Stevia extract containing selected Steviol Glycosides

as flavor, salty and sweetness profile modifier

This application is a continuation-in-part application of and claims the benefit of priority to U.S. patent applications Ser. No. 61/832,451, filed June 7, 2013 and Ser. No 61/942,331 , filed on February 20, 2014.

Field of the Invention

[0001] The invention relates to the use of stevia extracts as flavor modifiers that contain mixtures of steviol glycosides extracted from Stevia rebaudiana plant. This invention also relates to the application of the above-said stevia extracts as sweetness profile modifier, not a sweetener, with other natural and artificial sweeteners. This invention also relates to the production and use of the above-mentioned stevia extracts that can be used as flavor and sweetness profile modifier when used in food, beverage, and pharmaceutical products.

Description of the Related Art

[0002] High intensity sweeteners possess sweetness level many times exceeding that of sucrose. They are essentially non-caloric and used widely in manufacturing of diet and reduced calorie food. Although natural caloric sweetener such as sucrose, fructose, and glucose provide the most desirable taste to consumers, they are caloric. High intensity sweeteners do not affect the blood glucose level and provide little or no nutritive value.

[0003] However, high intensity sweeteners that generally are used as substitutes for sucrose possess taste characteristics different than that of sugar, such as sweet taste with different temporal profile, maximal response, flavor profile, mouthfeel, and/or adaptation behavior than that of sugar. For example, the sweet taste of some high-potency sweeteners is slower in onset and longer in duration than that of sugar and thus changes the taste balance of a food composition. Because of these differences, usage of high-potency sweetener in replacing such a bulk sweetener as sugar in a food or beverage causes imbalance in temporal and/or flavor profile. If the taste profile of high-potency sweeteners could be modified to impart desired taste characteristics, it can provide low calorie beverages and food products with taste characteristics more desirable for consumers. To attain the sugar-like temporal and/or flavor profile, several ingredients have been suggested in different publications.

[0004] Non-limiting examples of synthetic sweeteners include sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone synthetic derivatives, cyclamate, neotame, dulcin, suosan, N-[N-[3-(3-hydroxy-4- methoxyphenyl)propyl]-L-a-aspartyl]-L-phenylalanine 1 -methyl ester, N-[N-[3-(3-hydroxy-4- methoxyphenyl)-3-methylbutyl]-L-«-aspartyl]-L- phenylalanine 1 -methyl ester, N-[N-[3-(3- methoxy-4-hydroxyphenyl)propyl]-L-a-aspartyl]-L-phenylalanin e 1 -methyl ester, salts thereof, and the like.

[0005] Non-limiting examples of natural high intensity sweeteners include Stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside, mogrosides, brazzein, neohesperidin dihydrochalcone (NHDC), glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3 -acetate, neoastilibin, tr ra-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside and others.

[0006] High intensity sweeteners can be derived from the modification of natural high intensity sweeteners, for example, by fermentation, enzymatic treatment, or derivatization.

[0007] A growing number of consumers perceive the ability to control their health by enhancing their current health and/or hedging against future diseases. This creates a demand for food products with enhanced characteristics and associated health benefits, specifically a food and consumer market trend towards "whole health solutions" lifestyle. The term "natural " is highly emotive in the world of sweeteners and has been identified as one of key trust, along with "whole grains", "heart-healthy" and "low-sodium". 'Natural ' term is closely related to 'healthier'.

[0008] Stevia rebaudiana 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 beverages, foods and medicines.

[0009] 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.

[0010] Steviol glycosides have zero calories and can be used wherever sugar is used. They are ideal for diabetic and low calorie diets. In addition, the sweet steviol glycosides possess functional and sensory properties superior to those of many high potency sweeteners.

[0011] The extract of 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 CI 9. 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 (0.3%), Rebaudioside C (0.6%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other glycosides identified in Stevia extract include Rebaudioside B, C, D, E, and F, Steviolbioside and Rubusoside (FIG. 1).

