# Patent

## Title
Stevia blends containing rebaudioside B

## Abstract

The present invention relates to the use of sweet steviol glycoside compounds, particularly rebaudioside B, in sweetening compositions. Sweetening compositions comprising selected amounts of rebaudioside B have been shown to possess favorable flavor profiles when compared to other high intensity sweetener compounds and are useful in the preparation of consumables.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a non-provisional application claiming the benefit of U.S. Provisional Patent Application Ser. No. 61/437,390, filed Jan. 28, 2011, which is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable.
BACKGROUND
Natural caloric sweeteners, such as sucrose, glucose, and fructose, possess desirable taste characteristics, but they add to the caloric content of products. Therefore, there is great consumer interest in low or non-caloric sweeteners that are considered as healthier alternatives. Non-caloric natural and synthetic high-potency sweeteners are known, but they most often possess flavor profiles that are not as desirable to consumers as sugars. Thus, it is desirable to develop non-caloric sweeteners that can be substituted for sugar and that have a more desirable taste profile.
The species Stevia rebaudiana (“Stevia”) is the source of certain naturally occurring sweet steviol glycosides. Considerable research and development has been done to evaluate the use of sweet steviol glycosides of Stevia as non-caloric sweeteners. Sweet steviol glycosides that may be extracted from Stevia include the six rebaudiosides (i.e., rebaudioside A to F), stevioside (the predominant glycoside in extracts from wild type Stevia), and dulcosides.
Commercial low or non-caloric sweeteners based on rebaudioside A and other steviol glycosides tend to have bitter and licorice aftertastes. These characteristics are especially notable at concentrations above about 300 ppm. In food applications, preferred use levels (8-10% sugar equivalence values) are typically about 500 ppm to about 1000 ppm, above the range at which off tastes are first noticed. Thus a need continues to exist for reduced-, low-, and/or non-caloric sweeteners comprising sweet steviol glycosides that have taste profiles with reduced or no bitterness, undesirable flavors (e.g., licorice), or sweetness profiles more like natural caloric sweeteners, or combinations of such properties.
SUMMARY OF THE INVENTION
The present invention is directed to a Stevia extract comprising rebaudioside B at a concentration that is in the range of 10 to about 90% by weight of the Stevia extract.
The present invention is also directed to a sweetening composition comprising sweet steviol glycoside compounds, wherein the sweet steviol glycoside compounds comprise rebaudioside B at a concentration that is in the range of 10% to about 90% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.
The present invention is also directed to consumables such as beverages, foodstuffs, oral care products, tobacco products, pharmaceutical products, and nutraceutical products comprising the foregoing sweetening compositions and methods of the sweetening the same using the foregoing sweetening compositions.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the solubility of rebaudioside B in citric acid buffer at pH of 3 as a function of rebaudioside A and stevioside concentrations.
FIG. 2 shows the structures of steviol glycosides.
FIG. 3 is a graph showing the effect of rebaudioside B content (in a solution containing rebaudioside A and added rebaudioside B for a total concentration of 900 ppm in water) on various flavor attributes as scored by taste panelists.



DETAILED DESCRIPTION
I. Definitions
As used herein, the phrase “sweet steviol glycoside compounds” means any of a number of naturally occurring compounds with a general structure of the steviol diterpene ring system with one or more saccharide residues chemically attached to the ring.
II. Overview
It has unexpectedly been discovered that by including and/or controlling the concentration of rebaudioside B in Stevia extracts and sweetening compositions comprising sweet steviol glycosides tends to reduce or eliminate taste characteristics generally considered to be negative such as bitterness, licorice aftertaste, or result in a sweetness profile more like that of natural caloric sweeteners, or combinations of such properties. Specifically, it has been discovered that the foregoing benefits may be achieved by selecting a relatively high concentration of rebaudioside B with respect to the total concentration of sweet steviol glycosides in the Stevia extract and sweetening composition (e.g., at least 10% by weight of the total amount of sweet steviol glycoside compounds being rebaudioside B).
The aforementioned Stevia extracts and sweetening compositions of the present invention are useful as reduced-caloric, low-caloric, or non-caloric sweeteners in foodstuffs, i.e., edible or chewable compositions such as food, beverages, medicine, candy, chewing gum, and the like. It has been discovered that the Stevia extract and sweetening compositions of the present invention can possess a sweetness profile that is more sugar-like, reduced bitter aftertaste, reduced off flavors (e.g., licorice) than other mixtures of sweet steviol glycosides, such as commercially available blends and mixtures of steviol glycosides. Testing has shown that, in most cases, sweetening compositions of the present invention are preferred by test subjects over compositions that comprise 97% rebaudioside A, when tested at the same concentration. Adding Stevia extract and sweetening compositions of the present invention to foods and beverages is expected to result in better tasting foods and beverages compared to those prepared with known Stevia extracts and sweetening compositions containing sweet steviol glycosides, such as compositions having 97% rebaudioside A.
It is also contemplated that rebaudioside B may be added to other high intensity sweeteners. Representative examples of high intensity sweeteners suitable for embodiments of the invention include natural high intensity sweeteners such as:

    
    
        dulcoside A, dulcoside B (also known as rebaudioside C), rubusoside, siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, stevioside, rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside F, stevia, steviolmonosides, and steviolbiosides;

and artificial high intensity sweeteners such as:

        saccharin, aspartame, sucralose, neotame, acesulfame potassium.

Further, one of skill in the art will recognize that rebaudioside B can be added to caloric sweeteners, such as sugars (e.g., high fructose corn syrup, sucrose, fructose, etc.) and polyols (e.g., sorbitol, xylitol, lactitol, etc.) or other low-calorie sweeteners to produce sweetening compositions that are reduced in caloric value.

    
    


