GLUCOSYLATED STEVIOL GLYCOSIDE COMPOSITION AS A FLAVOR MODIFIER

BACKGROUND

Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. The leaves of the plant contain from 10 to 20% of diterpene glycosides, which are around 150 to 450 times sweeter than sugar. The leaves have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas and medicines.

At present there are more than 230 Stevia species with significant sweetening properties. The plant has been successfully grown under a wide range of conditions from its native subtropics to the cold northern latitudes.

The extract of the Stevia rebaudiana plant contains a mixture of different sweet diterpene glycosides, which have a single base—steviol—and differ by the presence of carbohydrate residues at positions C13 and C19. These glycosides accumulate in Stevia leaves and compose approximately 10%-20% of the total dry weight. Typically, on a dry weight basis, the four major glycosides found in the leaves of Stevia are Dulcoside A, Rebaudioside C, Rebaudioside A and Stevioside. Other glycosides identified in Stevia extract include Rebaudioside B, D, E, and F, Steviolbioside and Rubusoside.

Steviol glycosides differ from each other by sweetness power as well as other sensory features contributing to taste quality such as bitterness, lingering aftertaste, etc. Kinghorn, A. D., Stevia: The genus Stevia, Taylor & Francis, London (2002). The taste quality of a steviol glycoside is one of the major characteristics which is decisive for its usage in various food and beverage applications. The sweetness and taste quality of stevia glycosides are set forth below in Table 1.

TABLE 1

Steviol Glycoside molecules and their sweetness and taste quality

Sweetening

Steviol

power relative
Taste

Glycosides
R1 (C19)
R2 (C13)
to sucrose
Quality

Stevioside (G3)
β-Glc
β-Glc-β-Glc(2-1)
150-300
0

Rebaudioside A
β-Glc
β-Glc-β-Glc(2-1)-
200-400
+2

(G4)

β-Glc(3-1)

Rebaudioside B
H
β-Glc-β-Glc(2-1)-

300-350^a
0

(G3)

β-Glc(3-1)

Rebaudioside C
β-Glc
β-Glc-α-Rha(2-1)-
50-120
−1

(G4)

β-Glc(3-1)

Rebaudioside D
β-Glc-β-Glc(2-1)
β-Glc-β-Glc(2-1)-
200-300
+3

(G5)

β-Glc(3-1)

Rebaudioside E
β-Glc-β-Glc(2-1)
β-Glc-β-Glc(2-1)
250-300
+1

(G4)

Rebaudioside F
β-Glc
β-Glc-β-Xyl(2-1)-
Na
Na

(G4)

β-Glc(3-1)

Rubusoside (G2)
β-Glc
β-Glc
110
−2

Steviolmonoside
H
β-Glc
Na
Na

(G1)

Steviolbioside
H
β-Glc-β-Glc(2-1)
100-125
−3

(G2)

DulcosideA (G3)
β-Glc
β-Glc-α-Rha(2-1)
50-120
−2

Previous studies show a certain correlation between number of glycosidic residues and taste quality of a steviol glycoside. When comparing steviol glycosides, rebaudioside A (G4, having 4 glucose residues) clearly surpasses stevioside and rebaudioside B (G3, each having 3 glusose residues) in taste quality. Steviolbioside and rubusoside (G2, each having 2 glucose residues) have a taste quality that is proven to be significantly inferior to that of stevioside (G3). In addition, the taste quality of rhamnosylated glycosides is inferior compared to that of glucosylated glycosides. Tanaka, O., “Improvement of Taste of Natural Sweeteners,” Pure & Appl. Chem., Vol. 69, No. 4, pp. 675-683 (1997).

The sweetness and taste quality of steviol glycoside molecules containing only glucose units are plotted in FIGS. 1a-1b. Steviol glycosides with a greater number of glucose residues, for example, more than two glucose residues, show a better taste quality. Particularly mono- and di-glucosyl forms of stevioside (having 4 glucose residues (G4) and 5 glucose residues (G5) respectively) possess significantly better taste quality. Tanaka, O., “Improvement of Taste of Natural Sweeteners,” Pure & Appl. Chem., Vol. 69, No. 4, pp. 675-683 (1997).

It is known that the undesired taste attributes can be substantially reduced or eliminated by the reaction of intermolecular transglycosylation of various enzymes, upon which the attachment of new carbohydrates at positions C13 and C19 of steviol glycosides takes place. Tanaka (1997) evaluated the effect of adding glucose molecules to purified stevioside molecules by tranglycosylation. The resulting glucosylated steviosides were evaluated for their sweetness and taste quality. The improvement of taste quality was greater when the glucose units were added to the C19 position rather than at the C13 position.

Various enzymes were used to conduct such transglycosylation. Pullulanase, isomaltase (Lobov, S. V. et al., “Enzymic Production of Sweet Stevioside Derivatives: Transglucosylation by Glucosidases,” Agric. Biol. Chem., Vol. 55, No. 12, pp. 2959-2965 (1991)), β-galactosidase (Kitahata, S. et al., “Production of Rubusoside Derivatives by Transgalactosylation of Various β-Galactosidases,” Agric. Biol. Chem., Vol. 53, No. 11, pp. 2923-2928 (1989)), and dextrine saccharase (Yamamoto, K. et al., Biosci. Biotech. Biochem., Vol. 58, No. 9, pp. 1657-1661 (1994)) were used as enzymes with pullulan, maltose, lactose, and partially hydrolyzed starch being donors.

The transglucosylation of steviol glycosides was also achieved by the action of cyclodextrin glucanotransferases (CGTase). The obtained sweeteners possessed improved sweetness without bitterness and licorice taste (U.S. Pat. Nos. 4,219,571, 7,838,044, and 7,807,206).

With an increase in the number of glucose units in steviol glycoside molecules (for example, from stevioside to Rebaudioside A), the sweetness intensity increases and sweetness profile (taste) improves. However, the relative sweetness does not increase significantly beyond a certain level with a further increase of glucose units, as shown in FIG. 1a. The published data show that the sweetness quality improves with the addition of glucose units, but does not explicitly or implicitly mention that the addition of glucose units contributes to a reduction of sweetness.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a taste and flavor profile enhancing composition. The composition includes glucosylated steviol glycosides which can enhance the intensity of a taste and/or a flavor in a food or beverage product. In some embodiments, the glucosylated steviol glycosides may include a plurality of glucose units. For example, the glucosylated steviol glycosides may include three, four, five, or more than five glucose units.

The present invention is also directed to a food or beverage product having an intense taste and flavor profile, wherein the food or beverage product includes a taste and flavor enhancing composition comprising glucosylated steviol glycosides. A wide range of food and beverage products, such as, but not limited to, carbonated soft drinks, fruit juices, dairy foods, dairy beverages, baked goods, cereal products, and table top sweeteners, may be made in accordance with the present invention. The taste and flavor profile of a food or beverage product including a taste and flavor enhancing composition, wherein the taste and flavor enhancing composition includes glucosylated steviol glycosides, may be more intense than a comparative taste and flavor profile of a comparative food or beverage product which does not include the taste and flavor enhancing composition. Moreover, the mouthfeel of a food or beverage product including the taste and flavor enhancing composition, wherein the taste and flavor enhancing composition includes glucosylated steviol glycosides, may be improved in relation to a mouthfeel of a comparative food or beverage product which does not include the taste and flavor enhancing composition.