[0012] The chemical structures of the diterpene glycosides of Stevia rebaudiana are presented in FIG. l . The physical and sensory properties are well studied only for Stevioside and Rebaudioside A. The sweetness potency of Stevioside is around 210 times higher than sucrose, Rebaudioside A around 300 times, and Rebaudioside C and Dulcoside A around 30 times. The Stevia extract containing Rebaudioside A and Stevioside as major components showed sweetness potency around 250 times. Rebaudioside A and Rebaudioside D are considered to have most favorable sensory attributes of all major Steviol Glycosides (TABLE

1).

TABLE 1

[0013] In addition to the commercially known steviol glycosides (Table 1), several new steviol glycosides (Glycosylated diterpene) have been found in stevia leaf extracts (5,6,7) as shown in Table 2. Besides diterpene glycosides, a number of flavonoids, labdane diterpene, triterpenes, sterols, and volatile oils have also been reported in the extracts of Stevia rebaudiana [1, 2, 3, 4]

TABLE 2

Chemical Classes Chemical Components

Rebaudioside G, Rebaudioside H, Rebaudioside I, Rebaudioside J, Rebaudioside K, Rebaudioside L

Glycosylated Rebaudioside M, Rebaudioside N, Rebaudioside O, 13-[(2-0-(6-0-B- diterpene D-glucopyranosyl)-B-Dglucopyranosyl-B-D-glucopyranosyl)oxy] kaur- 16- derivatives en-18-oic acid Π-D-glucopyranosyl ester, 13-[(2-(9-B-D-glucopyranosyl-3-

O-B-D-fructofuranosyl-B-D-glucopyranosyl)

oxy] kaur-16-en-18-oic acid B-D-glucopyranosyl ester

Monoterpenoids Borneol

Diterpenoids Austroinulin, 6-0-acetyl austroinulin, 6-acetyl austroinulin

7-0-acetyl austroinulin, Sterebin A, B, C, D, E, F, G, H, Jhanol

Triterpenoids Amyrin beta acetate Sesquiterpenes a-bergamotene, Bisabolene, β-bourbonene, δ -cadinene, γ -cadinene

Essential oils β-caryophyllene, Trans β-tarnesene, a-humulene, δ-cadiene

caryophyllene oxide, Nerolidol, Linalol, a-terpineol, Terpinen-4-ol

Sterol derivatives Stigmasterol, β-sitosterol, Campesterol

Flavonoids Glucosyl-4'-0-apigenin, Glucosyl-7-O-luteolin, Rhamnosyl-3-O- kaempferol, Quercetin, Glucosyl-3-O-quercetin, Arabinosyl-3-O- quercetin, 5,7,3 '-methoxyflavone, 3,6,4'-methoxyflavone,

Centaureidin, avicularin

[0014] All steviol glycosides provide sweetness and other taste attributes at a higher than certain threshold level of concentrations in water. Below the threshold level of concentration, the steviol glycoside components and their mixtures as found in a typical non-limiting stevia extract as shown below has no recognizable sweetness taste. But such stevia extract below the threshold level of significant sweetness recognition show remarkable characteristics of sweet and flavor profile modification in food and beverage applications.

[0015] This invention relates to use of the following stevia extracts (Table 3) with the varying level of different steviol glycosides and other stevia plant-derived glycosides, the combination of which contributes no significant sweetness but modifies flavor and sweetness profile at certain concentration in typical food and beverage applications.

Table 3

*TSG or Total Steviol Glycosides contain nine Steviol Glycosides that are recognized by Codex Alimentarius (a commission of FAO and WHO) and major regulatory authorities

[0016] The present invention also relates to the stevia extracts that contain major steviol glycosides (Table 3) and other minor steviol glycosides and glycosylated diterpene derivatives (water soluble molecules). The non-limiting examples of such minor molecules are Reb E, Reb G, Reb H, Reb I, Reb K, Reb L, Reb M, Reb N, Reb O (Ohta et al, 2010).

[0017] The present invention is also directed to a method of making a specific stevia extract composition, including: extracting steviol glycosides and other water soluble molecules from leaves of a Stevia rebaiidiana plant, and separating the excess steviol glycosides than the amount and type of steviol glycosides required to contribute the taste and flavor modifying characteristics of the stevia extract.