Simple extraction of steviol glycosides from plants generally results in extracts that are less preferred in terms of taste than purified extracts higher in rebaudioside A. However, simple extracts are easier to produce and are generally less expensive to produce than high purity rebaudioside A. Therefore, a further advantage of the present invention may be a combination of a simple extract or partially purified product with rebaudioside B, so as to obtain a glycoside mixture that is less expensive to produce than purified rebaudioside A, yet possesses comparable or superior flavor characteristics. It is also contemplated that steviol glycoside processing streams that have been depleted of good tasting glycosides during the purification of rebaudioside A can be made to taste better by increasing the rebaudioside B content.
The compositions containing rebaudioside B may be further modified using known technology to modify particle size such as agglomeration, spray-drying, drum drying and other forms of physical processing commonly applied to adjust particle size in order to deliver better flow, hydration, or dissolution properties.
The compositions containing rebaudioside B may be further modified using known technology to provide liquid forms with preservative for ease-of-use in specific applications.
The compositions containing rebaudioside B may be further modified using known techniques to co-process with bulking agents such as maltodextrins and similar compounds to deliver products with controlled sweetness, dosing, potency, and handling properties. Further, it is to be noted that rebaudioside B and/or combinations of it and other steviol glycosides can be combined with other ingredients that may be desirable to include in a sweetening composition. For example, rebaudioside B may be spray coated or spray agglomerated onto rebaudioside A or other steviol glycosides and/or with other materials such as maltodextrins, sucrose, or any other desired functional carrier.
III. Stevia Extracts and Sweetening Compositions Comprising Rebaudioside B
It has been discovered that the taste of sweet steviol glycoside mixtures and blends (e.g., steviol glycoside mixtures and blends) can be improved by controlling and/or increasing the concentrations of rebaudioside B in steviol glycoside compositions in accordance with the present invention. It is believed that the improved taste is evident at pH values from about pH 2 to about pH 8.
The solubility limits of rebaudioside B were determined (see Example 5). For example, experimental results to date indicate that (i) rebaudioside B has relatively high solubility in neutral pH solution and (ii) the solubility of rebaudioside B is limited in a pH 3 citric buffer. Further experimental results to date indicate that the presence of rebaudioside A in solution increases the solubility of rebaudioside B. On the other hand, experimental results to date indicate that the presence of stevioside slightly reduces the solubility of rebaudioside B. This solubility information may be considered when formulating solutions of rebaudioside B and mixtures of rebaudiosides.
Rebaudioside B for mixing with other sweeteners can be obtained in various ways. For example, rebaudioside B can be isolated from plant extracts by chromatography, precipitation, or crystallization. Alternatively, rebaudioside B may be obtained by treating rebaudioside A with various hydroxides of mono, di, and trivalent cations under appropriate temperature and pH conditions. The rebaudioside B mixture with residual rebaudioside A can be used to increase the amount of rebaudioside B in another mixture, or the rebaudioside B can be isolated from the rebaudioside A/rebaudioside B mixture by chromatography, precipitation, or selective crystallization. Rebaudioside B can also be obtained in a similar manner by treating rebaudioside D with the same hydroxide compounds as mentioned above for rebaudioside A. The product mixture or isolated rebaudioside B can be used to prepare the above mentioned improved tasting steviol glycoside mixtures. As another alternative, rebaudioside B can be produced enzymatically from rebaudioside A or rebaudioside D.
In certain embodiments, a Stevia extract or sweetening composition comprises rebaudioside B and one or more additional sweet glycoside compounds. Representative examples of sweet glycoside compounds include rebaudioside A, rebaudioside B, rebaudioside C (dulcoside B), rebaudioside D, rebaudioside E, rebaudioside F, stevia, stevioside, dulcoside A, and rubusoside. In certain embodiments, the one or more sweet glycosides may be sweet steviol glycosides, including steviolbiosides and steviolmonosides. More specifically, representative examples of sweet steviol glycosides include rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and stevioside. For example, partially purified extractions of steviol glycosides from plants often comprise a mixture of rebaudioside B and additional steviol glycosides.
In certain embodiments where a Stevia extract or sweetening composition comprises rebaudioside B and one or more additional sweet steviol glycoside compounds, the amount of rebaudioside B is at a concentration that is at least about 10% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 15% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 20% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 25% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 30% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 35% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 40% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 45% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the rebaudioside B is at a concentration that is at least about 50% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition.
In certain embodiments where a Stevia extract or sweetening composition comprises rebaudioside B and one or more additional sweet steviol glycoside compounds and where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above (the term “consistent” rules out potential combinations wherein a lower limit of a range from above is selected that is greater than an upper limit of a range from below), the concentration of rebaudioside B may also be at a concentration that is not greater than about 90% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 80% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 70% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 60% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 50% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 40% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 35% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 30% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments where the concentration of rebaudioside B is at a concentration consistent with any of the embodiments described above, the concentration of rebaudioside B is not greater than about 25% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition.
In certain embodiments, the one or more additional sweet steviol glycoside compound comprises rebaudioside A. For example, partially purified extractions of steviol glycosides may comprise a mixture of rebaudioside B and rebaudioside A or rebaudioside B may be incorporated into purified preparations of rebaudioside A. In certain embodiments comprising rebaudioside A, the amount of rebaudioside A in the Stevia extract or sweetening composition is at a concentration that is at least about 1% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 5% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 10% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 20% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 30% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 40% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 50% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 60% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments the amount of rebaudioside A is at least about 70% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition.
In certain embodiments comprising rebaudioside A wherein the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 95% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 90% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 85% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 80% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 75% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 70% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 65% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 60% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 55% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments comprising rebaudioside A where the concentration of rebaudioside A is at a concentration consistent with any of the embodiments described above, the rebaudioside A is at a concentration that is not greater than about 50% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition.
Although sweetening compositions of the invention may comprise mixtures of various types of sweeteners in various quantities, in certain embodiments, a sweetening composition with rebaudioside B and one or more additional sweet steviol glycoside compounds consists essentially of sweet steviol glycoside compounds. For example, in such embodiments the total concentration of rebaudioside B and all other sweet steviol glycoside compounds present therein provide essentially all the sweetness functionality of the sweetening composition. The amount of other sweetening compounds that could be included in sweetening compositions that consists essentially rebaudioside and sweet steviol glycoside compounds will depend upon the type of other sweetening compound in question and its sweetness threshold concentration below which it is believed it does not appreciably contribute to the sweetness of a sweetening composition. Further, in certain embodiments, a sweetening composition with rebaudioside B and one or more additional sweet steviol glycoside compounds consists of sweet steviol glycoside compounds.
In certain embodiments the Stevia extract or sweetening composition comprising rebaudioside B and one or more additional sweet steviol glycoside compounds comprises stevioside. In certain embodiments, the concentration of stevioside is at least about 1% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is at least about 5% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is at least about 10% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is at least about 20% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is at least about 30% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is at least about 40% by weight, of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is not greater than about 95% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is not greater than about 90% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is not greater than about 80% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is not greater than about 70% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition. In certain embodiments, the concentration of stevioside is not greater than about 60% by weight of the total amount of sweet steviol glycoside compounds in the Stevia extract or sweetening composition.
IV. Products Comprising High Rebaudioside B Sweeteners
Certain embodiments of the invention are drawn to foodstuffs comprising Stevia extract or sweetening compositions with high concentrations of rebaudioside B. One of skill in the art would recognize any edible or chewable compositions may be sweetened in accordance with the present inventions, such as foodstuffs, (e.g., snacks, baked goods, soups, sauces, processed meats canned fruits, canned vegetables, dairy products, frozen confections, cakes, cookies, bars, and other sweet bakery items, cereals, cereal bars, yogurt, yogurt-containing drinks, energy bars, granola bars, hard candy, jelly candy, chocolate candy, and other sweet confections); beverages (e.g., carbonated soft drinks, ready to drink teas, sports drinks, dairy drinks, alcoholic beverages, energy drinks, coffees, flavored waters, vitamin drinks, fruit drinks, and fruit juices, powdered soft drinks), medicines or pharmaceutical products (e.g., tablets, lozenges, suspensions, etc.), nutraceutical products (e.g., supplements, vitamins, etc.), candy or confections; chewing gum; tobacco products (e.g., chewing tobacco); and the like. The addition of rebaudioside B or Stevia extracts or sweetening compositions comprising rebaudioside B and other optional sweeteners to foodstuffs is a process that will depend on the foodstuff and its preparation. Such preparation is known to those skilled in the art of preparing foodstuffs. Preferably, the sweetening composition is included in an effective amount that imparts the desired amount of sweetness to foodstuff. One of skill in the art would recognize that it is routine practice to determine the preferred amount of sweetener to add in the preparation of foodstuffs.
In certain embodiments, the foodstuff contains a sweetening composition comprising rebaudioside B and one or more additional sweet steviol glycoside compounds as described herein. In certain embodiments, steviol glycosides of the sweetening composition are at a total concentration that is less than their sweetening threshold (which is believed to be about 40 ppm). In such an embodiment, it is believed that at such low amounts the sweet steviol glycosides are functioning as a flavoring agent or a flavor enhancing agent rather than as a sweetener. In certain embodiments, the sweet steviol glycosides of the sweetening composition are at a total concentration that is at least about 50 ppm. In certain embodiments, the sweet steviol glycosides of the sweetening composition are at a total concentration that is at least about 200 ppm, or at a concentration that is at least about 500 ppm, or at a concentration that is at least about 1500 ppm.
In certain embodiments, the foodstuff is a beverage containing a sweetening composition comprising rebaudioside B and one or more additional sweet steviol glycoside compounds as described herein. In certain embodiments, the pH of the beverage is at least about pH 2 (and preferably at least about pH 4) and not greater than about pH 8. In certain embodiments, the sweet steviol glycosides of the sweetening composition are at a total concentration that is at least about 50 ppm. In certain embodiments, the sweet steviol glycosides of the sweetening composition are at a total concentration that is at least about 200 ppm, or at least about 500 ppm, or at least about 1500 ppm.
V. Production of Sweetening Composition
Sweetening compositions in accordance with the principles set forth herein may be produced according to any appropriate method of which there are a variety. One such method involves blending certain amounts of rebaudioside B with and one or more additional sweet steviol glycoside compounds such as rebaudioside A and/or other sweet steviol glycoside compounds. For example a blend of purified rebaudioside B and rebaudioside A and/or other sweet steviol glycoside compounds may be made by blending dry powders of the components. Alternatively, a mixture of sweet steviol glycoside compounds may be prepared in solution or suspension and co-dried to produce a powder.
Rebaudioside A is a commercially available material that is typically characterized as being, for example, >80% rebaudioside A, >95% rebaudioside A, or ≧97% of rebaudioside A. Such a purified form of rebaudioside A is typically achieved by reducing the amounts of other steviol glycosides by using solvent recrystallization, adsorption resins, or chromatographic fractionation.
Rebaudioside B may be obtained according to a variety of means. For example, rebaudioside B may recovered from process streams associated with processing and purifying rebaudioside A by using, for example, precipitation, recrystallization, chromatographic fractionation, adsorption resins. Additionally, rebaudioside B may be obtained by the alkaline or acid hydrolysis of rebaudioside A such as disclosed by Kohda, et. al., Phytochemistry, vol. 15, pp. 981-983 (1975) and JP52083731A. Rebaudioside B may also be produced by the enzymatic hydrolysis of rebaudioside A such as disclosed by Mizukani, H., et al., Phytochem vol. 21, pp. 1927-1930 (1982).
Because rebaudioside B may be formed from rebaudioside A, stevia extracts may have their rebaudioside B content increased by, for example, modifying the process parameters associated with the extraction of steviol glycosides from the stevia plant. For example, the amount of rebaudioside B may be increased by controlling the pH of a process stream, the temperature of a process stream, increasing process duration, or a combination of such modifications.
If desired, rebaudioside B may be separated from other steviol glycosides and related compounds using any appropriate method. For example, rebaudioside B may be precipitated from solutions by decreasing the solution pH. Rebaudioside B is typically transformed into its essentially insoluble, protonated form in room temperature water at pH values lower than about 4.5.
After the precipitation of rebaudioside B, it may be separated from the solution comprising solute compounds by any of common means of purifying a suspension. The precipitate can be centrifuged and the supernatant removed. The precipitate can be separated by filtration such as vacuum filtration or the use of a filter press. The soluble and insoluble phases can be separated by use of membranes. The filter cake, centrifuge pellet, or membrane retentate can be further purified by washing with water. Alternatively, the partially purified and recovered precipitate can be re-dissolved in water of pH greater than about 7.7, re-precipitated by the addition of acid to drop pH below about 4.5, and again separated from the impurity-containing liquid phase by any of the above techniques.
Alternatively, rebaudioside B may be precipitated by the addition of a solvent in which rebaudioside B has limited solubility or is insoluble. The specific solvent, amount added, and temperature are preferably to be selected such that essentially only rebaudioside B precipitates, not other compounds.
At neutral pH in water, soluble rebaudioside B may be separated from other soluble compounds by chromatographic fractionation, recrystallization, membrane separation using a membrane of appropriate pore size that retains rebaudiosides but allows smaller molecules to pass, or treatment with adsorptive resins that will either adsorb all impurities, eluting rebaudiosides, or adsorb rebaudiosides and elute all impurities. The resin would then be washed with an eluent having affinity for the adsorbed material, to regenerate the resin (in the first case) or recover the rebaudiosides (second case).
Separated rebaudioside B may be dried by any appropriate method and associated apparatus such as by belt drying, drum drying, tray drying, spray drying, freeze drying, flash drying, or drying with a fluidized bed. Alternatively, instead of drying rebaudioside B and then blending the dried rebaudioside B with rebaudioside A and/or other sweet steviolside compounds, one may blend the same while in solution and then dry the composition.
EXAMPLES
The following disclosed embodiments are merely representative of the invention which may be embodied in various forms. Thus, specific structural, functional, and procedural details disclosed in the following examples are not to be interpreted as limiting.
Example 1
Preference Testing of Commercial Mixtures of Steviol Glycosides and Rebaudioside B Enriched Glycoside Mixtures
A commercial blend of a steviol glycosides was dissolved in 0.0056 M citric acid buffer pH 3.1. A solution containing a commercially available sweetening composition containing 97% rebaudioside A was similarly prepared.







