Rebaudioside a composition and method for purifying rebaudioside a.

(54) Title: REBAUDIOSIDE A COMPOSITIONS AND METHOD TO PURIFY REBAUDIOSIDE A. (54) Title: REBAUDIOSIDE A COMPOSITION AND METHOD FOR PURIFYING REBAUDIOSIDE A.

(57) Summary

Exemplary embodiments of this invention encompass a method of purifying a low purity steviol glycoside composition. In particular, this invention relates to a method of purifying low purity spheviolgiucoside compositions to obtain substantially pure steviolgiucoside compositions comprising about 95% or more steviolgiucosides and about 75% or more rebaudioside A with a single crystallization step.

(57) Abstract

Exemplary embodiments of this invention encompass a method for purifying a low purity steviol glycoside composition. In particular, this invention relates to a method for purifying low purity steviol glycoside compositions to obtain substantially puree steviol glycoside compositions comprising approximately 95% or greater steviol glycosides and approximately 75% or greater rebaudioside A with a single crystallization step.

REBAUDIOSIDE COMPOSITION AND METHOD TO PURIFY THE

REDUCED TO

FIELD OF THE INVENTION

This invention relates generally to methods for purifying low purity steviolglucoside compositions to obtain substantially pure steviolglucoside compositions in high yield. In another aspect, this invention relates to methods for purifying low purity rebaudioside A and low purity steviolglucoside compositions to obtain substantially pure steviolglucoside compositions having acceptable levels of rebaudioside A in high yield.

BACKGROUND OF THE INVENTION

Rebaudioside A is a high potency diterpenoid glucoside sweetener that has the chemical structure:

1)11

110

OR

<img file="MX2012007644A_D0001.tif" />

on

110.

Rebaudioside A is isolated and extracted, along with other steviolglucosides, from the Stevia rebaudiana (Bertoni) (Stevia) plant, which is grown commercially in Japan,

Singapore, Taiwan, Malaysia, South Korea, China, Israel,

India, Brazil, Australia, and Paraguay. It is an alternative non-caloric sweetener with functional and organoleptic properties superior to that of many high potency sweeteners. Processed forms of Stevia can be 3 to 300 times more potent than sugar; however, Stevia also has a bitter component. Of the four main diterpenoid glycoside sweeteners present in Stevia, rebaudioside A has been identified as the least bitter, and with the least persistent aftertaste. Bitterness is often considerably due to impurities in the extracts.

The current standards of the Joint FAO / WHO Expert Committee on Food Additives (JECFA) require that the total amount of steviolglucosides be purified at 95% or more. Steviolglucoside compositions that are currently only available have 80-92% steviolglucosides by weight - with the cost of these compositions increasing substantially when the purity levels of steviolglucosides and / or rebaudioside A are increased. Furthermore, these compositions generally fail to provide sufficient purities of rebaudioside A among steviolglucosides (eg, they are generally only ^ 80% rebaudioside A by weight).

Previously reported efforts to purify rebaudioside A from mixtures of rebaudioside A and stevioside require numerous repeated purification steps. The

US Patent No. 5,962,678 describes recrystallization of rebaudioside A using an anhydrous methanol solution to obtain a pure 80% rebaudioside A. By repeating the recrystallization with anhydrous methanol numerous times, the purity of rebaudioside A can be increased to more than 95%. North American Patent Publication No.

2006/0083838 describes the purification of rebaudioside A by re-crystallization with a solvent comprising ethanol and between 4 and 15% water. The Patent Application

Japanese No. 55-23756 describes a method of purifying rebaudioside A and stevioside by crystallizing from aqueous ethanol (&gt; 70%) to obtain a 80% pure rebaudioside.

North American Patent Publication No. 2007/0082103 describes a method to purify rebaudioside A by recrystallizing from aqueous ethanol, maintaining a two-stage recrystallization of crude rebaudioside (60%) resulting in the formation of rebaudioside&gt; 98% pure to a

97% yield. These prior art methods, however, do not provide a substantially pure steviolglucoside composition or a rebaudioside A composition of sufficient purity using only a single recrystallization step which is capable of meeting current JECFA standards.

Accordingly, there is a need for a simple, efficient, and inexpensive method of preparing substantially pure steviolglucoside and rebaudioside A compositions.

BRIEF DESCRIPTION OF THE INVENTION

Exemplary embodiments of the invention address the above identified need by providing methods for purifying a low purity steviolglucoside composition to obtain a substantially pure steviolglucoside composition having high purity with high yield.

In a particular embodiment, the method of purifying a low purity steviolglucoside composition comprises simple crystallization. In particular, the method comprises combining a low purity steviolglucoside A composition and an organic solvent to form a steviolglucoside solution, the low purity steviolglucoside composition comprises up to 92% total steviolglucosides on a dry basis, and therefore the crystallization of the low purity steviolglucoside solution in a single step a substantially pure steviolglucoside composition comprising rebaudioside A and one or more other steviolglucosides selected from the group consisting of rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside

E, rebaudioside F, stevioside, dulcoside A, rubusoside, and steviolbioside. Steviolglucosides are present in the composition of substantially pure steviolglucoside in an amount of 95% or more by weight on a dry basis. In one embodiment, rebaudioside A is present in the composition of substantially pure steviolglucoside in an amount of

5% or more by weight on a dry basis. The total mass yield of the substantially pure steviolglucoside composition is 25% or more by weight.