[0018] This invention combine the different natural sweeteners, especially steviol glycosides in certain proportion along with other water soluble molecules to provide enhanced sweetness and flavor profile in food and beverage application, which can be blended with other natural caloric sweeteners to impart more desirable sweetness profile. Non-limiting examples of caloric sweeteners include dextrose, fructose, sucrose, maltose, lactose, corn syrup, gluco- syrup derived from different carbohydrates, cane syrup, flavored sugar, honey, molasses,

[0019] This invention combine the different natural sweeteners, especially steviol glycosides in certain proportion along with other water soluble molecules to provide enhanced sweetness and flavor profile in food and beverage application, which can be blended with other natural non-caloric sweeteners to impart more desirable sweetness profile. Non-limiting examples of natural high intensity sweeteners include steviol glycosides, brazzein, monatin and its salt, neohesperidin dihydrochalcone (NHDC), glycyrrhizic acid and its salts, thaumatin, mogrosides and lu han guo extracts, perillartine, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside and others.

[0020] This invention combine the different natural sweeteners, especially steviol glycosides in certain proportion along with other water soluble molecules to provide enhanced sweetness and flavor profile in food and beverage application, which can be blended with other synthetic non-caloric sweeteners to impart more desirable sweetness profile. Non-limiting examples of synthetic sweeteners include sucralose, potassium acesulfame, aspartame, alitame, advantame, saccharin, neohesperidin dihydrochalcone synthetic derivatives, cyclamate, neotame, dulcin, suosan, N-[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-D-aspartyl]-L- phenylalanine 1 -methyl ester, N-[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-D- aspartyl]-L- phenylalanine 1 -methyl ester, N-[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-D- aspartyl]-L-phenylalanine 1 -methyl ester, salts thereof, and the like.

Brief Summary of the Invention

[0001] The present invention is directed to a taste and flavor modifying composition. The composition includes different steviol glycosides with other water soluble molecules derived from Stevia leaf, such as non-limiting examples of plant glycosides, flavonoids, labdane diterpene, triterpenes, which can modify the intensity of a taste and/or a flavor in a food or beverage product.

[0002] 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 modifying composition comprising the stevia extract of steviol glycosides and water soluble molecules derived from stevia plant. 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, snack foods, 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 modifying composition, wherein the taste and flavor modifying composition comprising the stevia extract of steviol glycosides and water soluble molecules derived from stevia plant, 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 modifying composition. Moreover, the mouthfeel and overall taste perception of a food or beverage product including the taste and flavor modifying composition, wherein the taste and flavor enhancing composition includes the complex mixture of steviol glycosides and water soluble molecules, may be improved in relation to a mouthfeel and overall taste perception of a comparative food or beverage product which does not include the taste and flavor enhancing composition. [0003] 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 modifying composition comprising the stevia extract of steviol glycosides and water soluble molecules derived from stevia plant. 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 modifying composition to the food or beverage product. For example, adding the taste and flavor modifying 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).

[0004] The present invention is further directed to a method of increasing the taste and flavor intensity of a medical food and pharma product, including the step of adding a taste and flavor modifying composition to the food or beverage product, wherein the taste and flavor modifying composition comprising the stevia extract of selected steviol glycosides and water soluble molecules derived from stevia plant. The present invention is also directed to a method of improving the organoleptic properties of a medical food or pharma product containing functional food ingredients like vitamins, minerals and amino acids, including the step of adding the taste and flavor modifying composition to the food or beverage product. For example, adding the taste and flavor modifying composition may cause the off-taste due to vitamins, mineral, amino acids and other non-limiting functional ingredients, to improve taste and palatability.

[0005] The present invention is also directed to a method of making a taste and flavor enhancing composition, including: extracting steviol glycosides and other water soluble molecules from leaves of a Stevia rebaudiana plant, and separating the excess steviol glycosides than the amount and type of steviol glycosides required to contribute the taste and flavor modifying characteristics of the stevia extract. [0006] 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.