TABLE 1







97% Rebaudioside A and Commercial Blend Compositions.












97% rebaudioside
Commercial



Glycoside
A, mg
blend, mg/l














Rebaudioside A
473
437



Stevioside
1
35



Rebaudioside B
14
17



Rebaudioside C
0
9



Rebaudioside D
0
20



Total
498
518









Thirty-one Tate & Lyle employees participated in paired comparison tests for sweetness and preference. The products were made up in pH 3.1 citric acid buffer (0.9 grams anhydrous citric acid (Tate & Lyle, Decatur, Ill.) and 0.26 grams sodium citrate dihydrate (Tate & Lyle, Decatur, Ill.) per liter and tested at room temperature in two ounce soufflé cups label with random three-digit codes. The presentation order was rotated. The panelists were asked to identify the solution that was sweeter and which they like better. The ballot was presented and the data was collected with SIMS sensory software (Sensory Computer Systems, LLC, Morristown, N.J.). Bottled water, a 2% sucrose solution, and unsalted crackers were available for the panelists to clear their palates before and during testing.
The results of the sweetness and preference questions were analyzed with the binomial test and the Thurstonian d′ calculated. The p-value for a one-tailed binomial test is calculated as




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          -
          k
        
      
    
  




where c is the number of successes, n is the number of trials, and p0 is the chance probability. A test is considered statistically significant when the p-value is less than the a priori set alpha risk. The two-tailed p-value is double the one-tailed p-value as calculated above.

Thurstonian d′ is a linear measure of psychophysical difference. A d′ of 1 is generally considered to be a just-noticeable-difference (JND) where a stimulus will be judge stronger in 75% of the trials. The Thurstonian d′ is independent of test method and for paired comparison tests is calculated as

pc=Φ(d′/√{square root over (2)})

where pc is the proportion of successes, and Φ(•) is the cumulative distribution function of the standard normal distribution. These statistical terms are more fully defined in standard textbooks on the subject such as “Sensory Discrimination Tests and Measurements”, Jian Bi (Blackwell Publishing, 2006).

