BACKGROUND OF THE INVENTION

The invention is directed to the positive impact of minor steviol glycosides on the sweetness profile of stevia sweeteners.

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 banned or restricted in some countries due to 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; Chaturvedula et al., 201 1a,b,c; Chaturvedula and Prakash, 201 1a,b; Ohta et al., 2010). 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 generic structure of the steviol glycosides is presented in Figure 1.

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 : 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 1 10-270 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., 201 la,b,c; Chaturvedula and Prakash, 201 1 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- a or β-glycosidic linkages. The identity and grouping of various steviol glycosides is summarized in Table 1. Due to the large number of steviol glycosides, Table 1 lists the steviol glycosides in 5 groups depending on the sugar moieties connected to the steviol backbone and identified with abbreviated compositional names.

• Glucosyl steviol family: comprising only steviol and glucose residues. This group can be presented by general formula "SvGn", wherein Sv is the steviol and G is glucose.

• Rhamnosyl steviol family: comprising steviol, rhamose and glucose residues. This group can be presented by general formula "SvRIGn", wherein R is the Rhamnose.

• Xylosyl steviol family: comprising steviol, xylose and glucose residues. This group can be presented by general formula "SvXIGn", wherein X is the Xylose.

• Fructosyl steviol family: comprising steviol, fructose and glucose residues. This group can be presented by general formula "SvFlGn", wherein F is the Fructose.

• Deoxyglucose steviol family: comprising steviol, deoxyglucose and glucose residue. This group can be presented by general formula SvdGlGn.

Table 1

Common Abbr.

#

Name Formula Ri R ₂

2.2 Dulcoside B SvRlG2 H Rhaa( 1 -2)[GlcP( 1 -3)]GlcP 1 -

2.3 Rebaudioside C SvRlG3 Glcpl- Rhaa(l-2)[Glcp(l -3)]Glcpl-

2.4 Rebaudioside C SvRlG3 Rhaa(l-2)Glcpl- GlcP(l-3)Glcpl- (isomer)

2.5 Rebaudioside H SvRlG4 Glcpl- Glcp(l-3)Rhaa(l-2)[Glcp(l- 3)lGlcpi-

2.6 Rebaudioside K SvRlG4 Glcp(l-2)Glcpl- Rhaa(l-2)[GlcP(l-3)]Glcpl-

2.7 Rebaudioside J SvRlG4 Rhaa(l-2)Glcpl- GlcP(l-2)[GlcP(l-3)]Glcpl-

2.8 Rebaudioside N SvRlG5 Rhaa(l-2)[Glcp(l- Glcp(l-2)[GlcP(l-3)]Glcpl- 3)]Glcpl-

2.9 Rebaudioside 0 SvRlG6 Glcp(l-3)Rhaa(l- Glcp(l-2)[Glcp(l-3)]Glcpi-

2) [Glcp(l-

3) ]Glcpi-

3) Steviol + Xylose + Glucose (SvXl Gn)

3.1 Stevioside F SvXlG2 Glcpl- XyiP(l-2)Glcpl-

3.2 Rebaudioside F SvXlG3 Glcpl- Xylp(l-2)[Glcp(l-3)]Glcpl-

3.3 Rebaudioside SvXlG3 Glcpl- Glcp(l-2)[Xylp(l-3)]Glcpl- F2

3.4 Rebaudioside SvXlG3 Xylp(l-6)Glcpi- GlcP(l-2)Glcpl- F3

4) Steviol + Fructose + Glucose (SvFIGn)

4.1 Rebaudioside SvFlG3 Glcpl- Glcp(l-2)[Frup(l-3)]Glcpl- A3

5) Steviol + deoxyCilucose + Glucose (SvdGIGn)