The present invention is further directed to a method of increasing the taste and flavor intensity of a food or beverage product, including the step of adding a taste and flavor enhancing composition to the food or beverage product, wherein the taste and flavor enhancing composition includes glucosylated steviol glycosides. The present invention is also directed to a method of improving the organoleptic properties of a food or beverage product including a high fructose syrup, including the step of adding the taste and flavor enhancing composition to the food or beverage product. For example, adding the taste and flavor enhancing composition may cause the high fructose syrup, such as high fructose corn syrup, to taste more like sugar. Also, if the high fructose syrup is high fructose corn syrup 42 (HFCS 42), adding the taste and flavor enhancing composition may cause the HFCS 42 to taste more like high fructose corn syrup 55 (HFCS 55).

The present invention is also directed to a method of making a taste and flavor enhancing composition, including: extracting steviol glycosides from leaves of a Steviol rebaudiana Bertoni plant, and transglycosylating the steviol glycosides to add glucose units to the steviol glycosides. In some embodiments, transglycosylating the steviol glycosides includes enzymatic transglycosylation using an enzyme. Examples of enzymes which may be used in accordance with the present invention include, but are not limited to, pullulanase, isomaltase, β-galactosidase, dextrine saccharase, and cyclodextrin glucotransferase.

The present invention is further directed to a method of making a food or beverage product, including: adding a taste and flavor enhancing composition including glucosylated steviol glycosides, and adding a reduced amount of erythritol, wherein the reduced amount of erythritol is less than the amount of erythritol in a comparative food or beverage composition which does not include the taste and flavor enhancing composition. The mouthfeel of the food or beverage product is similar to the mouthfeel of the comparative food or beverage product, even though the comparative food or beverage product contains a higher level of erythritol.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features of the invention which form the subject of the claims of the invention will be described hereinafter. It should be appreciated by those skilled in the art that the specific embodiments disclosed may be readily utilized as a basis for modifying or designing other methods or structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a bar graph showing the relative sweetness of steviol glycoside molecules with increasing numbers of glucose units. FIG. 1b is a bar graph showing the taste quality of steviol glycoside molecules with increasing numbers of glucose units.

FIG. 2 shows the chemical structures of stevioside and glucosyl stevioside.

FIG. 3 is a graph of the sweetness potency, or sucrose equivalent value (SEV), of stevia glycosides at a 5% sugar sweetness level.

FIG. 4a is a graph of the relative ratings of the sweetness intensity and sweet onset of mango-passion fruit flavored water beverages to which different stevia ingredients were added, and which did not include sugar. FIG. 4b is a graph of the relative ratings of the passion fruit flavor and mango flavor of these mango-passion fruit flavored water beverages.

FIG. 5a is a graph of the relative ratings of the sweetness intensity and sweet onset of mango-passion fruit flavored water beverages to which different stevia ingredients were added, and which included sugar. FIG. 5b is a graph of the relative ratings of the passion fruit flavor and mango flavor of these mango-passion fruit flavored water beverages.

FIG. 6 is a graph of the SEV (sucrose equivalent value) contribution of NSF-02 plotted against the concentration of NSF-02 in ppm, in acidified water and in acidified sugar solutions. NSF-02 (Natural Sweet Flavor #2) contains glucosylated steviol glycosides and dextrin, and is discussed further below.

FIG. 7 is a graph showing the synergy of NSF-02 with stevia exract, in a plot of the reduction of stevia extract against the concentration of NSF-02.

FIG. 8 is a bar graph showing the relative ratings of various attributes in an alcoholic beverage.

FIG. 9 is a bar graph showing the relative ratings of various attributes in a savory snack product.

FIG. 10 is a bar graph showing the relative ratings of various attributes in a fat-based dressing.

DETAILED DESCRIPTION

It has been unexpectedly discovered that glucosylation of steviol glycosides beyond a certain number of glucose units effectively reduces sweetness. It has also been discovered that with the reduction of sweetness, the glucosylated steviol glycosides can contribute to the modification of flavor and sweetness profiles. Therefore, while sweetness decreases with glucosylation, flavor modification increases. The steviol glycosides mixture provides a certain amount of sweetness, but the present invention shows that the glucosylated steviol glycosides (hereinafter “GSG”) enhance the flavor and sweetness profile in a wide range of applications, such as those listed in, but not limited by, the categories shown below in Table 2.

TABLE 2

GSG Applications

Application
Product
Results

Beverages
Apple Blueberry
Significantly more berry flavor intensity than the

Juice Drink
Control. Directionally, more acceptable for overall

flavor.

Orange Pineapple
Significantly more orange flavor intensity, pineapple

Passion Fruit
flavor intensity, and sweetness intensity.

Carbonated Drink

Baked
Lemon Poppy
Significantly more overall flavor intensity and

Goods
Seed Muffins
sweetness intensity than the Control.

Directionally, the Test sample was more acceptable

overall and had more lemon flavor intensity.

Processed
Strawberry
Significantly less bitter than the control.

Fruits
Topping

Dairy
Banana Flavored
Significantly more overall flavor intensity, banana

Milk Drink
flavor intensity, sweetness intensity, tartness intensity

and bitterness intensity.

Similar taste and flavor improvements were found in other categories of products, including, but not limited to, table top sweeteners, sauces and gravies, confectionery products, baked goods, cereals, snacks, and fruit and vegetable preparations.

EXAMPLES

In the following examples, the percentages in the formulas refer to percentages by weight.

Example 1
Evaluation of Iso-Sweetness of Steviol Glycosides

To evaluate the iso-sweetness of steviol glycosides (SG) and glucosylated steviol glycosides (GSG), a series of samples were selected as shown below in Table 3. The GSG was produced by treating the raw materials, steviol glycosides extracted from the Stevia plant, and starch extracted from tapioca, with a natural enzyme. The enzyme transfers glucose units from starch to the steviol glycosides. The enzyme used to facilitate this transfer is produced by means of fermentation using non-GMO (non-genetically modified organism) bacteria.

FIG. 2 is an illustration of an example of glucosylation. Specifically, FIG. 2 illustrates the single glucosylation (G1) of a stevioside molecule. This process can yield multiple glucosylation (G2, G3, etc.) of different steviol glycosides (mainly stevioside and Rebaudioside A) present in stevia extract.

TABLE 3

Glucosylated Steviol

Non-glucosylated
Glycosides

Test
Molecular
Steviol
Steviol
G1 &

Ingredients
Wt (avg)
equivalent
Glycosides
G2
G3-G9
G10-G20

Stevioside
805
0.396
>99%

Reb A
967
0.329
>99%

Reb D
1129
0.282
>99%

GSG-S
1210
0.263
<10%
95%

GSG-M
1380
0.231
7.80%
42%
50%

GSG-L
1798
0.177
5%
19%
60%
16%

To evaluate the sweetness potency of various concentrations of stevia products in aqueous solutions, aqueous solutions of sugar, stevioside, Rebaudioside A (Reb A), Rebaudioside D (Reb D), GSG-S (contains mainly smaller GSGs with 1 or 2 glucose units added to SG), GSG-M and GSG-L at various concentrations were prepared using bottled water. Samples were evaluated by the judges at room temperature (70-72° F.).

The judges were 11 panelists that have been previously qualified for their taste acuity and trained in the use of a sweetness intensity rating scale. The evaluations were done in duplicate using the same panelists so that a total of 22 values were generated for each average data point. Prior to the conduct of the study, judges were trained with sugar solutions and the use of the ballot.

Results were statistically analyzed to generate a standard error value for each solution as well as a confidence level at a 95% level. By comparing the sweetness of each test ingredient to the sweetness of several sucrose solutions, the sweetness potency of different stevia ingredients was estimated as shown in FIG. 3. FIG. 3 is a graph of the sweetness potency, or sucrose equivalent value (SEV), of different stevia ingredients at a 5% sugar sweetness level (i.e. at a concentration equivalent to 5% sucrose). This figure shows the effect of glucosylation on the SEV of steviol glycosides. As the number of glucose units on steviol glycosides increases, the sweetness increases from stevioside to Reb A and then starts decreasing with additional glucose units.

