RA-A crystals (ST 1.6%, RA 90.4% and RC 1.4%) and 1.2 g of ST-RA crystals (ST 1.6%, RA 96.9% and RC 1.4%) respectively.

Example 5
Structural Determination of Steviol Glycosides

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-10Advp HPLC using a column of TSKgel Amide-80 (4.6×250 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 0.65 ml/min, the column temperature was 40° C. and the detection was performed at ultraviolet absorption of 210 nm.

In the measurement of molecular weight, Waters' Alliance HPLC System 2695 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 (2.0×250 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 40° C. Nitrogen gas was used as a desolvation gas and argon gas was used as a collision gas. As a capillary voltage, 15.0 kV were used in the Steviol Glycoside analysis in the negative mode and in the analysis of ABEE-oligosaccharides 13.5 kV were used in the positive mode. A voltage of 10V 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 100° C. and 400° C. respectively and the flow volume of cone gas and desolvation gas were 50 l/hr and 900 l/hr respectively.

The results of HPLC Analysis regarding each extract and crystal were illustrated in FIGS. 1-10.

The analytical results of each chromatography peak shown in FIGS. 2-10 were shown in the following Table 1-9.

TABLE 1

Crystals RA-A

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
4.857
6932
486

0.2192
Stev mono

2
8.817
1467
107

3
9.603
2029
124

0.0642
Rubuso

4
10.244
3630
214

5
11.065
12106
605

6
14.763
14947
397

0.4726
Rebau B

7
16.782
58617
2320

1.8535
Stev

8
18.901
35984
1440

1.1379
Rebau C

9
19.984
23401
820

0.7400
Rebau F

10
23.334
2971977
97370

93.9772
Rebau A

11
28.648
4872
191

0.1541
Rebau E + III + IV

12
34.608
5912
174

0.1869
V + VI + VII

13
35.531
28131
892
V

0.8895
Rebau D + VIII

14
37.222
5390
185

15
42.731
6943
222

0.2195
IX

16
51.166
2700
88

0.0854
X

TOT
3185038
105635

100.0000

TABLE 2

Crystals RA-B

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.098
1307
246

2
2.309
675
139
V

3
2.833
7713
1426

4
4.775
3286
244

5
13.518
91704
3965

2.8021
I

6
14.834
14854
462

0.4539
Rebau B

7
16.077
1125
54

8
16.8
747
88

9
17.034
9460
352
V

0.2891
Stev

10
18.601
2647
110

11
19.157
7685
314
V

0.2348
Rebau C

12
20.268
20421
652

0.6240
Rebau F

13
23.663
3106124
102260

94.9097
Rebau A

14
27.533
2882
95

15
29.043
5444
210

0.1663
III + Rebau E + IV

16
31.461
2828
91

0.0864

17
35.959
8128
282

0.2484
Rebau D + V + VI + VII

18
37.696
6165
183

19
43.225
4259
120

0.1301
IX

20
51.644
1807
53

0.0552
X

TOT
3299261
111346

100.0000

TABLE 3

Crystals RA-C

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.335
15257
2745

2
3.398
2074
247

3
4.436
3292
209

4
9.657
51969
2355

1.6822
Rubuso

5
14.351
4502
107

0.1457
Rebau B

6
16.175
5072
189

0.1642
Stev

7
18.192
2634
88

0.0853
Rebau C

8
19.366
13008
474

0.4211
Rebau F

9
22.682
3002254
98794

97.1825
Rebau A

10
34.744
4564
160

0.1477
V + VI + Rebau D + VII + VIII

11
36.455
3018
96

0.0977

12
41.927
1880
67

0.0609
IX

13
50.485
393
17

0.0127
X

TOT
3109917
105548

100.0000

TABLE 4

Extract RA-A

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.646
12581
1924

2
3.502
1786
313

3
3.715
5140
389
V

4
4.411
36010
2058
V

5
4.64
18976
2043
V

6
4.904
94086
5839
V

1.3702
Stev mono

7
5.683
42933
1608
V

8
6.108
9264
666
V

9
6.642
30742
778
V

10
7.192
23108
906
V

11
7.736
52867
2210
V

0.7699
Stev bio

12
8.292
22402
750
V

13
8.831
44958
2378
V

14
9.615
104428
4765
V

1.5208
Rubuso

15
10.262
92646
4531
V

16
10.917
33787
1420
V

17
12.778
63372
2978

0.9229
Dulco A

18
13.466
12776
658
V

0.1861
I

19
14.821
47594
1672

0.6931
Rebau B

20
16.812
2795705
107308

40.7133
Stev

21
18.95
683621
23300
V

9.9554
Rebau C

22
20.017
130330
3792
V

1.8980
Rebau F

23
22.017
4444
192

24