The instructions for the paired comparison test were:

    
    
        (i) It is important that you rinse before and between samples.
        (ii) Clear your palate with a bite of cracker. Then rinse with the sugar water. Finally rinse with plain water.
        (iii) Taste the samples in the order presented, from left to right.
        (iv) Taste at least half of the first sample and note the sweetness.
        (v) Rinse with the sugar water followed by rinsing with plain water.
        (vi) Now taste at least half of the second sample.
        (vii) Do not re-taste the first sample.
        (viii) Evaluate the samples for preference and sweetness. Pick the sample that you like more and pick the sample that is sweeter. They may or may not be the same sample. If you are not sure or don't have a preference then pick either one.

The questions for the paired comparison test were:

        (i) Which of these two samples do you like more?
        (ii) Which of the two samples is sweeter?
    
    


The results of this test and the psychophysical d′ value are shown in Table 2 below.







TABLE 2







Commercial Blend vs. 97% rebaudioside A











Sample
# preferring
# sweeter














Commercial Blend
11
16



97% rebaudioside A
20
15



Totals
31
31



Binomial p-value,
0.07
0.72



two tailed





d′ value
−0.53
0.16









The results show that the commercial blend was found to be slightly less preferred and about as sweet as the 97% rebaudioside A.

In a subsequent test performed the same way, an addition of rebaudioside B was made to the foregoing Commercial Blend to make a mixture of glycosides that is an embodiment of the present invention having a concentration of rebaudioside B relative to the total amount of sweet steviol glycosides of about 21% and a ratio of rebaudioside A to rebaudioside B of about 3:1.
The glycoside blend of a steviol glycosides was dissolved in 0.0056 M citric acid buffer pH 3.1. A solution containing a commercially available sweetening composition containing 97% rebaudioside A was similarly prepared.







TABLE 3







97% rebaudioside A and Glycoside Mixture Compositions.












97% rebaudioside
Mixture,



Glycoside
A, mg
mg/l














Rebaudioside A
473
350



Stevioside
1
32



Rebaudioside B
14
104



Rebaudioside C
0
10



Rebaudioside D
0
0



Total
498
496









The two solutions were presented to a panel of Tate & Lyle employees and they were asked to identify the solution that was sweeter and which they like better using the same instructions and questions as set forth above. The results of the sweetness and preference questions were analyzed with the binomial test and the Thurstonian d′ calculated as set forth above. The results and the psychophysical d′ value are presented in Table 4 below.







TABLE 4







Rebaudioside B blend vs. 97% Rebaudioside A











Sample
# preferring
# sweeter














Rebaudioside B blend
27
27



97% rebaudioside A
11
11



Totals
38
38



Binomial p-value,
<0.01
<0.01



two tailed





d′ value
0.79
0.79









The rebaudioside B blend was both preferred and found to be sweeter than 97% rebaudioside A.

Example 2
Preference Testing of 800 ppm Mixtures of Rebaudioside A or B Mixed with Stevioside
A taste panel was asked to compare a commercial mixture of rebaudioside A and stevioside to a 500 ppm sample of 97% rebaudioside A. The glycoside blend of steviol glycosides was dissolved in 0.0056 M citric acid buffer pH 3.1. A solution containing a commercially available sweetening composition containing 97% rebaudioside A was similarly prepared. Sample presentation was rotated and the samples of the two solutions (at room temperature) were presented to a panel of Tate & Lyle employees and they were asked to identify the solution that was sweeter and which they like better using the same instructions and questions as in Example 1. The results of the sweetness and preference questions were analyzed with the binomial test and the Thurstonian d′ calculated as in Example 1.







TABLE 5







97% Rebaudioside A and Rebaudioside A


and Stevioside Mixture Compositions.












97% rebaudioside
Mixture,



Glycoside
A, mg
mg/l














Rebaudioside A
473
374



Stevioside
1
403



Rebaudioside B
14
11



Rebaudioside C
0
0



Rebaudioside D
0
0



Total
498
789









Another panel was asked to compare a mixture of rebaudioside B and Stevioside. The glycoside blend of steviol glycosides was dissolved in 0.0056 M citric acid buffer pH 3.1. A solution containing a commercially available sweetening composition containing 97% rebaudioside A was similarly prepared. The two solutions were presented to a panel of Tate & Lyle employees and they were asked to identify the solution that was sweeter and which they like better using the same instructions and questions as in Example 1.







TABLE 6







97% Rebaudioside A and Rebaudioside B


and Stevioside Mixture Compositions.












97% rebaudioside
Mixture,



Glycoside
A, mg
mg/l














Rebaudioside A
473
4



Stevioside
1
397



Rebaudioside B
14
400



Rebaudioside C
0
0



Rebaudioside D
0
0



Total
498
801









The results of the sweetness and preference questions were analyzed with the binomial test and the Thurstonian d′ calculated as in Example 1. The results of the two panels and the psychophysical d′ values are shown in Table 7 and Table 8 respectively.







TABLE 7







Rebaudioside A & stevioside blend vs. 97% Rebaudioside A











Sample
# preferring
# sweeter














Rebaudioside A and
4
22



stevioside





97% rebaudioside A
37
19



Totals
41
41



Binomial p-value,
<0.01
0.27



two tailed





d′ value
−1.83
0.13









The results show that the rebaudioside A—stevioside blend was much less preferred as compared to 97% rebaudioside A and was found to be nearly equally sweet.








TABLE 8







Rebaudioside B & stevioside blend vs. 97% Rebaudioside A











Sample
# preferring
# sweeter














rebaudioside B and
21
30



stevioside





97% rebaudioside A
22
13



Binomial p-value,
0.38
<0.01



two tailed





Totals
43
43



d′ value
−0.04
0.73









The results show that the rebaudioside B—stevioside blend was equally preferred to the 97% rebaudioside A (500 ppm) and the rebaudioside B—stevioside blend found to be sweeter than the 97% rebaudioside A.

Example 3
Preference Testing Mixtures of Rebaudioside A or B
A taste panel was asked to compare a commercial mixture of rebaudioside A and rebaudioside B and a 900 ppm sample of 97% rebaudioside A. Tate & Lyle employees participated in paired comparison tests for sweetness and preference. Samples were tested at room temperature in two ounce soufflé cups label with random three-digit codes. The presentation order was not rotated because of carryover of the off-flavor of rebaudioside A at 900 ppm. The panelist evaluated the test sample first and then the control 900 ppm rebaudioside A sample. The panelists were instructed not to re-taste the samples. Additionally, the panelists were required to wait one minute between testing samples and instructed to clear their palates with a 2% sucrose solution, an unsalted cracker, and bottled water. The panelists were asked to identify the solution that was sweeter and which they like better. The ballot was presented and the data was collected with SIMS sensory software (Sensory Computer Systems, LLC, Morristown, N.J.).







TABLE 9







97% Rebaudioside A and Rebaudioside A and


Rebaudioside B Mixture Compositions.











Ingredient
Test
Control














Hinkley Spring Water
98.88
98.88



Rebaudioside A
0.0536
0.0900



Rebaudioside B
0.0310
0.0000



Phosphoric Acid, 85%
0.0361
0.0361



Total
100
100









The instructions for the paired comparison test were:

    
    
        (i) It is important that you rinse before and between samples.
        (ii) Clear your palate with a bite of cracker. Then rinse with the sugar water. Finally rinse with plain water.
        (iii) Taste the samples in the order presented, from left to right.
        (iv) Taste at least half of each sample and note the sweetness.
        (v) Rinse with the sugar water followed by rinsing with plain water.
        (vi) Evaluate the samples for preference and sweetness. Pick the sample that you like more and pick the sample that is sweeter. They may or may not be the same sample. If you are not sure or don't have a preference then pick either one.
        (vii) Taste the sample on the left now.
        (viii) Wait one minute before tasting the next sample (60 second timer started in SIMS)
        (ix) Taste the sample on the right now.