Example 2
Effect of Glucosylation on Flavor Modification

A mango-passion fruit flavored water formula was developed to evaluate the effect of different stevia ingredients on the sweetness and flavor profile of the beverage. A total of 9-10 panel members participated in this sensory test, where they assigned relative values to sweetness, onset of sweetness, mango fruit flavor, passion fruit flavor, acidity, overall taste, etc.

Example 2A
Effect of Glucosylation on Flavor Modification of No-Sugar-Added Beverage

Table 4 shows the no-sugar added beverage formula that used mainly Reb A, Reb D, GSG-S, or GSG-L. The amount of each ingredient (Reb A: 150 ppm; Reb D: 165 ppm; GSG-S: 190 ppm; and GSG-L: 300 ppm) was selected to have around 50 ppm of steviol in each formula.

TABLE 4

Mango-Passion Fruit Flavored Beverage (No Sugar Added)

Stevia Ingredient
Reb A
Reb D
GSG-S
GSG-L

Water
95.82
95.82
95.82
95.82

SG Content
0.015
0.0165
0.019
0.03

Citric Acid
0.078
0.078
0.078
0.078

Sodium Citrate
0.056
0.056
0.056
0.056

Mango flavor
0.031
0.031
0.031
0.031

Passion fruit flavor
0.014
0.014
0.014
0.014

FIGS. 4a-4b show the modification of flavor and sweetness profiles caused by glucosylation. The sweetness intensity decreased and sweet onset delayed with glucosylation. Mango flavor was enhanced and the passion fruit flavor reduced with glucosylation.

Example 2B
Effect of Glucosylation on Flavor Modification of a Beverage with Sugar

Table 5 shows the same mango-passion fruit flavored beverage formula with 4% sugar and stevia ingredients that contribute an additional 4% sugar-equivalent sweetness. The formula used Reb A, Reb D, GSG-S, or GSG-L in the amount of 50, 55, 73, and 200 ppm respectively as outlined in the formula below. As the number of glucose units increased more flavor modification (whether enhancement or suppression) was observed as shown in FIGS. 5a-5b.

TABLE 5

Mango-Passion Fruit Flavored Beverage (Sugar Added)

Reb A
Reb D
GSG-S
GSG-L

Stevia Ingredient
(%)
(%)
(%)
(%)

Water
95.82
95.82
95.82
95.82

Sugar
4.00
4.00
4.00
4.00

SG Content
0.005
0.0055
0.0073
0.02

Citric Acid
0.078
0.078
0.078
0.078

Sodium Citrate
0.056
0.056
0.056
0.056

Mango flavor
0.031
0.031
0.031
0.031

Passion fruit flavor
0.014
0.014
0.014
0.014

FIGS. 5a-5b show the effect of glucosylation of SG on the modification of flavor and sweetness profiles of a beverage sweetened with sugar and stevia (SG). The sweetness intensity decreased and sweet onset delayed with glucosylation. Both Mango and passion fruit flavors were somewhat suppressed with glucosylation. A key point is that GSG modified the flavor profile.

Example 3
Juice Drink with Glucosyl Steviol Glycosides

In this and the following examples, the GSG may be any glucosyl steviol glycoside composition, such as, but not limited to, a combination of GSG-S, GSG-M, and GSG-L.

To evaluate the flavor modification in a juice drink, a range of GSG concentrations (0 to 1000 ppm) was used with a typical apple blueberry juice drink formula. The objective was to assess whether the addition of GSG has an effect on key flavor attributes in various beverage applications. Specifically, the objective was to determine whether the flavor profile and overall acceptance of a Control sample of apple blueberry juice (containing no GSG) differs from a 30% reduced sugar Test sample of the same beverage (containing GSG). After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 50 to 750 ppm, and most preferably in the range of about 50 to 500 ppm.

For detailed sensory tests, two samples were selected to test against the control sample. The methodology of the detailed sensory tests is set forth below in Table 6. The formulas of the control and test samples are set forth below in Table 7.

TABLE 6

Methodology

Number of Sessions
1

Number of Participants:
20

Test Design:
Balanced, randomized within pair. Blind

Sensory Test Method:
Intensity and acceptance ratings

Environmental Condition
Standard booth lighting

Attributes and Scales:

Overall Acceptance on a 6-pt hedonic scale where 5 = Like Extremely,

2 = Neither Like Nor Dislike, and 0 = Dislike Extremely

Overall Flavor, Apple Flavor, Berry Flavor, Sweetness, Tartness,

Bitterness and Lingering Sweet Aftertaste Intensity on a 6-pt continuous

intensity scale where 0 = Imperceptible and 5 = Extremely Pronounced

Open Ended General Comments

Serving Temperature
Refrigerated temperature (~45° F.)

Serving/Panelists Instruction:
Samples served simultaneously. Panelists

instructed to read ingredient statement,

evaluate each sample.

TABLE 7

Samples

Test-1
Test-2

Description Flavor
Control
Low GSG Level
Hi GSG Level

Apple Juice Concentrate
6.21%
6.21%
6.21%

Blueberry Juice
0.76%
0.76%
0.76%

Concentrate

Apple and blueberry flavor
0.12%
0.12%
0.12%

Sugar
5.60%
5.60%
5.60%

GSG

0.025%
0.05%

Water
Balance
Balance
Balance

In this study, twenty consumer panel members evaluated three samples of apple blueberry flavored juice drink for overall acceptance and attribute intensities of apple and berry flavors, onset of flavor, sweetness, and aftertaste (includes tartness, bitterness and lingering sweet aftertaste intensity). The three samples included a full sugar control sample containing no Glucosyl Steviol Glycosides (GSG) and two test samples containing low (0.025%) and high (0.05%) levels of GSG. The objective of the test was to determine if the addition of Glucosyl Steviol Glycosides affects the flavor profile of a juice drink. The results indicated:

- The Test samples had significantly higher overall acceptability and more apple flavor intensity than the Control sample (at >90% confidence).
- The sweetness intensity of the Test sample with low GSG was not significantly different from the control. GSG enhances sweetness and flavor at high level of usage (p=0.047).
- There was no significant difference in aftertaste intensities between the test and control samples (at 90% confidence).

Example 4
Flavored Carbonated Soft Drink with Glucosyl Steviol Glycosides

To evaluate the flavor modification in a carbonated soft drink, a range of GSG concentrations (0 to 1000 ppm) was used with an orange-pineapple flavored carbonated soft drink formula. The objective was to assess whether the addition of GSG has an effect on key flavor attributes in various carbonated soft drink (CSD) beverage applications. Specifically, the objective was to determine if the flavor profile and overall acceptance of a Control sample of orange pineapple carbonated drink differs from Test samples of the same beverage containing GSG. After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 25 to 750 ppm, and most preferably in the range of about 50 to 500 ppm.

For detailed sensory tests, two samples were selected to test against the control sample. The methodology of the detailed sensory tests is set forth below in Table 8. The formulas of the control and test samples are set forth below in Table 9.

TABLE 8

Methodology

Number of Sessions
1

Number of Participants:
24

Test Design:
Balanced, randomized within pair. Blind.