EXAMPLES

Example 1 A: Detection of concentration threshold for sweetness recognition

[0007] To detect the sweetness recognition level of PCS-5001 (stevia extract), the test method outlined by Harman, et al (Food Technology, 1 1/2013) was used with ten trained panelists that have been previously qualified for their taste acuity and trained in the use of a sweetness intensity rating scale, evaluated a series of aqueous solutions of sucrose and the stevia extract (PCS-5001 or PCS-1015) at room temperature; the sucrose solutions of 1.5% concentration and the stevia extract solutions with concentrations ranging between 100 and 120 ppm for PCS-5001 and 70-80 ppm for PCS-1015 were prepared with filter water. The objective of the test was to determine the sweetness recognition level of the stevia extract. The evaluations were done in triplicate using the same panelists so that a total of 30 values were generated for each average data point.

[0008] The samples were coded and presented in random order to panel members to taste and determine which sample was sweeter (ASTM E2164-08: Standard Method for Directional Difference Test). Panelists were asked to focus only on sweet attribute of those samples and to use warm water and salt solution in order to cleanse the palate between samples. [0009] The results were tallied and significance was calculated by SIM 2000 (Sensory Computer System, NJ). Results are presented in Table 4. The overall sweetness of those samples was barely detectable. The 2-AFC shows that 100 ppm PCS-5001 and 70 ppm of PCS-1015 solution were the least sweet sample and were significantly less sweet then the 1.5% sugar control. The sample with 120 ppm PCS-5001 and 80 ppm PCS-1015 were the sweetest samples showing significantly higher sweetness than the 1.5% sugar control (Table 4). The recognition threshold concentration of STEVIA EXTRACT (PCS-5001) in water was determined to be 100 ppm. The recognition threshold concentration of STEVIA EXTRACT (PCS-1015) in water was determined to be 70 ppm.

Table 4: Sweetness perception of Stevia Extract in different concentration against 1.5% sugar solution.

Example IB: Sweetness detection of concentration threshold for sweetness detection

[0010] The ten panel members evaluated a series of lemon-lime flavored carbonated soft drink (CSD) sweetened with sucrose and STEVIA EXTRACT at room temperature; the evaluations were done in triplicate using the same panelists so that at least 30 values were generated for each average data point. The lemon lime flavored carbonated soft drink control sample had 1.5% sucrose concentration and the test sample contained STEVIA EXTRACT (PCS-5001) with concentrations at 1 10 and 120 ppm or STEVIA EXTRACT (PCS-1015) with concentrations of 70 and 90 ppm. Other ingredients in the CSD samples were citric acid, lemon-lime flavor, sodium benzoate, potassium citrate and xanthan gum. The objective of the test was to determine the sweetness detection limit of STEVIA EXTRACT. Tests were conducted as outlined in Example 1 A.

[0011] The samples with 120 ppm PCS-5001 (STEVIA EXTRACT) and 90 ppm PCS- 1015(STEVIA EXTRACT) showed no significant difference in sweetness than the 1.5% sugar control. The recognition threshold concentration of PCS-5001 (STEVIA EXTRACT) in a lemon-lime flavored carbonated soft drink water was determined to be 110 ppm. The recognition threshold concentration of PCS-1015 (STEVIA EXTRACT) in a lemon-lime flavored carbonated soft drink water was determined to be 70 ppm. Results are shown in table 5.

Table 5: Sweetness perception of STEVIA EXTRACT in different concentrations against 1.5% sugar solution in a typical carbonated soft drink (CSD)

EXAMPLE 2: Effect of Stevia Extract on Flavor Modification in a typical carbonated soft drink application

[0012] A cola flavored carbonated soft drink was developed to evaluate the effect of PCS-5001 and PCS-1015 (stevia extract) on the sweetness and flavor profile of the beverage that was sweetened with sugar and stevia sweetener to achieve 30% sugar reduction (Table 6). The samples with and without PCS-5001 or PCS-1015 were evaluated by thirty consumer panel members, who assigned relative values to each sample for overall Liking, sweetness, vanilla flavor, brown note, and aftertaste on a 10-pt continuous intensity scale as outlined in Table 7. Table 6: Cola flavored Soft drink for sensory evaluation

Table 7: Sensory evaluation of Cola flavored carbonated soft drink

FIGURE 2 shows the modification of flavor and sweetness profiles caused by the addition of stevia extract (PCS-5001). The results indicated the sample containing stevia extract PCS- 5001 and the sample containing PCS-1015 had significantly higher cola flavor, vanilla flavor, brown spice notes and overall liking compared to the control samples (at 95% confidence). The sample containing PCS-5001 had directionally lower bitterness, and bitter aftertaste intensity compared to the control samples (at 90% and 95% confidence respectively). The sample containing PCS-1015 had directionally lower bitterness, and sweet aftertaste intensity compared to the control samples (at 80% confidence). In addition, the sample with stevia extract (PCS-1015) had significantly lower bitter aftertaste compared to the control sample (at 95% confidence).