The questions for the paired comparison test were:

        (i) Which of these two samples do you like more? Carefully check the 3-digit code before marking your answer. They may not appear in the same order as the samples were presented.
        (ii) Which of the two samples is sweeter?

(120 second timer started in SIMS between tests). The results of the sweetness and preference questions were analyzed with the binomial test and the Thurstonian d′ calculated as in Example 1.

    
    


The results of the two panels and the psychophysical d′ values are shown in Table 11.







TABLE 11







Rebaudioside A & Rebaudioside B vs. 97% Rebaudioside A















p-value


p-value





two-


one-



sweetness
tailed
d′
preference
tailed
d′

















Test 1 536 ppm
23
0.14
0.38
36
<0.01
2.29


Reb A


310 ppm


Reb B


control
15


2


900 ppm


Reb A









The results show that the rebaudioside A—Rebaudioside B blend was preferred as compared to 97% rebaudioside A and was found to be nearly equally sweet.

Example 4
Preference Testing of Mixtures of Steviol Glycosides
This study was performed to determine the preference for blends of rebaudioside A and rebaudioside B relative to rebaudioside A alone at a sweetness of approximately 10 SEV with the sensory methodology changed to reduce panelist confusion with the ballot and requiring the panelist testers to consume approximately 2 ounces of each sample.
Panelists were used in paired comparison tests for sweetness and preference. The products were tested at refrigerated temperature in two ounce soufflé cups label with three-digit codes. The samples were poured immediately before serving. The panelists were asked to identify the beverage that is sweeter and which they like better. Bottled water, a 2% sucrose solution, and unsalted crackers were available for the panelists to clear their palates before and during testing.
The panelists evaluated the test sample first and then evaluated the control 900 ppm rebaudioside A sample second. The presentation order in this test was not rotated because of carryover of the off-flavor of rebaudioside A at 900 ppm. The panelists were instructed to consume all of the samples and not to re-taste the samples. The panelists were alerted to the fact that the order of the samples on the ballot may not be the same as the order the samples are presented. The panelists were instructed to mark the sample they prefer with an adhesive-backed note and these results were compared to the results from the ballot. There was an enforced rest of one minute between samples and two minutes between tests where the panelists were instructed to clear their palates with 2% sucrose, cracker, and water.
The products tested were lemon-lime carbonated soft drinks comprising one part syrup and four parts carbonated water, wherein the syrups had the compositions set forth in Table 12, below.







TABLE 12







Syrup Formulations











Ingredient
Test 1
Control














Hinkley Spring Water
98.20
98.18



Sodium Benzoate
0.10
0.10



REB A
0.268
0.450



REB B
0.155
0.000



Sodium Citrate Dihydrate
0.15
0.15



Citric Acid Anhydrous
0.63
0.63



Givaudan Natural Lemon
0.50
0.50



Flavor #881337





Total
100
100









The results were analyzed with the binomial test at an alpha risk of 0.05 as a one-tailed test for preference and two-tailed test for sweetness. The results of the test are set forth in Table 13, below.







TABLE 13







Test 1 Beverage compared Control Beverage















p-value


p-value





two-


one-



sweetness
tailed
d′
preference
tailed
d′
















Test 1 536 ppm
20
0.76
−0.04
26
0.03
0.48


RebA


310 ppm


RebB


control
21


15


900 ppm


RebA









The tests show that both of the test lemon-lime carbonated soft drinks did not significantly different in sweetness from the rebaudioside A sweetened lemon-lime carbonated soft drink and that they were significantly preferred to the rebaudioside A sweetened lemon-lime carbonated soft drink.
The analysis suggests that a blend of rebaudioside A and rebaudioside B should be more pleasant than rebaudioside A alone especially at higher sweetness levels.
Example 5
Solubility
To determine the solubility of rebaudioside A and rebaudioside B in certain solutions, four stock solutions were prepared. A 10× concentrated citric acid/sodium citrate pH 3 stock buffer solution was prepared by dissolving 0.9 g anhydrous citric acid and 0.26 g of sodium citrate dihydrate in water to make 100 mL of buffer (0.047 M citric acid+0.0088 M sodium citrate). A 2500 ppm (nominal) solution of rebaudioside A was prepared by dissolving 0.125 g of GLG RA 97 to make 50 mL of solution. A 2500 ppm (nominal) solution of stevioside was prepared by dissolving 0.125 g of GLG STV 97 to make 50 mL of solution. A 1000 ppm (nominal) solution of rebaudioside B was prepared by diluting 56 mL of a solution assayed at 1790 ppm to make 100 mL of solution.
These solutions were mixed as 1 mL total amounts by micropipetting the volumes, in microliters, into 1.5 mL microcentrifuge tubes As shown in Table 14. The resulting nominal concentrations of each of the three glycosides, in ppm, are also listed in Table 14.







TABLE 14







Summary of Test Solutions










Microliters of stock solution added















10X

2500 ppm
2500 ppm
1000 ppm
PPM (nominal)















Tube
citric acid
Water
Reb A
stevioside
Reb B
Reb A
stevioside
Reb B


















1
100
200
0
200
500
0
500
500


2
100
250
0
150
500
0
375
500


3
100
300
0
100
500
0
250
500


4
100
350
0
50
500
0
125
500


5
100
400
0
0
500
0
0
500


6
100
150
50
200
500
125
500
500


7
100
200
50
150
500
125
375
500


8
100
250
50
100
500
125
250
500


9
100
300
50
50
500
125
125
500


10
100
350
50
0
500
125
0
500


11
100
100
100
200
500
250
500
500


12
100
150
100
150
500
250
375
500


13
100
200
100
100
500
250
250
500


14
100
250
100
50
500
250
125
500


15
100
300
100
0
500
250
0
500


16
100
50
150
200
500
375
500
500


17
100
100
150
150
500
375
375
500


18
100
150
150
100
500
375
250
500


19
100
200
150
50
500
375
125
500


20
100
250
150
0
500
375
0
500


21
100
0
200
200
500
500
500
500


22
100
50
200
150
500
500
375
500


23
100
100
200
100
500
500
250
500


24
100
150
200
50
500
500
125
500


25
100
200
200
0
500
500
0
500









Immediately after mixing, all of the solutions were clear (vs. cloudy) and showed no precipitation. The tubes were then allowed to stand undisturbed at room temperature in the lab ( ̃25° C.) for around 100 hours, by which time all of them showed at least some precipitation. After standing for five days ( ̃100 hours), the tubes were spun in a bench top microcentrifuge to pelletize the precipitate. The clear supernate was sampled into vials and assayed for glycosides using a reverse phase high performance liquid chromatograph (HPLC) gradient method with UV detection (Waters 2695 Separations Module equipped with a Waters 2487 Dual λ Absorbance Detector or equivalent instrumentation) that is summarized in Table 15. The HPLC conditions were as follows:

    
    
        column—Waters Atlantis T3 4.6×250 mm; 4μ with a Phenomenex Security Guard AQ C18 guard cartridge, 4×3.0 mm;
        buffer—0.0284% ammonium acetate; 0.0116% acetic acid;
        flow rate—1.0 mL/min;
        detector—UV Detector with analysis at 203 nm;
        inj. vol.—20 μL or as desired to conform to standard concentration; and
        col. temp.—40° C.
    