Sensory Test Method:
Intensity and acceptance ratings

Environmental Condition
Standard booth lighting

Attributes and Scales:

Overall Acceptance on a 6-pt hedonic scale where 5 = Like and

0 = Dislike

Overall Flavor, Orange Flavor, Pineapple Flavor, Aftertaste, and

Sweetness 6-pt continuous intensity scale where 0 = Imperceptible and

5 = Extremely Pronounced

Statistical Analysis:
ANOVA (by Block) with Post Hoc

Duncan's Test

Sample Size
~1.5 oz. in a clear capped plastic cup

Serving Temperature
Refrigerated temperature (~45° F.)

Serving/Panelists Instruction:
Samples served simultaneously. Panelists

instructed to read ingredient statement,

evaluate each sample.

TABLE 9

Samples

Ingredients
Control (%)
Test 1 (%)
Test 2 (%)

Water
90.8
90.8
90.8

Sugar
8.93
8.93
8.93

Acids
0.142
0.142
0.142

Orange Flavor
0.069
0.069
0.069

Pineapple Flavor
0.050
0.050
0.050

SG95
0.006
0.006
0.006

GSG

0.025
0.05

In this study, twenty-four consumer panel members evaluated three samples of orange pineapple fruit flavored carbonated drink for overall acceptance and attribute intensities (overall flavor, orange flavor, pineapple flavor, sweetness, and aftertaste). The three samples included: a reduced sugar control sample containing SG95 (a stevia extract) and two test samples that were the same as the Control sample plus GSG added at 0.025% (low) and 0.05% (High) levels. SG95 is a high purity stevia sweetener available from PureCircle, 915 Harger Road, Suite 250, Oak Brook, Ill. 60523, USA. The objective of the test was to determine if the addition of stevia extract solids affects the flavor profile of a reduced sugar carbonated drink. The results indicated:

- The Test sample with low GSG had significantly more orange flavor intensity than the Control sample (95% confidence). The Test sample with high GSG also displayed more orange flavor intensity than the Control sample (p=0.089).
- There was no significant difference in overall acceptance, pineapple flavor intensity, or aftertaste intensity between the control and two test samples (at 90% confidence).

Example 5
Flavored Milk Drink with Glucosyl Steviol Glycosides

To evaluate the flavor modification in a flavored dairy beverage, a range of GSG concentrations (0 to 1000 ppm) was used with a banana flavored beverage formula. The objective was to determine if the addition of Glucosyl Steviol Glycosides (GSG) has an effect on key flavor attributes and/or improves flavor perception in various beverage applications, specifically in dairy beverages. Specifically, the objective was to determine if the flavor profile and overall acceptance of a Control sample of banana flavored milk drink (containing no GSG) differs from two Test samples of the same drink containing two levels of GSG. Though the test was conducted with banana flavor, the findings are also pertinent with all fruit, vegetable, chocolate, coco flavored beverages and energy drinks. After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 25 to 750 ppm, and most preferably in the range of about 50 to 500 ppm.

For detailed sensory tests, two samples were selected to test against the control sample. The methodology of the detailed sensory tests is set forth below in Table 10. The formulas of the control and test samples are set forth below in Table 11.

TABLE 10

Methodology

Number of Sessions
1

Number of Participants:
32

Test Design:
Balanced, randomized within pair. Blind.

Sensory Test Method:
Intensity and acceptance ratings

Environmental Condition
Standard booth lighting

Attributes and Scales:

Overall Acceptance on a 9-pt hedonic scale where 9 = Like Extremely,

5 = Neither Like Nor Dislike, and 1 = Dislike Extremely

Overall Flavor, Banana Flavor, Dairy Flavor, Sweetness, Tartness

and Bitterness Intensity on a 11-pt continuous intensity scale where

0 = Imperceptible and 10 = Extremely Pronounced

Statistical Analysis:
ANOVA (by Block) with Post Hoc

Duncan's Test

Sample Size
~2.0 oz. in a clear capped 3.5 oz. plastic

drinking cup

Serving Temperature
Refrigerated temperature (~45° F.)

Serving/Panelists Instruction:
Samples served simultaneously. Panelists

instructed to read ingredient statement,

evaluate each sample.

TABLE 11

Samples

Control: Sugar and Stevia Extract (Reb A)

Test: Sugar, Stevia Extract (Reb A) & GSG

Test-1
Test-2

Ingredients
Control
Low GSG Level
High GSG Level

MILK (2% fat)
95.6%
95.6%
95.6%

SUGAR
4.2
4.2
4.2

BANANA FLV
0.20
0.20
0.20

Reb A
0.006
0.006
0.006

GSG

0.0175
0.035

TOTAL
100
100
100

In this study, thirty-two consumer panel members evaluated three samples of reduced sugar banana flavored milk drink for overall acceptance and attribute intensities (overall flavor, banana flavor, dairy flavor, sweetness, and aftertaste intensity). The three samples included: a control sample sweetened with sugar and stevia extract (Reb A) and containing no Glucosyl Steviol Glycosides (GSG) and two test samples sweetened with sugar and stevia extract (Reb A) containing Glucosyl Steviol Glycosides (GSG). One of the test samples contained a low (0.0175%) amount of GSG and the other one contained a high (0.035%) amount of GSG. The objective of the test was to determine if the addition of Glucosyl Steviol Glycosides affects/improves the flavor profile of a banana flavored milk drink. The results indicated:

- The Test sample containing a high level of GSG had significantly more banana flavor intensity, sweetness intensity, and delayed onset of dairy flavor note than the Control sample (at 95% confidence).
- The test sample containing a low level of GSG also contributed to higher banana flavor intensity (at >90% confidence).
- There was no significant difference in overall acceptance, dairy flavor intensity, onset of banana flavor and aftertaste between the test samples and control.

Example 6
Baked Goods with Glucosyl Steviol Glycosides

To evaluate the flavor modification in baked goods, a range of GSG concentration (0 to 5000 ppm) was used with a lemon poppy seed muffin formula. The objective was to determine if the addition of Glucosyl Steviol Glycosides has an effect on key flavor attributes and/or improves flavor perception in various food applications, specifically in various baked goods. Specifically, the objective was to determine if the flavor profile and overall acceptance of a Control sample of lemon poppy seed muffin (containing no Glucosyl Steviol Glycosides) differs from a Test sample of the same muffin (containing Glucosyl Steviol Glycosides). Though the test was conducted with muffins, the findings are also pertinent with all baked goods, not limited to cookies, cakes, pastries, bread, etc. After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 5000 ppm, more preferably in the range of about 100 to 3000 ppm, and most preferably in the range of about 100 to 2000 ppm.

For detailed sensory tests, two samples were selected to test against the control sample. The methodology of the detailed sensory tests is set forth below in Table 12. The formulas of the control and test samples are set forth below in Table 13.

TABLE 12

Methodology

Number of Sessions
1

Number of
35

Participants:

Test Design:
Balanced, randomized within pair. Blind.

Sensory Test Method:
Intensity and acceptance ratings

Environmental
Standard booth lighting

Condition

Attributes and Scales:

Overall Acceptance on a 9-pt hedonic scale where 9 =

Like Extremely, 5 = Neither Like Nor Dislike, and

1 = Dislike Extremely

Overall Flavor, Lemon Flavor, Sweetness, Tartness and

Bitterness Intensity on a 11-pt continuous intensity scale

where 0 = Imperceptible and 10 = Extremely Pronounced

Open Ended General Comments

Gender and Age

Statistical Analysis:
ANOVA (by Block) with Post Hoc Duncan's Test

Sample Size
¼ muffin (~14 grams) in a 3.5 oz. plastic soufflé

cup

Serving Temperature
ambient temperature (~68° F.)

Serving/Panelists
Samples served simultaneously. Panelists

Instruction:
instructed to read ingredient statement, evaluate

each sample.