5.1 Stevioside D SvdGlG Glcpl- 6-deoxyGlcP(l-2)Glcpl- 2

5.2 Stevisoide E SvdGlG Glcpi- 6-deoxyGlcp(l-2)[Glcp(l- 3 3)lGlcpi-

5.3 Stevioside E2 SvdGlG 6-deoxyGlcpi- Glcp(l-2)[Glcp(l-3)]Glcpl- 3

SUMMARY OF THE INVENTION

The present invention is directed to improving the sweetness profile of steviol glycoside sweeteners, such as stevioside and Rebaudioside A (Reb A). It was unexpectedly discovered that certain minor steviol glycosides have a favorable impact on the sweetness profile of other steviol glycosides, including Rebaudioside D (Reb D) and Rebaudioside M (Reb M). These minor steviol glycosides, although present in small amounts, demonstrate statistically significant positive impacts on the sweetness profile of stevia sweeteners. As used herein, a minor steviol glycoside is one that is present in a composition in an amount less than 5%, or less than 3%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the generic structure of steviol glycosides.

FIG. 2a shows the structure of Rebaudioside N. FIG. 2b shows the structure of

Rebaudioside O.

FIG. 3 a is a bar graph showing the results of a taste panel. The first bars represent the results from tasting a highly purified (at least 97% pure) Rebaudioside A composition in acidified water. The second bars represent the results from tasting a fructosylated Rebaudioside A composition in acidified water.

FIG. 3b is a bar graph showing the results of a taste panel. The first bars represent the results from tasting a diet lemon-lime carbonated soft drink including a highly purified Reb M/Reb D blend, wherein the blend includes 50% Reb M and 50% Reb D by weight. The second bars represent the results from tasting a diet lemon-lime carbonated soft drink including a fructosylated Rebaudioside A composition.

FIG. 4a is a bar graph showing the results of a taste panel. The first bars represent the results from tasting a highly purified (at least 97% pure) Rebaudioside A composition in acidified water. The second bars represent the results from tasting a glycosylated minor steviol glycoside composition in acidified water, wherein the glycosylated minor steviol glycoside composition contains 19% glycosylated Reb Q by weight and 18% glycosylated Reb R by weight.

FIG. 4b is a bar graph showing the results of a taste panel. The first bars represent the results from tasting a diet lemon-lime carbonated soft drink including a highly purified Reb M/Reb D blend, wherein the blend includes 50% Reb M and 50% Reb D by weight. The second bars represent the results from tasting a diet lemon-lime carbonated soft drink including a glycosylated minor steviol glycoside composition in acidified water, wherein the glycosylated minor steviol glycoside composition contains 19% glycosylated Reb Q by weight and 18% glycosylated Reb R by weight.

DETAILED DESCRIPTION Steviol glycoside compositions including minor glycosides (SG compositions), such as

Rebaudioside N and Rebaudioside O, were found to have a positive impact on the sweetness profile of stevia sweeteners. An exemplary steviol glycoside composition is described in Table 2.

Table 2: Steviol Glycoside Composition (SG Composition)

Comparison of Reb D/Reb M Blends to Steviol Glycoside Compositions Example 1 : Acidified Water - 5 Brix Target

15 panelists tested the sample solutions, as summarized in Table 3:

Table 3; Acidified water 5 brix target - SG Composition vs Reb D and Reb M blends

Summary of Mean-Scores, P-Values, and Signi cance

200 ppm of

200 ppm of

70/30 Reb

Attribute 5% Sugar SG P-Value Sig

D/Reb M

Composition

blend

Sweetness 4.91 4.86 4.98 0.0997

b ab a

Bitterness 0.75 0.98 1.23 0.0083 ***

b b a

Astringency 0.84 1.02 1.77 0.0035 ***

c b a

Acidity 0.99 1.41 1.99 0.0012 **#

b b a

Off-Note (Metallic/ Licorice) 0.52 0.61 1.13 0.0081 ***

c b a

Sweet Aftertaste 0.56 0.99 1.38 0.0046 ** *

Bitter Aftertaste 0.33 0.37 0.53 0.5487 NS

a b b

Overall Liking 4.96 4.53 4.46 0.1964 *

*= 80% confidence interval, **= 90% Confidence interval, ***=95% Confidence interval As can be seen, the steviol glycoside composition (SG composition) has many attributes that are more similar to a 5% sugar solution than a Reb D/Reb M blend, and the overall liking is greater than the Reb D/Reb M blend. Steviol glycoside compositions with minor glycosides and a 70/30 Reb D/M blend were parity in sweetness. The Reb D/Reb M 70/30 blend had significantly higher astringency, acidity, off-notes, and sweet aftertaste compared to the SG composition. The two samples have significantly different flavor profiles.