23.385
2759980
90911

40.1931
Rebau A

25
26.082
15240
516

26
28.592
5443
144

0.0793

27
29.425
6048
234
V

0.0881
III + Rebau E

28
30.066
29426
961
V

0.4285
IV

29
33.96
10732
419

0.1563
V + VI + VII

30
35.559
51215
1667

0.7458
Rebau D + VIII

31
42.755
15644
462

0.2278
IX

32
51.175
3535
108

0.0515
X

TOT
7260819
267900

100.0000

TABLE 5

Extract RA-B

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.136
1244
231

2
2.862
8119
1484

3
4.411
37288
2536

4
4.634
25374
2584
V

5
4.889
113717
7669
V

3.2793
Stev mono

6
5.766
57184
1940
V

7
6.666
16138
503
V

8
7.319
34921
927
V

0.3964
Stev bio

9
7.917
13888
628
V

10
8.533
19410
741
V

11
8.887
44852
2585
V

12
9.35
16370
1111
V

0.4751
Rubuso

13
9.71
81099
3752
V

14
10.339
81250
3739
V

15
12.183
4543
172

16
12.941
26992
1213
V

0.5187
Dulco A

17
13.658
35796
1693
V

0.7239
I

18
15.012
62182
2088

0.8928
II

19
17.039
1579831
61299

26.2115
Stev

20
19.212
630967
21477
V

9.1836
Rebau C

21
20.303
129387
3817
V

1.6322
Rebau F

22
23.721
3953586
128495
S

54.9446
Rebau A

23
26.44
11802
359
T

24
29.088
9888
346

25
29.817
9663
299
V

26
30.449
17347
602
V

0.2574
Rebau E + III + IV

27
32.984
10425
255

28
34.415
15693
537

29
35.159
18016
574
V

0.2454
V + VI + VII

30
36.025
68454
2067
V

0.8839
Rebau D + VIII

31
37.749
4538
157

32
43.275
20100
626

0.2677
IX

33
51.757
7320
205

0.0877
X

TOT
7167384
256711

100.0000

TABLE 6

Extract RA-C

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.183
616
134

2
2.597
10175
1537

3
3.437
1414
323

4
4.46
13740
1017

5
4.642
6308
820
V

6
4.906
54449
4098
V

0.8853
Stev mono

7
5.721
9947
485
V

8
6.06
3281
277
V

9
8.264
25348
701

10
8.844
16133
1093
V

11
9.66
34116
1479

12
10.275
34888
1821
V

0.5673
Rubuso

13
10.799
10225
529
V

14
11.829
144125
5701

15
12.826
9263
377
V

0.1506
Dulco A

16
14.874
129304
2644

2.1024
Rehau B

17
16.871
437696
17109
V

7.1167
Stev

18
19.001
472663
16510

7.6852
Rebau C

19
20.078
99978
2988
V

1.6256
Rebau F

20
23.439
4700591
156131

76.4290
Rebau A

21
26.161
6575
216

22
28.759
19933
692

23
29.523
16030
496
V

0.2606
Rebau E

24
30.192
4314
195
V

0.0701
III + IV

25
32.915
22341
361

0.0000

26
34.044
20210
662
V

0.3286
V

27
34.784
37467
1171
V

0.6092
VI + VII

28
35.637
82080
2479
V

1.3346
Rebau D + VIII

29
42.833
32965
977

0.5360
IX

30
51.265
18371
536

0.2987
X

TOT
6474546
223559

100.0000

TABLE 7

Extract ST

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.626
7139
1183

2
3.325
824
127

3
3.683
3069
423
V

4
3.836
9867
1104
V

5
4.1
20804
2418
V

6
4.432
158950
6323
V

7
4.898
244425
14003
V

3.2613
Stev mono

8
5.679
171018
7277
V

9
6.067
36443
2486
V

10
6.567
84143
3356
V

11
6.833
59479
3255
V

12
7.702
364999
10439
V

4.8701
Stev bio

13
8.831
50633
2395
V

14
9.622
121367
5308
V

1.6194
Rubuso

15
10.283
61202
2776
V

16
10.651
82679
3615
V

17
12.789
172505
7512

2.3017
Dulco A

18
13.476
39377
1734
V

0.5254
I

19
14.117
9126
325
V

20
14.843
56080
1517
V

0.7483
Rebau B

21
15.883
18828
574
V

0.2512
II

22
16.837
4190811
160109
SV

55.9165
Stev

23
18.317
837
72
T

24
18.961
471940
16634
V

6.2969
Rebau C

25
20.03
96159
2847
V

1.2830
Rebau F

26
22.021
11777
430

27
23.407
1635704
52768
SV

21.8246
Rebau A

28
26.087
18075
573
T

29
30.114
37321
1256

0.4980
Rebau E + III + IV

30
34.028
11847
362

0.1581
V + VI + VII

31
35.633
28790
851
V

0.3841
Rebau D + VIII

32
42.867
4616
125

0.0616
IX

TOT
8280834
314177

100.0000

TABLE 8

Crystals ST-ST

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.64
20368
3347

2
3.342
792
150

3
8.16
199715
8553

4
9.579
28792
1602

0.7198
Rubuso

5
13.436
6892
362

0.1723
I

6
16.763
3809949
146224
S

95.2514
Stev

7
19.017
1413
63
T

0.0353
Rebau C

8
21.883
3078
108

0.0770
Rebau F

9
23.323
138456
4564
V

3.4615
Rebau A

10
26.007
6455
215

11
30.02
11309
367

0.2827