TABLE 13

Samples

Ingredients
Control (%)
Test (%)

AP Flour
24.37
24.35

Milk
24.01
23.99

Sugar
23.16
23.14

Vegetable Oil
14.79
14.78

Eggs
9.54
9.53

Poppy Seeds
1.00
1.00

Lemon Flavor
0.83
0.83

Salt
0.72
0.71

Lemon Juice
0.59
0.59

Vanilla Extract
0.52
0.52

Baking Powder
0.48
0.48

GSG

0.07

TOTAL
100.00
100.00

In this study, thirty-five consumer panel members evaluated two samples of lemon poppy seed muffins for overall acceptance and attribute intensities (overall flavor, lemon flavor, sweetness, tartness and bitterness intensity). The two samples included: 1) a Control sample containing no Glucosyl Steviol Glycosides (GSG) and 2) a Test sample containing GSG. The objective of the test was to determine if the addition of Glucosyl Steviol Glycosides affects/improves the flavor profile of a lemon poppy seed muffin. The results indicated:

- The Test sample (containing Glucosyl Steviol Glycosides) had significantly more overall flavor intensity and sweetness intensity than the Control (at 90% confidence).
- There was no significant difference in overall flavor acceptance, lemon flavor intensity, tartness intensity or bitterness intensity between the two samples (at 90% confidence). Directionally, the Test sample was more acceptable overall and had more lemon flavor intensity than the Control (p values=0.124 and 0.190 respectively).
- Based on panelist comments, the Control sample had less lemon flavor than the Test sample.

Example 7
Spreads & Fruit/Vegetable Preparations with Glucosyl Steviol Glycosides

To evaluate the flavor modification in fruit/vegetable spreads and fruit preparations, a range of GSG concentration (0 to 5000 ppm) was used with a strawberry topping formula for spread. The objective was to determine if the addition of Glucosyl Steviol Glycosides has an effect on key flavor attributes and/or improves flavor perception in various food applications, specifically in fruit or vegetable preparations. Specifically, the objective was to determine if the flavor profile and overall acceptance of a Control sample of strawberry topping (containing no GSG) differs from a Test sample of the same topping (containing GSG). Though the test was conducted with strawberry spread/fruit prep/topping, the findings are also pertinent with fruit and vegetable preparations, not limited to fruits (banana, all berries, mango, etc.) and vegetables (celery, artichoke, squash, avocado, etc). After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 5000 ppm, more preferably in the range of about 1000 to 4000 ppm, and most preferably in the range of about 2000 to 3000 ppm.

For detailed sensory tests, two samples were selected to test against the control sample. The methodology of the detailed sensory tests is set forth below in Table 14. The formulas of the control and test samples are set forth below in Table 15.

TABLE 14

Methodology

Number of Sessions
1

Number of Participants:
28

Test Design:
Balanced, randomized within pair. Blind.

Sensory Test Method:
Intensity and acceptance ratings

Environmental Condition
Standard booth lighting

Attributes and Scales:

Overall Acceptance on a 9-pt hedonic scale where 9 = Like Extremely,

5 = Neither Like Nor Dislike, and 1 = Dislike Extremely

Overall Flavor, Fresh Strawberry Flavor, Sweetness, Tartness and

Bitterness Intensity on a 11-pt continuous intensity scale where

0 = Imperceptible and 10 = Extremely Pronounced

Open Ended General Comments

Gender and Age

Statistical Analysis:
ANOVA (by Block) with Post Hoc

Duncan's Test

Sample Size
~0.5 oz. in a clear capped 1 oz. plastic cup

Serving Temperature
Refrigerated temperature (~45° F.)

Serving/Panelists Instruction:
Samples served simultaneously. Panelists

instructed to read ingredient statement,

evaluate each sample.

TABLE 15

Samples

Ingredients
Control (%)
Test (%)

Sugar
39.23
39.18

Water
29.43
29.39

Diced Strawberries
15.44
15.42

Strawberry Juice
15.44
15.42

Pectin
0.26
0.26

Potassium Benzoate
0.10
0.10

RebA
0.06
0.06

Citric Acid
0.05
0.05

GSG

0.022

TOTAL
100.00
100.00

In this study, twenty-eight consumer panel members evaluated two samples of strawberry flavored reduced sugar topping for overall acceptance and attribute intensities (overall flavor, fresh strawberry flavor, sweetness, tartness and bitterness intensity). The two samples included: 1) a control sample sweetened with sugar and Rebaudioside A containing no Glucosyl Steviol Glycosides (GSG) and 2) a test sample sweetened with sugar and Rebaudioside A containing GSG. The objective of the test was to determine if the addition of GSG affects/improves the flavor profile of strawberry flavored topping. The results indicated:

- There was no significant difference in overall flavor acceptance, overall flavor intensity, fresh strawberry flavor intensity, sweetness intensity or tartness intensity (at 90% confidence).
- The test sample was significantly less bitter than the control (at 90% confidence).

Example 8
Yogurt with GSG

To evaluate the flavor modification in flavored and unflavored yogurt, a range of GSG concentration (0 to 1000 ppm) was used with a vanilla flavored yogurt bought from a local store. Though the test was conducted with vanilla flavored yogurt, the findings are also pertinent with all other unflavored and flavored fermented dairy products like cheese, yogurt (with fat or no-fat), drinkable yogurts, smoothies, yogurt with fruit preparations not limited to fruit (banana, all berries, mango, etc.). After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 50 to 500 ppm, and most preferably in the range of about 100 to 400 ppm.

Three vanilla-flavored yogurt samples (generic brand) were sweetened with sugar, sugar+Reb A and Sugar+Reb A+GSG. The amount of GSG was 220 ppm. The twelve member panel found that GSG enhanced the sweetness profile and positively impacted the flavor profile. GSG helped in rounding the sweetness profile and directionally helped in reducing the aftertaste of Reb A.

Example 9
Lemon-Lime Carbonated Soft Drink with GSG

To evaluate the flavor modification in a flavored carbonated soft drink (CSD), a range of GSG concentration (0 to 1000 ppm) was used with a lemon-lime flavored CSD. Though the test was conducted with reduced sugar lemon-lime flavored CSD, the findings are also pertinent with all other flavored CSDs (cola, orange, grape fruit, passion fruit, berries fruit group, mango, etc.) with all levels of sugar and diet (no sugar) products. After preliminary sensory tests, it was apparent that GSG modifies the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 100 to 500 ppm, and most preferably in the range of about 200 to 400 ppm.

Three CSD samples were made with sugar, Sugar+Reb A, and Sugar+Reb A+GSG. The amount of GSG was 310 ppm. The sample with GSG had directionally improved sweetness profile, enhanced lemon flavor note and reduced bitterness and aftertaste compared to the sample sweetened with sugar and Reb A only.

Example 10
Chocolate Milk with GSG

To evaluate the flavor modification in flavored dairy beverages, a range of GSG concentration (0 to 1000 ppm) was used with chocolate milk formulations sweetened with sugar and/or high fructose corn syrup (HFCS) and stevia. Though the test was conducted with a reduced sugar chocolate flavored dairy beverage, the findings are also pertinent with all other flavored dairy beverages with different levels of fat or no-fat with different flavors (strawberry, blueberry, mango, etc.) with different levels of sugar including diet (no sugar) products. After preliminary sensory tests, it was apparent that GSG modifies the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 1000 ppm, more preferably in the range of about 25 to 500 ppm, and most preferably in the range of about 50 to 400 ppm.

One of the test samples was sweetened with a mixture of HFCS42, sugar and Reb A; the other test sample also included 337 ppm of GSG. The GSG enhanced the chocolate flavor, dairy note and sweetness profile.