EXAMPLE 3: Peach Flavored Tea Beverage for sensory evaluation

[0013] A peach flavored black tea drink was developed to evaluate the effect of STEVIA EXTRACT on the sweetness and flavor profile of the beverage that was sweetened with sugar and stevia sweetener to achieve 30% sugar reduction (Table 8). The samples with and without STEVIA EXTRACT were evaluated as outlined in EXAMPLE 2 by thirty consumer panel members, who assigned relative values to sweetness, bitterness, peach flavor, tea flavor, acid intensity, astringency, and aftertaste on 10-pt continuous intensity scale where 0 = Imperceptible and 10 = extremely pronounced.

Table 8: Peach Flavored Tea Beverage samples for sensory evaluation

Reduced Sugar Reduce Sugar 1 ea Reduce Sugar Tea

>vith PCS-5001 w ith PCS-1015

Water 95.71 95.70 95.71

Sucrose 3.850 3.850 3.850

Black Tea Powder 0.275 0.275 0.275

Citric Acid 0.0880 0.0880 0.0880

Peach Flavor 0.0330 0.0330 0.0330

Sodium Citrate 0.0150 0.0150 0.0150

Potassium Sorbate 0.0150 0.0150 0.0150

Stevia Glycoside 0.0140 0.0140 0.0140

Stevia Extract PCS-5001 0.0120

Stevia Extract PCS-1015 0.0080

Xanthan Gum - TIC 0.0013 0.0013 0.0013 FIGURE 3 shows the modification of flavor and sweetness profiles contributed by the addition of STEVIA EXTRACT (PCS-5001) in peach flavored ice tea beverage. The results indicated that the test sample containing PCS-5001 had significantly higher peach flavor, and overall liking (at 95%, confidence). The sample containing PCS-5001 had significantly lower astringency than the control sample (at 95% confidence). The results shown in Figure 4 indicated that the test sample containing PCS-1015 had significantly higher peach flavor, black tea flavor, and overall liking (at 95%, confidence). The sample PCS-1015 also had significantly lower astringency, sweet intensity, bitter intensity, and bitter aftertaste than the Control sample (at 95% confidence). In addition, the PCS- 1015 sample had lower sweet aftertaste intensity than the Control sample at 90% confidence).

EXAMPLE 4: Effect of Stevia Extract on Flavor modification of Savory applications

[0014] A seasoning blend was developed to determine the flavor modification effect of stevia extract in a seasoning blend on reduced sugar roasted peanut samples. Thirty consumer panel members evaluated two samples of the peanuts for overall acceptance and attribute intensities (overall flavor, saltiness, sweetness, smoke flavor, spice/heat intensity, peanut flavor, chili powder flavor, bitterness and lingering sweet aftertaste intensity). The two samples (Table 9) included: 1) 50%sugar reduced control sample containing stevia glycosides, and 2) 50% reduced sugar test sample containing steviol glycoside and stevia extract, PCS-5001 or PCS-1015.

[0015] The objective of the test was to determine if the addition of stevia extract affects the flavor profile of a savory snack food. The results indicated that the addition of PCS-5001 at 110 ppm and PCS-1015 at 70 ppm provided flavor modification (FIGURE 5). The test samples containing 1 10 ppm PCS-5001 had significantly higher salt intensity, smoke flavor, and bitter intensity compared to the control (95% confidence). The test sample also had lower sweet intensity than the control (95%> confidence). In addition, the test sample containing stevia extract had directionally higher spice and chili notes (90% confidence). The test sample containing PCS-1015 had significantly higher salt intensity than the control sample (at 95% confidence). The test sample showed an increase in heat/spice intensity, and chili flavor compared to the control.