Example 2: Acidified Water - 10 Brix Target

The same test was done as in Example 1, but with a 10 Brix target. 13 panelists tested the solutions, as summarized in Table 4.

Table 4: Acidified water 10 brix target - SG Composition vs Reb D and Reb M blends

*= 80% confidence interval, **= 90% Confidence interval, ***=95% Confidence interval

In this test, the steviol glycoside composition and the Reb D/Reb M blend were parity in sweetness. The 70/30 blend also had significantly higher sweet aftertaste compared to the steviol glycoside composition. While not intending to be bound by theory, it is believed that Rebaudioside N and

Rebaudioside O, while present in minor amounts in the steviol glycoside composition, have a favorable impact on the flavor and sweetness profile of the steviol glycoside composition, particularly at the 5 Brix target level. The structures of Rebaudioside N and Rebaudioside O are shown in Figs. 2a and 2b. However, certain advantages were observed even at the 10 Brix target, making these minor steviol glycosides relevant in the improvement of sweetness profiles.

Example 3 : Fructosylated Steviol Glycosides It was discovered that adding one or more fructose molecules to rebaudioside A provides flavor and sweetness profile advantages. It is believed that fructosylation of rebaudioside A or other steviol glycosides, including minor steviol glycosides, can provide characteristics that are similar to or even better than highly purified steviol glycosides. Rebaudioside A was subjected to a glycosylation process to add fructose to the Gl and G2 positions, and compared to a highly purified rebaudioside A composition in acidified water, and a highly purified Reb M/Reb D blend in a diet lemon-lime carbonated soft drink. The results from a trained test panel are shown in Figures 3a and 3b.

Example 4: Glycosylated Minor Steviol Glycosides It was discovered that in addition to their favorable impact on the flavor and sweetness profiles of steviol glycoside compositions, the impact of minor glycosides can be improved when they are glycosylated. Glycosylated steviol glycosides (GSG) have one or more glycoside moieties attached to the steviol glycoside backbone, and have been found to have a favorable impact on attributes such as bitterness, sweetness aftertaste, etc. on steviol glycoside compositions. Glycosylated minor steviol glycosides can improve the flavor and sweetness profiles of steviol glycoside compositions to be similar to or even surpass the flavor and sweetness profiles of highly purified steviol glycosides, thereby providing an efficient and effective alternative to highly purified steviol glycosides.

The glycoside moiety can be any monosaccharide, disaccharide, or oligosaccharide, such as glucose, fructose, rhamnose, xylose, and the like. The glycoside moiety can be a single molecule, or can be a chain of molecules, for example, 2, 3 or 4 glucose units attached to the steviol glycoside. The glycosylation process typically results in the glycoside moiety attaching to the C-13 or C-19 position on the steviol glycoside. In one embodiment, a rebaudioside A and minor steviol glycoside composition was glycosylated. The minor steviol glycoside content included glycosylated rebaudioside Q at about 19% by weight, and glycosylated rebaudioside R at about 18% by weight.

Acidified water and diet lemon-lime carbonated soft drink samples containing various amounts of glycosylated minor steviol glycoside composition were tested by a trained panel, and the results are shown in Figures 4a and 4b. As can be seen, the glycosylated minor steviol glycoside-containing composition performed nearly the same as, and in some cases better than, highly purified steviol glycoside compositions (Reb A and Reb M/Reb D) across a number of sweetness and flavor attributes.

It is to be understood that the foregoing descriptions 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.