Example 11
Baked Good Frosting with GSG

To evaluate the flavor modification in baked good frostings and spreads, a range of GSG concentration (0 to 0.5%) was used with vanilla flavored cake frosting formulations sweetened with sugar and/or high fructose corn syrup (HFCS) and stevia. Though the test was conducted with a reduced sugar frosting, the findings are also pertinent with all other flavored frostings with different levels of fat or no-fat and different flavors (chocolate, strawberry, blueberry, mango, etc.) with different levels of sugar including no-sugar-added products. After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 0.5%, more preferably in the range of about 0.1 to 0.4%, and most preferably in the range of about 0.2 to 0.3%.

As an example, a typical sensory test was conducted where GSG was added to a reduced sugar cake frosting. The amount of GSG was 0.23%, which enhanced the sweetness profile as more sugar-like as well as promoted the vanilla flavor.

Example 12
Snacks with GSG

To evaluate the flavor modification in snacks and cereal/nut products, a range of GSG concentration (0 to 0.5%) was used with cinnamon flavored coated almonds sweetened with sugar and/or high fructose corn syrup (HFCS). Though the test was conducted with full sugar coated nuts, the findings are also pertinent with all other coatings used for snacks, cereal, confectionery with different level of moisture and fat (or no-fat) and different flavors (chocolate, cinnamon, hazelnut, maple, brown-sugar, strawberry, blueberry, mango, etc.) with different levels of sugar including no-sugar-added products. After preliminary sensory tests, it was apparent that GSG modified the flavor and sweetness profile at all concentrations. The GSG concentration is preferably in the range of about 0 to 0.5%, more preferably in the range of about 0.05 to 0.4%, and most preferably in the range of about 0.1 to 0.3%.

As an example, two coated almond snacks were prepared where the test sample had reduced sugar. GSG was added in the amount of 0.19% to the test sample. The GSG provided the rounded sweetness and enhanced cinnamon flavor to the test sample.

GSG may also be added to confectionery formulations in order modify the flavor of confectionery, such as, but not limited to, hard boiled candy, soft textured confectionery, and chocolates.

Example 13
Sweetness Synergy of GSG with Sugar

To evaluate the sweetness detection of GSG, a series of samples was made with NSF-02 (Natural Sweet Flavor #2) in acidified water (pH=3.3, pH was adjusted using citric acid). NSF-02 contains GSG and about 15% to 20% dextrin, and is available from PureCircle, 915 Harger Road, Suite 250, Oak Brook, Ill. 60523, USA.

Aqueous solutions of NSF-02 at various concentrations were prepared using bottled water that was acidified to a pH of about 3.5. The pH was adjusted using 1% citric acid solution within a narrow range. The concentrations of NSF-02 ranged between 0 to 1000 ppm. Samples were evaluated by the judges at room temperature (70-72° F.).

The judges were 10 panelists that have been previously qualified for their taste acuity and trained in the use of a sweetness intensity rating scale. The evaluations were done in duplicate using the same panelists (N=20). Prior to the conduct of the study, judges were presented with sugar controls prepared with the acidified water for the intensity rating on the ballot referencing 2, 4, 6 and 8 on the evaluation scale. These solutions were provided to the judges in order to refresh the judge's memory with the intensity ratings.

Samples were given to the judges sequentially and coded with triple digit numbers. The order of sample presentation was randomized to avoid order of presentation bias. A rest period of five minutes was provided between samples. Water and unsalted crackers were provided in order to cleanse the palate. The judges could not detect any sweetness below 150 ppm of NSF-02. The sweetness equivalence value (SEV) of different solutions of NSF-02 is shown in FIG. 6.

Similarly, to quantify the synergy between NSF-02 and sugar at 8% sucrose equivalent sweetness in acidified water (about 3.5 pH) at various sugar reduction levels, a sensory test was conducted. The pH was adjusted using 1% citric acid solution within a narrow range. Sugar was reduced by adding the required level of NSF-02 to attain 8% sugar equivalent sweetness. As shown in FIG. 6, the sensory evaluation shows that the addition of NSF-02 contributes additional sweetness in the presence of sugar, even though at a lower level of NSF-02 (0 to 150 ppm of NSF-02 in acidified water, without sugar), it did not contribute any detectable level of sweetness. The synergy between sugar and NSF-02 is the difference in SEV contribution of NSF-02 with and without sugar. The NSF-02 concentration is preferably greater than about 25 ppm, more preferably greater than about 100 ppm, and most preferably in the range of about 500 ppm to 1,000 ppm.

Accordingly, the present invention shows that GSG not only modifies flavor, but it can also enhance sweetness in the presence of other sweeteners. This enhancement of sweetness is caused by the synergy between GSG and other sweeteners.

Example 14
GSG Synergy with HFCS

The objective of this experiment was to develop the same sweetness and mouthfeel of High Fructose Corn Syrup (HFCS) 55 in water solution using an equal amount of HFCS 42 plus GSG. HFCS 55 contains a total of 77% dry solid, and 55% of the dry solid is fructose. HFCS 42 contains 71% dry solid, and 42% of the dry solid is fructose. The sweetness equivalence values (SEVs) of HFCS 55 and HFCS 42 are 0.99 and 0.91, respectively.

To match a similar sweetness profile and mouthfeel of HFCS 55, samples were tested including different amounts of GSG from 0 to 500 ppm and different biogums (Xanthan, Gum Arabic, CMC, guar, Locust bean gum, pectin), or polysaccharides (maltodextrin, oligosaccharides, resistant maltodextrin), or polyols. All combinations of GSG and bulking agent (to aid mouthfeel) provided the desired sweetness and mouthfeel that matched HFCS 55. The GSG concentration is preferably in the range of about 0 to 500 ppm, more preferably in the range of about 25 to 300 ppm, and most preferably in the range of about 50 ppm to 200 ppm. However, the best solution was the combination of Xanthan gum and GSG (shown in Table 16). A similar analysis was carried out in a lemon-lime CSD application, which had a combination of GSG and Xanthan gum to provide the similar sweetness and mouthfeel with HFCS 42 as found in the formulation with HFCS 55.

TABLE 16

Ingredients
HFCS 55
HFCS 42

Water
87.02
87.00

HFCS 55
12.98
—

HFCS 42
—
12.98

Xanthan Gum
—
0.0085

GSG
—
0.0068

Brix
10
9.30

Total
100 g
100 g

- It was noticed that when Xanthan gum was added together with GSG the mouth feel perception was improved as well as the overall flavor profile.
- The addition of GSG made the sample taste more like a sugar-based product.
  
  Sensory results:

Sensory analysis (discrimination test) was conducted to determine the difference between the beverage samples with HFCS 55 and HFCS 42. A triangle test was conducted with 19 panelists. Only 4 identified the correct sample, two of whom were guessing. The two that correctly identified the odd sample indicated the differences were due to the HFCS 42 being less acidic and more flavorful; they both also mentioned that HFCS 55 was slightly less sweet than HFCS 42.

Although the tests were conducted using high fructose corn syrups, the results are not limited to high fructose syrups made from corn. The results are also applicable to high fructose syrups made from other carbohydrate sources, such as, but not limited to, wheat, barley, tapioca, rice, and potatoes.