Table 9: Effect of STEVIA EXTRACT on snack and seasoning applications

Table 10: Sensory evaluation of snack and seasoning applications

Nature of Participants : Company employees

Number of Sessions 1

Number of Participants: 30

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 ledonic scale where 9 = Like Extremely, 5 = Neither Like Nor Dislike, and 1 = Dislike E xtremely

Overall Flavor, Saltiness, Sweetness, Smoke Intensity, Heat/spice intensity, peanut flavor, chili powder and Aftertaste Intensity (sweet and bitter) on a 10-pt continuous intensity scale where 0= Imperceptible and 10= Extremely Pronounced

Open Ended General Comments

• 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 (~70°F)

• Serving/Panelists Samples served simultaneously. Panelists evaluate each Instruction: sample once. EXAMPLE 5: Flavor modification of sauce and vegetable preparation

[0016] A tomato ketchup preparation was developed to determine the flavor modification effect of stevia extract (PCS-1015). A panel of thirty company employees evaluated the overall acceptance and attribute intensities (tomato, onion, vinegar, sweet, saltiness, bitterness and aftertaste) of each sample. The sensory evaluation methodology outlined in Example 4 was adopted for the sauce samples as presented in Table 1 1.

Table 11 : Effect of PCS-1015 (stevia extract) on tomato ketchup

FIGURE 6 shows the modification of flavor and sweetness profiles caused by the addition of stevia extract (PCS- 1015). The results indicate the test samples containing stevia extract, PCS- 1015, had a significant increase in herbal notes, and savory (onion/garlic) notes at a 95% confidence interval. The test sample containing PCS-1015 had directional]}' lower bitterness, bitter aftertaste and overall liking at a 90% confidence interval compared to the control sample.

Example 6: Effect of PCS-1015 (Stevia Extract) on Flavor modification of Dairy applications

[0017] A chocolate flavored dairy beverage was developed to determine the flavor modification effect of stevia extract (PCS-1015) in dairy beverage. The panel evaluated samples of chocolate milk for overall acceptance and attribute intensities (chocolate flavor, dairy notes, sweetness, bitterness and aftertaste). The two samples (Table 12) included: 1) 50% sugar reduced control sample containing stevia glycosides, and 2) 50% reduced sugar test sample containing stevia glycoside and 80 ppm of stevia extract, PCS-1015.

Table 12: Effect of PCS-1015 (stevia extract) on flavored dairy beverage

Table 13: Sensory evaluation of Dairy beverage

• Nature of Participants: Company employees

• Number of Sessions 1

• N umber of Participants: : 30

• Test Design: Balanced, randomized within pair. Blind

• Sensorv Test Method:; Intensity and acceptance ratings

• Environmental

Standard booth lighting

Condition

• Attributes and Scales:

• Overall Acceptance on a 10-pt hedonic scale where 10 = Extremely Like and 0 =

Extremely Dislike

• Overall Liking, sweetness, bitterness, dairy notes, chocolate, 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 blear capped plastic cup

• Serving Temperature Refrigerated temperature (~45°F)

• Serving/Panelists Samples served simultaneously. Panelists ^" instructed to read

Instruction: ingredient statement, evaluate each sample. [0018] FIGURE 7 shows the modification of flavor and sweetness profiles caused by the addition of stevia extract (PCS-1015). The results indicate the 50% sugar reduced sample containing steviol glycoside sweetener and stevia extract, PCS-1015, had significantly higher chocolate flavor.

EXAMPLE 7: Effect of Stevia Extract (PCS-5001) on Flavor modification of Baked Goods applications

[0019] A lemon poppy seed flavored muffin formulation was developed to determine the flavor modification effect of stevia extract (PCS-5001) in baked good applications. To test the contribution of PCS-5001 in baked goods, lemon flavored poppy seed muffins were baked with a 45% sugar reduced formulation with steviol glycoside as control, and sugar reduced formulation with steviol glycoside and stevia extract (PCS-5001) as a test sample as shown in Table 14. A thirty member consumer panel evaluated two samples of lemon poppy seed muffins for several attributes (lemon, vanilla flavors, brown notes, sweet & bitter aftertaste).