Rebau E + III + IV

TOT
4227219
165555

100.0000

TABLE 9

Crystals ST-RA

CH
PKNO
T
A
H
MK
IDNO
CONC
N

1
1
2.567
11398
2039

2
3.19
846
174

3
11.629
43279
1893

4
14.703
13970
400

0.4290
Rebau B

5
15.927
1715
79

6
16.885
7321
220

0.2248
Stev

7
18.961
4537
192

0.1393
Rebau C

8
20.055
9706
413

0.2981
Rebau F

9
23.428
3204919
106088

98.4203
Rebau A

10
28.744
5579
170

0.1713
Rebau E + III + IV

11
33.32
4707
121

0.1445
V + VI + VII

12
35.603
4198
146

0.1289
Rebau D + VIII

13
42.8
1422
45

0.0437
IX

TOT
3313597
111980

100.0000

The abbreviations used in the tables above are as follows:

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

The concentration is calculated from the total area of the ultraviolet absorption spectrum at 210 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.

Example 6
Evaluation of Taste Quality

0.05% aqueous solution of each extract and 0.03% aqueous solution of the crystals were evaluated by 10 people familiar with a sensory test of Stevia, and the results of averaged evaluation are shown in Table 10 below: Evaluation 5: Excellent, 4: Good, 3: Ordinary, 2: Bad, 1: Worst

TABLE 10

Subject matter of

the sensory test
1)
2)
3)
4)
5)
6)

RA-A Extract
4.1
3.8
4.1
4.9
3.4
3.8

RA-A Crystal
5.0
4.9
4.9
3.2
4.9
4.9

RA-B Extract
4.2
3.8
4.1
4.9
3.5
3.7

RA-B Crystal
5.0
5.0
4.9
3.5
4.9
4.9

RA-C Extract
4.2
4.0
4.5
4.8
3.5
3.8

RA-C Crystal
5.0
5.0
5.0
3.8
5.0
5.0

ST Extract
1.2
1.3
1.2
4.0
2.0
2.0

ST-RA Crystal
4.8
4.6
4.6
3.0
4.8
4.3

1) Sweetness Quality

2) Taste remaining in the mouth

3) Astringency

4) Delicate taste

5) Refreshing feeling

6) Sweetness running out

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 novel Steviol Glycosides II-X influence the delicate taste.

Example 7
Determination of Variety

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.

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

According to the HPLC condition described in Example 5, 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

1) Tabletop sugar was prepared by mixing 1 g of RA-A Crystals and 99 g of powder sugar.

2) Tabletop sugar was prepared by mixing 1 g of RA-B Crystals and 99 g of erythritol.

3) Tabletop sugar was prepared by mixing 1 g of RA-C Crystals and 99 g of high-fructose corn syrup.

Example 10
Candies

Candy was prepared from 0.3 g of RA-C extract, 100 g of palatinit and an appropriate volume of spices.

Example 11
Milk Jelly

Milk jelly was prepared form 15 g of sugar, 0.08 g of RA-B extract, 250 g of milk, 5 g of gelatin and an appropriate volume of milk flavor.

Example 12
Sports Drinks

Sports drink was prepared from 0.075% of RA-B Crystals, 0.11% of calcium lactate, 0.045% of citric acid, 0.03% of trisodium citrate, 0.015% of magnesium chloride, 0.0055% of glutamic acid and 99.72% of water.

Example 13
Carbonate Drinks

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, 0.0025% of caffeine, 0.0034% of calcium pantothenate, 0.003% of niacin amide, 0.002% of vitamin B6, 0.00009% of vitamin B2, 0.000002% of vitamin B12, and appropriate volumes of spices and water to adjust 100% of whole ingredients, and then introducing carbon dioxide gas.

INDUSTRIAL APPLICABILITY

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

[FIG. 1] It shows the HPLC Analysis Chart for the extract RA-C.

[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. 5] It shows the HPLC Analysis Chart for the extract RA-A.

[FIG. 6] It shows the HPLC Analysis Chart for the extract RA-B.

[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.

[FIG. 9] It shows the HPLC Analysis Chart for Crystals ST-ST.

[FIG. 10] It shows the HPLC Analysis Chart for Crystals ST-RA.

	Number	Date	Country
Parent	14197636	Mar 2014	US
Child	14937329		US
Parent	13122232	Apr 2011	US
Child	14197636		US

STEVIOL GLYCOSIDE

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