Example 15
GSG Synergy with Other Non-Caloric Sweeteners

GSG was tested with several natural (Reb A, SG95 and PureCircle Alpha derived from stevia extract) and synthetic sweeteners (Sucralose, Acesulfame-K, cyclamate and aspartame) to investigate the synergy between GSG and high intensity sweeteners. While GSG modifies the flavor profile, it also shows a different degree of synergy with high intensity sweeteners. As an example, to estimate the synergy between GSG and stevia sweeteners, NSF-02 (GSG+dextrin) was mixed with a required quantity of PureCircle Alpha or Reb A 97 in acidic solution (pH=3.8) to attain 8% sugar equivalent sweetness as shown in Table 17. Alpha is a blend of selected steviol glycosides, as described in International Patent Application No. PCT/US2012/024722 filed Feb. 10, 2012, entitled “Stevia Composition,” and is available from PureCircle, 915 Harger Road, Suite 250, Oak Brook, Ill. 60523, USA. Reb A 97 is also available from PureCircle. Synergy was calculated as the reduction of stevia sweeteners (Alpha or Reb A) for the addition of different levels of NSF-02 as shown FIG. 7.

TABLE 17

Control
ppm
ppm
ppm
ppm
ppm
ppm

NSF-02
0
25
50
100
150
200
250

Reb A
345
340
340
338
286
272
260

Alpha
400
350
345
320
280
252
235

Synergy with
0
5
5
7
59
73
85

Reb A

Synergy with
0
50
55
80
120
148
165

Alpha

It was discovered that Alpha shows enhanced sweetness in the presence of a very small amount of NSF-02 (25 ppm or less), whereas more than 100 ppm NSF-02 had to be added to attain any synergy with Reb A as shown in FIG. 7. Note that the detection level of NSF-02 is around 150 ppm as shown in FIG. 6. In a solution with Alpha, the NSF-02 concentration is preferably greater than about 10 ppm, more preferably greater than about 25 ppm, and most preferably greater than about 100 ppm. In a solution with Reb A, the NSF-02 concentration is preferably greater than about 100 ppm, more preferably greater than about 150 ppm, and most preferably greater than about 200 ppm.

Example 16
GSG as a Mouthfeel Modifier

GSG works with sugar, HFCS and other natural sweeteners to provide a better mouthfeel and sweetness profile in beverages. Erythritol is used with stevia in beverages to provide some sweetness, but mainly to contribute mouthfeel that is lacking when a high amount of sugar is replaced with high intensity sweetener. A study was conducted to investigate the amount of erythritol that can be replaced with GSG in still beverages.

Sample preparation: A number of acidified beverage samples were targeted to 8 Brix sweetness level with 200 ppm of Reb A and the combination of erythritol and GSG as shown in Table 18.

TABLE 18

Erythritol Conc (%)

3.5
2.5
1.5
3
2.75

E
E6
E7
E8
E9

Reb A
0.1
0.1
0.1
0.1
0.1

GSG

0.044
0.0875
0.022
0.033

Erythritol
17.5
12.5
7.5
15
13.75

Citric
0.25
0.25
0.25
0.25
0.25

Water
482.2
487.1
492.1
484.6
485.9

Total
500
500
500
500
500

Sensory Evaluation:

- 1. In a comparison of E, E6 and E7 samples, E6 was found to be very close to control (E) on flavor and overall mouthfeel, and slightly less sweet than control. E7 was very watery.
- 2. A triangle test with E and E6 samples was conducted over two days with a total of 12 panel members. Five of them detected the correct sample. The conclusion was that panel members could detect the difference; thus the test failed.
- 3. A preliminary test with E, E8, E9 was then conducted, a triangle test with E and E9 was run. Three out of ten panel members could identify the correct sample.

Conclusion: GSG can reduce erythritol usage by 20-30% (from 3.5% to 2.75-3%) in beverages without any sacrifice of taste or mouthfeel. In the flavor system studied, the GSG concentration is preferably greater than about 10 ppm, more preferably greater than about 20 ppm, and most preferably in the range of about 30 ppm to about 200 ppm. In some flavor systems, GSG may replace more erythritol to provide a balanced, rounded sweetness flavor.

Example 17
Effect of Glucosylated Steviol Glycoside on Belgian White Wheat Ale

Flavor modification from glucosylated steviol glycoside (GSG) on an alcoholic beverage was tested using a commercially available Belgian white wheat ale. The two samples (Table 19) included: 1) the unaltered ale as purchased (Control), and 2) the test sample with the addition of 175 ppm glucosylated steviol glycoside. A 30 member panel evaluated the samples described for overall acceptance and attribute intensities (sweet intensity, bitterness, orange flavor, alcohol note, sweet aftertaste, bitter aftertaste). Table 20 describes the testing protocol.

Table 21 shows the sensory results for the control and test products. The test product with 175 ppm glucosylated steviol glycoside was lower in bitterness, higher in orange flavor and higher in sweet aftertaste at 95% confidence, higher in sweet intensity and higher in the alcohol note at 90% confidence, and directionally higher in overall liking (80% confidence). FIG. 8 illustrates this comparison.

TABLE 19

Commercially Produced Belgian White Wheat Ale

Market Product
Market Product with

(Control)
GSG

Belgian White Wheat Ale
100.00
99.9825

Glucosylated Steviol Glycoside
—
0.0175

Total
100
100

TABLE 20

Sensory evaluation of Belgian White Wheat Ale

Nature of Participants:
Company employees

Number of Sessions
1

Number of
15

Participants:

Test Design:
Balanced, randomized within set. Blind

Sensory Test
Intensity and acceptance ratings

Method:

Environmental
Standard booth lighting

Condition

Attributes and Scales:

Overall Acceptance on a 10-pt hedonic scale where

10 = Extremely Like and 0 = Extremely Dislike

Sweetness, bitterness, orange flavor, alcohol note, overall

liking and aftertaste. 10-pt continuous intensity scale where

0 = Imperceptible and 10 = Extremely Pronounced

Statistical Analysis:
ANOVA (by Block) with Post Hoc Duncan's Test

Sample Size
~1.5 oz. in a clear capped plastic cup

Serving Temperature
Refrigerated temperature (~45° F.)

Serving/Panelists
Samples served simultaneously. Panelists

Instruction:
instructed to evaluate each sample.

TABLE 21

Results from sensory evaluation of Belgian White Wheat Ale

Summary of the overall acceptance and mean attribute

intensity results for Belgian white wheat ale with

glucosylated steviol glycoside

Market Product
Market Product with

Attribute
(Control)
GSG
p-value
Sig

Sweet intensity
4.89
5.50
0.0972
**

Bitterness
4.45
3.13
0.0031
***

Orange flavor
2.89
4.20
0.0001
***

Alcohol note
2.15
2.80
0.0774
**

Sweet aftertaste
0.62
1.08
0.0036
***

Bitter aftertaste
1.74
0.89
0.0032
***

Overall liking
4.85
5.62
0.1032
*

* = 80% CI,

** = 90% CI,

*** = 95% CI

From this Example, it can be seen that the addition of a glucosylated steviol glycoside flavor modifier ingredient unexpectedly reduced the bitterness and bitter aftertaste in an alcoholic beverage as compared to the control, and improved the alcohol note and other taste and flavor attributes as well as the overall liking as compared to the control. The useful usage level of glucosylated steviol glycoside flavor modifier ingredients ranges from about 125 ppm to about 175 ppm in alcoholic beverages.

Example 18
Effect of Glucosylated Steviol Glycosides on BBQ Seasoned Peanuts

A BBQ seasoning for peanuts was developed to determine the impact of glucosylated steviol glycosides on salty snacks. The two samples (Table 22) included: 1) control sample of BBQ seasoned peanuts, and 2) test sample of BBQ seasoned peanuts with 175 ppm glucosylated steviol glycoside. A 30 member panel evaluated both samples for overall acceptance and attribute intensities (sweet intensity, saltiness, smoked flavor, heat/spice, peanut flavor, chili powder, bitterness, bitter aftertaste and sweet aftertaste). Table 23 describes the sensory protocol.