Table 14: Effect of PCS-5001 (stevia extract) on baked goods

Steviol Glycoside Steviol glycoside w/

Ingredients (400 ppm) Control 120 ppm stevia extract

DRY Ingredients

Sucrose 12.3722 12.3682

All Purpose Flour 17.6434 17.6434

Whole Wheat Flour 5.8763 5.8763

Poppy Seeds 1.0648 1 .0648

Maltodextrin - 10DE 2.1368 2.1368

Fibersol2 (ADM/Matsutani) 1.0648 1.0648

Modified Starch - Inscosity 656 1.0648 1.0648

Lemon Flavor - Firmenich 0.8860 0.8860

Salt (Sodium Chloride) 0.7479 0.7479

Baking Powder 1.0648 1.0648

Baking Soda 0.3205 0.3205

Steviol Glycoside 0.0400 0.0400

Stevia extract (PCS-5001) 0.0120

Wet Ingredients Steviol Glycoside Steviol glycoside >v7

Ingredients (400 ppm) Control 120 ppm stevia extract

Milk, 2% 27.2444 27.2444

Soybean Oil 1 1.7525 1 1.7525

Whole Eggs 8.5473 8.5473

Water 5.3420 5.3420

Yogurt, Plain Nonfat 1.6026 1.6026

Lemon Juice, 100% 0.6410 0.6410

Vanilla Extract 0.5342 0.5342

100 100

[0020] FIGURE 8 shows the modification of flavor and sweetness profiles caused by the addition of stevia extract (PCS-5001). The panel found that the addition of stevia extract provided an increase in brown note than control sample without stevia extract (at 90% confidence).

EXAMPLE 8: Effect of Stevia Extract (PCS-5001) on Flavor modification of reduced sodium applications

[0021] A 30% salt reduced tortilla chip formulation was developed to determine the flavor modification effect of stevia extract (PCS-5001) in a salt reduced applications. To test the contribution of PCS-5001 in a salt reduced application, cheddar cheese flavor tortilla chips were coated with a control salt formulation, and a 30% salt reduced formulation with stevia extract (PCS-5001) as a test sample as shown in Table 15. A sixteen member consumer panel evaluated two samples of cheddar cheese flavored tortilla chips for different attributes (sweet intensity, saltiness, cheese flavor, dairy notes, corn flavor, bitterness, and sweet & bitter aftertaste).

[0022] FIGURE 9 shows the modification of flavor and salt perception caused by the addition of stevia extract (PCS-5001). The panel found the addition of stevia extract in a 30% salt reduced formulation provided an increase in salt perception, parity to the full sodium control. In addition, stevia extract provided an increase in sweet intensity and dairy note higher than control sample without stevia extract (at 95% confidence). Table 15: Tortilla Chi s with Cheddar Cheese 30% less sodium

EXAMPLE 9: Effect of Stevia Extract (PCS-5001) on Flavor modification of dried meat applications

[0023] A beef jerky formulation was developed to determine the flavor modification effect of stevia extract (PCS-5001) in a dried meat applications. To test the contribution of PCS-5001 in a dried meat application, flank steak was marinated with a reduced sugar control formulation, and a 30% sugar reduced formulation with steviol glycosides and stevia extract (PCS-5001) as a test sample as shown in Table 16. A twenty member consumer panel evaluated two samples of beef jerky for different attributes (sweet intensity, saltiness, black pepper, teriyaki flavor, fat-like intensity, beef flavor and sweet aftertaste).

[0024] FIGURE 10 shows the modification of flavor and salt perception caused by the addition of stevia extract (PCS-5001). The panel found the addition of stevia extract in a 30% sugar reduced formulation provided an increase in salt perception. Table 16; 30% su ar reduced Beef Jerk

EXAMPLE 10: Effect of Stevia Extract (PCS-5001) on Flavor modification of reduced sodium applications in brown gravy

[0025] A 30% sodium reduced brown gravy formulation was developed to determine the flavor modification effect of stevia extract (PCS-5001) in a salt reduced applications. To test the contribution of PCS-5001 in a salt reduced application, a 30% sodium reduced brown gravy formulation, and a 30% salt reduced formulation with stevia extract (PCS-5001) as a test sample. A thirty member consumer panel evaluated two samples of brown gravy for different attributes (sweet intensity, saltiness, black pepper, beef flavor, and onion/savory notes, bitterness, and sweet & bitter aftertaste).