    









TABLE 15







HPLC Gradient Method: Mobile Phase:


Acetonitrile/Buffer gradient












Time (min)
% Water
% Buffer
Waters Curve















0
70
30
na



15
65
35
6



20
65
35
6



25
20
80
6



30
20
80
1



35
70
30
1









The supernatant data collected from the HPLC were processed using DESIGN EXPERT 8 software. Briefly, the data were entered into the program as “factor” data as a 2 factor-5 level general factorial design as the nominal data which were then converted to numerical data and replaced by the actual HPLC results. The software then selected a model that predicted the concentration (solubility) of rebaudioside B as a function of the concentration of rebaudioside A and stevioside in the supernatant. The software also calculated a number of statistical factors that indicated the significance of the model parameters. In this case, the modeling indicated that models with and without a small rebaudioside A—stevioside interaction parameter were about equally valid so we selected the simpler (non-interacting) model for further processing. The analysis of variance (ANOVA) for this model obtained from the program is shown in Table 16.







TABLE 16





ANOVA Results - Design Expert


Response 1 Reb B


ANOVA for Response Surface Linear Model


Analysis of variance table


[Partial sum of squares - Type III]





















Sum of

Mean
F
p-value


Source
Squares
df
Square
Value
Prob > F





Model
61253.89
2
30626.95
443.84
<0.0001


A—Reb A
61056.01
1
61056.01
884.81
<0.0001


B—Stevioside
363.55
1
363.55
5.27
0.0316


Residual
1518.11
22
69.00




Cor total
62772.00
24















Std. Dev.
8.31
R-Squared
0.9758



Mean
301.80
Adj R-Squared
0.9736



C.V. %
2.75
Pred R-Squared
0.9691



PRESS
1939.25
Adeq Precision
52.665

















Coefficient

Standard
95% CI
95% CI



Factor
Estimate
df
Error
Low
High
VIF





Intercept
297.61
1
1.67
294.15
301.08



A—Reb A
78.09
1
2.63
72.65
83.54
1.00


B—Stevioside
−5.66
1
2.47
−10.78
−0.55
1.00





Final Equation in Terms of Coded Factors:


Reb B = +297.61 + 78.09 * A − 5.66 * B


Final Equation in Terms of Actual Factors:


Reb B = +225.17918 + 0.31238 * Reb A − 0.02264 * Stevioside






Table 17 shows the experimentally determined compositions of the supernatants from each of the test solutions.







TABLE 17







Raw Assay Data from the HPLC


Assayed glycoside concentrations by HPLC ppm











Tube
Reb A
stevioside
Reb B
steviolbioside














1
38.8
478
234
4.39


2
38.5
351
231
3.61


3
37.6
241
229
2.28


4
36.2
112
233
1.59


5
34.9
0
226
0.73


6
154
480
265
5.03


7
154
360
255
3.35


8
151
241
284
2.86


9
149
121
273
2.03


10
146
0
271
0.77


11
274
476
300
5.1


12
273
363
293
4.51


13
269
248
305
3.38


14
260
119
286
1.49


15
260
0
308
0


16
375
473
336
4.87


17
381
357
344
3.78


18
372
245
340
2.61


19
370
125
342
2


20
365
0
342
0


21
487
475
361
4.74


22
477
356
363
4.35


23
481
245
358
2.72


24
498
120
375
1.25


25
484
0
391
0









The rebaudioside B used in this study contained about 6-7% rebaudioside A which explains the rebaudioside A found in tubes 1-5. It is also evident from the data that the concentrations of stevioside and rebaudioside A in the samples are consistent with the belief that these compounds are completely soluble in this buffer at this concentration, i.e., only the rebaudioside B was precipitating in the experiment. The data shown in Table 17 were entered into DESIGN EXPERT 8 and a two factor linear regression model was generated, which is set forth in Equation 1, below.

ConcB=225.18+0.312*concA−0.02264*concSs  (Eq. 1)

The equation shows that the concentration of B in the supernatant (concB, i.e., the solubility limit) was found to be influenced by both the concentration of rebaudioside A (concA) and the concentration of stevioside (concSs). The rebaudioside B solubility was increased substantially by increases in the concentration of rebaudioside A, and slightly decreased by the increases in the concentration of stevioside.

The linear-linear model represented by Equation 1 plots as a plane in a 3-D plot as shown in FIG. 1. The plane represents the maximum solubility of rebaudioside B in citric acid buffer (pH 3.1), ranging from a low of about 225 ppm when in 478 ppm stevioside (according to the model) to about 380 ppm in 480 ppm rebaudioside A.
Using the solubility limit (Equation 1) and the constraint that the sum of the mass fractions must add to 1 allows one to find an equation that relates the mass fraction of rebaudioside B (XB) at the solubility limit to the mass fraction of rebaudioside A (XA), the total rebaudioside concentration (rebaudioside B+rebaudioside A+stevioside=Ctot), and the coefficients (α0=225.18 ppm, α1=0.312, α2=−0.0226) of the regression equation (Equation 2, below).




  
    
      
        
          X
          B
        
        =
        
          
            
              [
              
                
                  
                    α
                    0
                  
                  
                    C
                    tot
                  
                
                +
                
                  α
                  2
                
              
              ]
            
            
              (
              
                1
                +
                
                  α
                  2
                
              
              )
            
          
          +
          
            
              [
              
                
                  
                    α
                    1
                  
                  -
                  
                    α
                    2
                  
                
                
                  1
                  +
                  
                    α
                    2
                  
                
              
              ]
            
            ⁢
            
              X
              A
            
          
        
      
    
    
      
        (
        
          eq
          .
          
            
          
          ⁢
          2
        
        )
      
    
  




It is believe that Equation 2, therefore, defines the edge of the stable solution solubility region in ternary mixtures of rebaudioside A, stevioside, and rebaudioside B. Thus, the size of the stable region able region will depend on the total concentration (Ctot).

This study clearly shows that the solubility of rebaudioside B is limited in pH 3 citric buffer, even though rebaudioside B has high solubility in neutral pH solutions. In addition the presence of rebaudioside A increases the solubility of rebaudioside B, but the presence of stevioside slightly reduces the solubility of rebaudioside B. This solubility information should be noted when attempting to formulate solutions of rebaudioside B and mixtures of rebaudiosides.
Example 6
Evaluation of Sweetener Taste as a Function of Rebaudioside B Content
A descriptive panel was used to quantify flavor attributes and intensities of varying levels of rebaudioside B added to rebaudioside A (which was 97% pure and contained 0.62% rebaudioside B). Specifically, the sweeteners were evaluated by the panelists at 900 ppm rebaudioside A+rebaudioside B solutions, wherein the amount of added rebaudioside B was such that the total content of rebaudioside B was 0.6%, 3.6%, 6.5%, 11.4%, 22.3%, 37.5%, and 52%. An 8% sucrose solution as a control. The solutions were prepared in neutral pH water. Other high intensity sweeteners included in the testing were Aspartame at 500 ppm, ASK at 750 ppm, Sucralose at 250 ppm, and Stevia at 500 ppm.
Before conducting the testing the ten panelists were extensively trained in the use of standardized vocabulary to describe the appearance, aroma, flavor, and texture of a wide variety of products in order to rate the samples for sweetness, bitterness, off flavor, chemical or artificial sweetener flavor, anise, and mouth coating. Each such attribute was evaluated on a first and second sip, and on after-taste. The testing began with a session to orient the panelists during which they tasted the samples and discussed the flavor characteristics. They also tasted and discussed references for “sweet”, “bitter”, and “anise”. The definitions of flavor terms are set forth in Table 18 below.