Other objects, aspects, and advantages of the invention will be apparent from the following detailed description, drawings, and claims. Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning that is commonly understood by someone of ordinary skill in the art to which this invention belongs. Although methods and compositions similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and compositions are described without proposing that any such methods and compositions limit the invention herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic representation of the formation and conversion of the polymorphic and amorphous forms of rebaudioside A in accordance with one embodiment of this invention.

DETAILED DESCRIPTION OF THE INVENTION

Steviolglucoside compositions can be used as high potency natural sweeteners. The rebaudioside

A is one of the steviolglucosides that can be found in varying amounts in steviolglucoside compositions.

When the amount of total steviolglucosides (including rebaudioside A) is increased, the cost of the composition is increased substantially as well. Similarly, when the amount of rebaudioside A in steviolglucoside compositions is increased, the cost of the composition is further increased.

In the North American Patent Application Serial No.

11 / 751,627, Applicants describe simple and inexpensive methods for preparing substantially pure rebaudioside A compositions for use as a high potency natural sweetener. The phrase "substantially pure rebaudioside A" was used to refer to compositions of rebaudioside A including at least about 85% by dry weight of rebaudioside A, at least about 90% by dry weight of rebaudioside A, from about 95% to about 98% by weight dry of rebaudioside A, and from about 99% to about 100% by weight dry of rebaudioside A.

Applicants have now discovered that the sweet taste and quality of these high potency natural sweetener compositions is impacted not only by the amount of rebaudioside A present in the composition, but also by the total combined amount of steviolglucosides present in the composition. Accordingly, there is a need to provide a method of preparing substantially pure steviolglucoside compositions in an economical manner. In particular, there is a need to provide a method of preparing substantially pure steviolglucoside compositions having a high purity of rebaudioside A in an economical manner.

Exemplary embodiments of this invention meet these needs by providing a method of purifying a low purity steviolglucoside composition to a substantially pure steviolglucoside composition. The constitution and performance of the resulting substantially pure steviolglucoside composition can be controlled through the appropriate selection of parameters such as the type and amount of organic solvent, the temperature of the solution, the temperature of precipitation, the mixing time, the time of precipitation, and seeding of the solution.

The method generally comprises the crystallization of a substantially pure steviolglucoside composition from a low purity steviolglucoside composition and an organic solvent in one step. Other exemplary embodiments of this invention encompass a substantially pure steviolglucoside composition comprising a high purity of rebaudioside A.

Method to Purify Steviolglucoside Compositions from

Low purity

Low purity steviolglucoside compositions that are commercially available comprise steviolglucosides in purities of up to about 92% by weight. When used herein, a crude steviolglucoside composition 'a low purity steviolglucoside composition are used interchangeably to refer to compositions comprising steviolglucosides in amounts of from about -80 to about 92% by weight.

In particular embodiments, the low purity steviolglucoside composition comprises up to about 60% rebaudioside A by weight on a dry basis. When used herein, a crude rebaudioside A composition or a low purity rebaudioside A composition are used interchangeably to refer to compositions comprising rebaudioside A in amounts of from about 15% to about 60% by weight, of about 30% up to about 60% by weight, or about 40% up to about 60% by weight. The remainder of the crude rebaudioside A composition generally comprises other steviolglucoside and impurities. In general, commercially available low purity rebaudioside A compositions contain from about 80% to about 92% steviolglucoside by weight.

It is also conceived that the compositions of low purity steviolglucoside and crude rebaudioside A in its natural form, when extracted from Stevia plants, can be purified using the methods provided herein.

In an exemplary embodiment for purifying a low purity steviolglucoside composition, the low purity steviolglucoside composition can be combined with an organic solvent to form a low purity steviolglucoside solution. The organic solvent optionally may further comprise water in an amount of up to about 25% by weight. For example, in particular embodiments, the organic solvent may further comprise water in an amount of from about 3% to about

20% by weight, from about 5% to about

15% by weight, from about 5% to about 10%, or any amount between them. Non-limiting examples of organic solvents include alcohol, acetone, acetonitrile, and ethyl acetate. Alcohol, when used herein, refers to any straight, branched, or cyclic alkyl, alkenyl, or alkynyl group;

substituted or unsubstituted, attached to at least one hydroxyl moiety. Non-limiting examples of alcohols include ethanol, methanol, isopropanol, 1-propanol, 1-butanol, 2butanol, tert-butanol, and isobutanol.

In an exemplary embodiment, the organic solvent comprises a mixture of water and at least one organic solvent.

In another embodiment, the at least one organic solvent comprises an alcohol, the alcohol comprises ethanol, methanol, or mixtures thereof. In exemplary embodiments where the at least one organic solvent comprises a mixture of ethanol and methanol, the ethanol and methanol can be combined in the organic solvent in a weight ratio ranging from about 20 parts to about 1 part of ethanol to about 1 part of methanol. In another exemplary embodiment, ethanol and methanol can be combined in the organic solvent in a weight ratio ranging from about 3 parts to about 1 part ethanol to about part of methanol.