[0026] Figure 1 1 shows the modification of flavor and salt perception caused by the addition of stevia extract (PCS-5001). The panel found the addition of stevia extract in a 30% salt reduced formulation provided an increase in salt perception compared to 30% sodium reduced control. In addition, stevia extract provided an increase in savory and black pepper note higher than control sample without stevia extract (at 95% confidence). There was also a decrease in bitter aftertaste. EXAMPLE 11: Effect of Stevia Extract on Flavor modification of Dairy Product

[0027] To evaluate the contribution of PCS- 1015 (MLD- 1 ), a stevia extract, to a dairy product, two 50% reduced sugar chocolate milk samples were prepared and tested by a consumer panel of 30 company employees. The consumer panel evaluated those two samples of chocolate milk for overall acceptance and attribute intensities (chocolate flavor, dairy notes, sweetness, bitterness and aftertaste) in two sessions. In session one, the two samples included: 1) a 50% sugar reduced control sample containing PureCircle Alpha (steviol glycoside sweetener) and 2) 50% sugar reduced test sample containing PureCircle Alpha and 70 ppm PCS-1015 (MLD-1). In session two, the two samples included: 1) a 50% sugar reduced control sample containing PureCircle Alpha (steviol glycoside sweetener) and 2) 50% sugar reduced test sample containing PureCircle Alpha and 80 ppm PCS-1015 (MLD-1). Tables 17 shows the formula of the control and test samples of 50% reduced sugar.

Table 17: 50% sugar reduced Chocolate Milk with PCS-1015

[0028] Table 18 shows the sensory results with the two test samples. Both test samples showed the impact of the stevia extract (PCS 1015) on the Chocolate flavor notes and dairy note. At 80 ppm use level, the chocolate milk sample showed better sweetness profile and overall liking than the control sample. Figure 12 shows the comparison of the taste profile between the control and the test sample with 80 ppm stevia extract PCS 1015. Table 18: Summary of the overall acceptance and mean attribute intensity results for each reduced sugar chocolate milk samples tested by 30 panel members.

^* = 80% CI, ^** = 90% CI, ^*** = 95%CI

EXAMPLE 12: Effect of Stevia Extract on Desserts (Vanilla Custard)

[0029] To test the contribution of the stevia extract, PCS-1015 in gelatin and puddings, two 30% calorie reduced vanilla custard samples were tested: 1) sweetened with PureCircle Alpha, a PureCircle stevia sweetener, 2) sweetened with PureCircle Alpha and PCS-1015 (MLD-1). Table 19 shows the formulation of the control and test samples. A panel of 30 trained panelists with extensive experience in profiling sensory attributes tasted both samples. [0030] To prepare the sample, blend the PureCircle Alpha and the test ingredient (PCS- 1015) with the dry ingredients. Add the dry ingredients to the milk using good agitation. Heat on low until all ingredients are dissolved. Heat up to 95 °C for 10 minutes to cook up the starches. Add flavors, stir it, cool, stir it before place it in the refrigerator. Serve at chilled in 1 oz cups.

Table 19: Reduced su ar dessert Vanill Custard with PCS- 015

[0031] The trained panel found that the test sample had stronger sweet intensity, vanilla, dairy flavor notes and overall liking at 80% confidence. The sample containing stevia extract also had significantly higher egg note at 95% confidence. Figure 13 shows the pictorial rendition of the sensory difference between the control and test dessert samples

Table 20: Summary of the overall acceptance and mean attribute intensity results for reduced sugar dessert (Vanilla Custard) with PCS-1015

[0032] Although various embodiments of the present invention have been disclosed here for purposes of illustration, it should be understood that a variety of changes, modifications and substitutions may be incorporated without departing from either the spirit or the scope of the invention.