TABLE 18







Definitions








Term
Definition





Total Flavor
The total intensity of all the aromas or flavors in the



product.


Sweet
One of the four basic tastes, perceived primarily on the



tip of the tongue; common to sucrose and other sugars.


Total Off
All of the flavors of the sample that are not sweet and


Flavor
would be considered unintended in the sample.


Artificial
The flavor reminiscent of artificial sweeteners or


Sweetener/
chemical tastes not intended to be in foods and


Chemical
beverages in the sample.


True Bitter
One of the four basic tastes, perceived primarily on



the back of the tongue; common to caffeine and quinine.


Anise
The total flavor reminiscent of anise or licorice.


Mouthcoating
The feeling of any type of coating on the soft tissues



of the mouth.









During the second and third days of testing, the panelists evaluated the samples for the various attributes and rated them on a scale from none to extreme that encompasses all food ingredient products, not just sweeteners. The products and solutions set forth in Table 19 were used to “anchor” the panelist to the scales.








TABLE 19







Flavor Anchors











Flavor
Scale Value
Reference






Anise
NR
Anise on blotter



Sweet
5.0
5% Sucrose in Water




10.0
10% Sucrose in Water



Bitter
2.0
0.025% Caffeine in Water




5.0
0.04% Caffeine in Water









Eight samples were evaluated per session. A 7 minute rest break was given between each sample and a 15 minute break was given after the first 4 samples had been evaluated. Two evaluations (i.e. replicates) were obtained from each panelist for each product; therefore, a total of 20 judgments were obtained for each product. During data collection, the panelists were instructed to indicate the intensity of each sensory characteristic by placing a vertical slash on 15-cm line scales. The serving order was balanced, with products seen approximately an equal number of times in each possible position. In the waiting room, ambient Alhambra drinking water, unsalted soda crackers, and celery were provided for cleansing the palate between samples.
Slash marks on the line scales were converted to numbers ranging from 1 to 15 by SIMS, the computerized sensory data collection system. Mean intensities were calculated for each sensory characteristic. Analysis of Variance and Duncan's Multiple Range Test, where appropriate, were used to determine significant differences among the samples for each attribute. When panelist-by-product interactions were significant, the mean square of the interaction term, instead of the mean square of the error term, was used in calculation of the product F values. The results are set forth in Tables 20 and 21, below.







TABLE 20







QUANTITATIVE DESCRIPTIVE EVALUATIONS OF BITTER MASKERS


n = 18 (9 Panelists, 2 Evaluations Each)



















0.6%
3.6%
6.5%
11.4%
22.3%
37.5%
52%
P-
Conf.



Sucrose
Reb B
Reb B
Reb B
Reb B
Reb B
Reb B
Reb B
Value
Level





TOTAL












FLAVOR:


First Sip
d
abc
a
ab
bc
bc
abc
c



8.67
9.94
10.16 
10.04 
9.69
9.61
9.76
9.47
0.0008
**


Second Sip
c
ab
a
ab
bc
c
c
c



7.86
8.62
8.79
8.56
8.28
8.07
8.00
7.97
0.0047
**


Aftertaste
d
ab
a
bc
bc
bc
c
bc



4.32
5.96
6.34
5.77
5.59
5.80
5.45
5.63
0.0001
**


SWEET:


First Sip
d
abc
ab
a
abc
bc
abc
c



8.06
9.15
9.34
9.56
9.13
9.01
9.21
8.80
0.0057
**


Second Sip
7.43
7.27
7.66
7.36
7.40
6.92
6.91
7.02
0.2088
NSD


Aftertaste
c
bc
a
ab
ab
ab
ab
ab



3.92
4.44
5.15
4.70
4.58
5.01
4.61
4.75
0.0184
**


TOTAL OFF


FLAVOR:


First Sip
e
ab
a
abc
cd
cd
bcd
d



0.89
7.69
7.79
7.34
6.83
6.72
6.98
6.48
0.0001
**


Second Sip
c
a
a
a
b
b
b
b



0.49
7.01
7.02
6.88
6.09
5.67
5.83
5.68
0.0001
**


ARTIFICIAL


SWEETENER/


CHEMICAL:


First Sip
d
a
ab
ab
c
c
bc
c



0.59
7.74
7.45
7.37
6.67
6.54
6.87
6.52
0.0001
**


Second Sip
c
a
a
a
b
b
b
b



0.41
6.79
6.88
6.71
5.73
5.31
5.48
5.64
0.0001
**


Aftertaste
f
ab
a
bc
cd
de
e
de



0.12
5.00
5.22
4.66
4.39
3.99
3.79
3.91
0.0001
**





Analysis of Variance Confidence Levels:


* = 90%,


** = 95%


NSD: Not significantly different at confidence levels of 90% or higher.


Mean ratings with different superscripts differ significantly at the 90% confidence level (Duncan's Multiple Range Test).













TABLE 21







QUANTITATIVE DESCRIPTIVE EVALUATIONS OF BITTER MASKERS


(continued)


n = 18 (9 Panelists, 2 Evaluations Each)



















0.6%
3.6%
6.5%
11.4%
22.3%
37.5%
52%
P-
Conf.



Sucrose
Reb B
Reb B
Reb B
Reb B
Reb B
Reb B
Reb B
Value
Level





















TRUE












BITTER:


First Sip
e
a
ab
abc
ab
cd
be
d



0.29
3.78
3.63
3.28
3.37
2.80
3.11
2.49
0.0001
**


Second Sip
d
a
ab
ab
b
c
c
c



0.24
3.89
3.71
3.46
3.36
2.81
2.62
2.74
0.0001
**


Aftertaste
e
a
a
b
bc
bcd
cd
d



0.15
3.39
3.45
2.78
2.57
2.26
2.08
1.89
0.0001
**


ANISE:


First Sip
c
ab
ab
ab
a
ab
ab
b



0.52
1.73
1.73
1.45
1.79
1.59
1.58
1.34
0.0001
**


Second Sip
b
a
a
a
a
a
a
a



0.41
1.53
1.64
1.36
1.62
1.34
1.36
1.29
0.0001
**


Aftertaste
d
a
a
abc
ab
c
abc
bc



0.13
1.42
1.46
1.27
1.34
0.98
1.12
1.03
0.0001
**


MOUTH-


COATING:


First Sip
c
b
b
b
a
b
b
b



0.76
1.47
1.48
1.40
1.74
1.49
1.49
1.37
0.0001
**


Second Sip
b
a
a
a
a
a
a
a



0.69
1.48
1.46
1.29
1.56
1.42
1.40
1.42
0.0001
**





Analysis of Variance Confidence Levels:


* = 90%,


** = 95%


NSD: Not significantly different at confidence levels of 90% or higher.


Mean ratings with different superscripts differ significantly at the 90% confidence level (Duncan's Multiple Range Test).