Table 24 shows that the seasoned peanut sample containing GSG had significantly higher smoked flavor, heat/spice, and chili powder intensity at 95% confidence and significantly higher saltiness at 90% confidence. In addition, the test sample GSG had directionally higher peanut flavor, and sweet aftertaste compared to the control at 80% confidence. FIG. 9 illustrates this comparison.

TABLE 22

BBQ Seasoned Peanuts

Seasoned Peanuts
Seasoned Peanuts with

(Control)
175 ppm GSG

Dry Roasted Unsalted
93.649
93.6315

Peanuts

Vegetable Oil
2
2

Sodium Chloride
2
2

Cumin Powder
0.286
0.286

Chili Powder
0.173
0.173

Garlic Powder
0.156
0.156

Ground Cayenne Pepper
0.156
0.156

Smoke Flavor
0.08
0.08

Sucrose
1.5
1.5

Glucosylated Steviol
—
0.0175

Glycoside

Total
100
100

TABLE 23

Sensory evaluation of BBQ seasoned peanuts

Nature of Participants:
Company employees

Number of Sessions
1

Number of
30

Participants:

Test Design:
Balanced, randomized within set. Blind

Sensory Test
Intensity and acceptance ratings

Method:

Environmental
Standard booth lighting

Condition

Attributes and Scales:

Overall Acceptance on a 10-pt hedonic scale where

10 = Extremely Like and 0 = Extremely Dislike

Sweetness, saltiness, smoked flavor, heat/spice, peanut flavor,

chili powder, bitterness, and aftertaste. 10-pt continuous intensity

scale where 0 = Imperceptible and 10 = Extremely Pronounced

Statistical Analysis:
ANOVA (by Block) with Post Hoc Duncan's Test

Sample Size
~1.5 oz. in a clear capped plastic cup

Serving Temperature
Room temperature

Serving/Panelists
Samples served simultaneously. Panelists

Instruction:
instructed to evaluate each sample.

TABLE 24

Summary of the overall acceptance and mean attribute intensity

results for BBQ seasoned peanuts with glucosylated steviol

glycoside

Seasoned Peanuts

Seasoned Peanuts
with 175 ppm

Attribute
(Control)
GSG
p-value
Sig

Sweet Intensity
1.98
1.94
0.8548
NS

Saltiness
5.05
5.92
0.0688
**

Smoked Flavor
1.28
2.23
0.0373
***

Heat/Spice
1.28
2.42
0.0207
***

Peanut Flavor
8.57
8.97
0.16
*

Chili Powder
3.04
4.01
0.0226
***

Bitterness
0.27
0.31
0.5419
NS

Bitter Aftertaste
0.28
0.26
0.6509
NS

Sweet Aftertaste
0.35
0.56
0.1673
*

Overall Liking
6.40
7.61
0.0058
***

* = 80% CI,

** = 90% CI,

*** = 95% CI

From this Example, it can be seen that the addition of a glucosylated steviol glycoside flavor modifier ingredient unexpectedly enhanced savory attributes such as saltiness, smoked flavor, and heat/spiciness, while having an insignificant impact on bitterness or bitter aftertaste, as compared to the control, and improved the overall liking as compared to the control. The useful usage level of glucosylated steviol glycoside flavor modifier ingredients ranges from about 133 ppm to about 175 ppm in snack foods and nut products.

Example 19
Effect of Glucosylated Steviol Glycosides on Ranch Dressing

A ranch dressing was formulated to determine the impact of glucosylated steviol glycosides in a fats and oils application. The two samples (Table 25) included: 1) control sample of ranch dressing, and 2) test sample of ranch dressing with 190 ppm glucosylated steviol glycoside. A 30 member panel evaluated both samples for overall acceptance and attribute intensities (sweetness, bitterness, sourness, dill flavor, garlic, vinegar, creaminess, balanced, sweet aftertaste and bitter aftertaste). Table 26 describes the sensory protocol.

Table 27 shows that the ranch dressing sample containing GSG had significantly lower bitter intensity and significantly higher dill flavor, creaminess, and was more balanced at 95% confidence. In addition, the test sample GSG had directionally higher garlic, and overall liking compared to the control at 80% confidence. FIG. 10 illustrates this comparison.

TABLE 25

Ranch Dressing

Ranch Dressing
Ranch Dressing with

(Control)
190 ppm GSG

Sour Cream
38.200
38.200

Low Fat Cultured Buttermilk
38.200
38.181

Mayonnaise
19.100
19.100

Minced Dried Garlic
0.309
0.309

Salt
0.955
0.955

Black Pepper
0.191
0.191

Dried Dill Weed
0.525
0.525

Freeze Dried Chives
0.102
0.102

100% Organic Lemon Juice
2.418
2.418

GSG
—
0.019

Total
100
100

TABLE 26

Sensory evaluation of Ranch Dressing

Nature of Participants:
Company employees

Number of Sessions
1

Number of
30

Participants:

Test Design:
Balanced, randomized within set. Blind

Sensory Test
Intensity and acceptance ratings

Method:

Environmental
Standard booth lighting

Condition

Attributes and Scales:

Overall Acceptance on a 10-pt hedonic scale where

10 = Extremely Like and 0 = Extremely Dislike

Sweetness, bitterness, sourness, dill flavor, garlic, vinegar,

creaminess, balanced, and aftertaste 10-pt continuous intensity

scale where 0 = Imperceptible and 10 = Extremely Pronounced

Statistical Analysis:
ANOVA (by Block) with Post Hoc Duncan's Test

Sample Size
~1.5 oz. in a clear capped plastic cup

Serving Temperature
Refrigerated temperature

Serving/Panelists
Samples served simultaneously. Panelists

Instruction:
instructed to evaluate each sample.

TABLE 27

Summary of the overall acceptance and mean attribute intensity

results for ranch dressing with glucosylated steviol glycoside

Ranch
Ranch Dressing with

Attribute
Dressing
190 ppm GSG
p-value
Sig

Sweetness
2.01
1.98
0.906
NS

Bitterness
1.05
0.63
0.0001
***

Sourness
2.13
2.01
0.5471
NS

Dill Flavor
4.28
5.09
0.0163
***

Garlic
2.01
2.34
0.1359
*

Vinegar
2.96
3.06
0.6633
NS

Creaminess
4.94
5.75
0.0015
***

Balanced
2.40
3.38
0.0066
***

Sweet Aftertaste
0.38
0.47
0.3144
NS

Bitter Aftertaste
0.35
0.32
0.6531
NS

Overall Liking
5.64
6.11
0.1922
*

* = 80% CI,

** = 90% CI,

*** = 95% CI

From this Example, it can be seen that the addition of a glucosylated steviol glycoside flavor modifier ingredient unexpectedly reduced the bitterness and bitter aftertaste in a fat or oil-based consumable product, as compared to the control, and improved the creamy mouthfeel and other taste and flavor attributes as well as the overall liking as compared to the control. The useful usage level of glucosylated steviol glycoside flavor modifier ingredients ranges from about 125 ppm to about 190 ppm in fat- or oil-based food products.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the invention described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, the compositions, devices, processes, methods, and steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention.

	Number	Date	Country
Parent	14855767	Sep 2015	US
Child	15478732		US
Parent	14005852	Sep 2013	US
Child	14855767		US

	Number	Date	Country
Parent	15478732	Apr 2017	US
Child	15625365		US
Parent	PCT/US2011/033912	Apr 2011	US
Child	14005852		US

GLUCOSYLATED STEVIOL GLYCOSIDE COMPOSITION AS A FLAVOR MODIFIER

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