FIG. 3 shows the magnitude of the mean responses of the panelists of each flavor attribute for which statistically significant differences were measured as a function of rebaudioside B content. Because the scale used encompasses “the universe”, the magnitude of differences between the 97% rebaudioside A solution and increasing levels of rebaudioside B is not as large as other informal testing determined. For example, several informal testers believed that adding about 20% rebaudioside A reduced the bitterness by about 80% whereas the results from above-described panel of tasters showed that adding about 20% rebaudioside A reduced bitterness by about 30%.
The flavor attributes were also compared between the samples. An attribute is mentioned as ‘highest’ or ‘lowest’ if the sample's attribute was significantly higher or lower than all of the other samples. An attribute is mentioned as ‘high’ or ‘low’ if the sample's attribute was the highest or lowest, respectively, but not significantly higher or lower than all of the other samples. Attributes discussed herein were found to be significant at the 95% confidence level.
Compared to the high intensity sweetener samples, sucrose was the Lowest in the following: Total Flavor (although not significantly on the second sip vs. 22.3%, 37.5%, and 52% rebaudioside B); Sweetness on the first sip and in the aftertaste (although not significantly lower than the bitter control); Total Off Flavor; Artificial Sweetener/Chemical; True Bitter; Anise; and Mouthcoating.
Among the high intensity sweetener samples the 97% rebaudioside with 0.6% rebaudioside B was High in the following: Total Off Flavor on the second sip; Artificial Sweetener/Chemical on the first and second sips; True Bitter; and Anise in the aftertaste.
Among the high intensity sweeteners samples, the sample with 3.6% of rebaudioside B added was High in the following: Total Flavor; Sweetness in the aftertaste; Total Off Flavor; Artificial Sweetener/Chemical on the second sip and in the aftertaste; True Bitter in the aftertaste; and Anise in the aftertaste.
Among the high intensity sweeteners samples, the sample with 6.5% of rebaudioside B added was High in the following: Sweetness in the first sip; Total Off Flavor on the second sip; and Artificial Sweetener/Chemical on the second sip.
Among the high intensity sweeteners samples, the sample with 11.4% of rebaudioside B added was Low in Total Off Flavor on the second sip and Artificial Sweetener/Chemical on the first and second sips and High in Anise on the first sip and Mouthcoating on the first sip.
Among the high intensity sweeteners samples, the sample with 22.3% of rebaudioside B added was Low in the following: Total Flavor on the second sip Total; Total Off Flavor on the second sip; Artificial Sweetener/Chemical on the first and second sips; True Bitter on the second sip; and Anise in the aftertaste.
Among the high intensity sweeteners samples, the sample with 37.5% of rebaudioside B added was Low in the following: Total Flavor on the second sip and in the aftertaste; Total Off Flavor on the second sip; Artificial Sweetener/Chemical on the second sip and in the aftertaste; and True Bitter on the second sip.
Among the high intensity sweeteners samples, the sample with 52% of rebaudioside B added was Low in the following: Total Flavor on the first and second sips; Sweet on the first sip; Total Off Flavor on the first and sips; Artificial Sweetener/Chemical on the first and second sips; True Bitter; and Anise on the first sip.
The results indicate that the addition of 3.6% of rebaudioside B resulted in worse flavor attributes and the addition of 6.5% of rebaudioside B had little impact on the Total Off Flavor and Artificial Sweetener/Chemical Flavor. The addition of greater amounts of rebaudioside B tended to result in the samples scoring more closely to sucrose in most characteristics with decreasing off flavors, particularly bitterness. That said, the results show that the increases the rebaudioside B content above about 20% had little further impact on undesirable flavor attributes. Surprisingly, the sweetness remained fairly unaffected by the rebaudioside B content, which is contrary to previous reports regarding rebaudioside B that found it to be one-half to two-thirds as sweet as rebaudioside A. Although the Sweetness and Total Flavor of the high intensity sweetener solutions were similar to the 8% sucrose solution, and similar to each other, the Artificial Sweetener/Chemical tastes were much higher than the sucrose solution, with a greater spread among high intensity sweetener samples, which indicates that the rebaudioside B concentration had a significant impact on these tastes.
Advantageously, the present invention may be used to produce high intensity sweetener compositions that can be used to provide the “full” sweetness needed for many applications, which typically cannot be achieved with rebaudioside A alone because of its bitterness at concentrations above about 200 ppm. More specifically, because the present invention allows for the production of high intensity sweeteners that may be added to consumables such that the consumables comprises about 800 to about 1000 ppm of rebaudiosides without the consumable having unacceptable levels of bitterness, sweetener compositions of the present invention may be used to provide the entire sweetness needed for many consumables (food applications).

## Claims

1. A sweetening composition comprising sweet steviol glycoside compounds, wherein the sweet steviol glycoside compounds comprise rebaudioside B at a concentration that is in the range of 10% to about 60% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition and rebaudioside A at a concentration that is in the range of about 40% to about 90% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

2. The sweetening composition of claim 1, wherein the concentration of the rebaudioside B is in the range of about 10% to about 40% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

3. The sweetening composition of claim 1, wherein the rebaudioside B is at a concentration that is at least about 15% and no greater than about 30% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

4. The sweetening composition of claim 1, wherein the rebaudioside B is at a concentration that is at least about 20% and no greater than about 25% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

5. A consumable comprising a sweetening composition that comprises sweet steviol glycoside compounds, wherein the sweet steviol glycoside compounds comprise rebaudioside B at a concentration that is in the range of 10% to about 60% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition and rebaudioside A at a concentration that is in the range of about 40% to about 90% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

6. The consumable of claim 5, wherein the sweet steviol glycosides are at a total concentration that is less than their sweetening threshold.

7. The consumable of claim 5, wherein the sweet steviol glycosides are at a total concentration that is in the range of about 50 ppm to about 2000 ppm and the rebaudioside B is at a total concentration of at least about 20 ppm.

8. The consumable of claim 7, wherein the rebaudioside B is at a total concentration of at least about 50 ppm and no greater than about 500 ppm.

9. The consumable of claim 7, wherein the rebaudioside B is at a total concentration of at least about 100 ppm and no greater than about 400 ppm.

10. The consumable of claim 7, wherein the rebaudioside B is at a total concentration of at least about 200 ppm and no greater than about 300 ppm.

11. The consumable of claim 5, wherein the consumable is selected from the group consisting of a beverage selected from the group consisting of carbonated soft drinks, powdered soft drinks, ready to drink teas, sports drinks, dairy drinks, yogurt-containing drinks, alcoholic beverages, energy drinks, flavored waters, vitamin drinks, fruit drinks, and fruit juices; a foodstuff selected from the group consisting of baked goods, soups, sauces, processed meats, canned fruits, canned vegetables, dairy products, frozen confections, confections, chewing gums, cakes, cookies, bars, and other sweet bakery items, cereals, cereal bars, yogurt, energy bars, granola bars, hard candy, jelly candy, chocolate candy, and other sweet confections; an oral care product selected from the group consisting of toothpastes, mouthwashes, and oral rinses; a tobacco product selected from the group consisting of snuff and chewing tobacco; a pharmaceutical selected from the group consisting of tablets, lozenges, and suspensions; a nutraceutical product selected from the group consisting of supplements and vitamins.

12. The sweetening composition of claim 1, wherein the rebaudioside A is at a concentration that is no greater than about 85% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

13. The sweetening composition of claim 1, wherein the rebaudioside A is at a concentration that is no greater than about 80% by weight of the total amount of sweet steviol glycoside compounds in the sweetening composition.

14. The sweetening composition of claim 1, wherein the sweetening composition consists essentially of sweet steviol glycoside compounds, wherein the sweet steviol glycoside compounds provide essentially all of the sweetness functionality of the sweetening composition.