In an exemplary embodiment, the low purity steviolglucoside solution comprises the organic solvent and the composition of low purity steviolglucoside in a weight ratio ranging from about 10 to about 4 parts of organic solvent to about 1 part of low purity steviolglucoside . In another exemplary embodiment, the low purity steviolglucoside solution comprises the organic solvent and the composition of low purity steviolglucoside in a weight ratio ranging from about 5 to about 3 parts of organic solvent to about 1 part of low purity steviolglucoside .

The method for preparing the low purity steviolglucoside solution comprises the organic solvent and the composition of the low purity steviolglucoside can be carried out at any suitable temperature. Such temperatures generally can range from about 20 ° C to about 85 ° C. For example, in one embodiment the low purity steviolglucoside solution can be prepared at about room temperature (about 2225 ° C). In another embodiment, the low purity steviolglucoside solution can be prepared by heating the steviolglucoside solution to a temperature in a range of from about 20 ° C to about 70 ° C, from about 20 ° C to about 60 ° C, from about 20 ° C to about 40 ° C, or from about 40 ° C to about 60 ° C. In yet another embodiment, the low purity steviolglucoside solution can be prepared by heating the low purity steviolglucoside solution to approximately reflux temperature (approximately 85 ° C). The step of preparing (or mixing) the low purity steviolglucoside solution can be carried out for a suitable length of time to substantially suspend the low purity steviolglucoside in the organic solvent. For example, the low purity steviolglucoside solution can be prepared by mixing the low purity steviolglucoside solution for about 5 minutes to about 60 minutes, for about 5 to about 30 minutes, for about 10 to about 15 minutes, or for any duration of time between them.

The method of purifying the low purity steviolglucoside composition further comprises crystallizing from the low purity steviolglucoside solution in a single step a substantially pure steviolglucoside composition comprising rebaudioside A. In particular embodiments, the crystallization of the low steviolglucoside solution Purity may further comprise cooling the low purity steviolglucoside solution.

Generally, the low purity steviolglucoside solution can be cooled to a temperature suitable for its precipitation (precipitation temperature) of the steviolglucoside and rebaudioside A from the low purity steviolglucoside solution. Non-limiting examples of such precipitation temperatures may be in a range from about 4 ° C to about 35 ° C, from about 15 ° C to about 25 ° C, or any temperature between them. Crystallization of the low purity steviolglucoside solution can allow it to be carried out for a sufficient length of time (precipitation time or cooling time) to obtain a desirable yield of the substantially pure steviolglucoside composition of the low purity steviolglucoside solution. . For example, in particular embodiments, crystallization of the low purity steviolglucoside solution can continue from about 0.5 hours to about 120 hours (5 days), from about hours to about 96 hours (4 days), from about 24 hours (1 day) up to approximately 72 hours (3 days), for approximately 48 hours (2 days), or for any length of time between them.

After crystallization of the low purity steviolglucoside solution, a substantially pure steviolglucoside composition comprising rebaudioside A can be obtained. A substantially pure steviolglucoside composition is used herein to refer to compositions comprising approximately 95% or more by weight (on a dry basis) of steviolglucosides. Steviolglucosides can include rebaudioside A in combination with one or more other steviolglucosides selected from the group consisting of rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside

E, rebaudioside F, stevioside, steviolbioside, rubusoside, and dulcoside. In particular embodiments, the composition of substantially pure steviolglucoside comprises rebaudioside

A in an amount of about 7 0% or more by weight. In particular embodiments, the composition of substantially pure steviolglucoside further comprises rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, stevioside, and steviolbioside. The generally substantially pure steviolglucoside composition comprises rebaudioside A in an amount of about 70% or more by weight (on a dry basis), about 75% or more by weight, about 80% or more by weight, about 85% or more by weight, about 90% or more by weight, about 95% or more by weight, or about 97% or more by weight.

In particular embodiments , the total yield of the composition of substantially pure steviolglucoside and rebaudioside A can be about 25% or more and about 20% or more, respectively. Yield is generally used herein to refer to the mass obtained in relation to the initial mass. Accordingly, the yield of steviolglucosides can be determined by comparing the mass of the nine substantially pure steviolglucosides in composition to the initial mass of the nine steviolglucosides in the low purity steviolglucoside. Similarly, the yield of the rebaudioside A composition can be determined by comparing the mass of rebaudioside A present in the substantially pure steviolglucoside composition compared to the initial mass of the rebaudioside A present in the low purity steviolglucoside. In exemplary embodiments, the total yield of the composition of substantially pure steviolglucoside and rebaudioside A can be about 15% or more, about 40% or more, or about 60% or more.

In an exemplary embodiment, the method for purifying the low purity steviolglucoside composition may optionally further comprise seeding the low purity steviolglucoside solution after crystallization of the low purity steviolglucoside solution begins. Seeding can generally be done at the same temperature at which crystallization is allowed to continue. For example, in particular modalities the sowing will be carried out at temperatures in the range of approximately 18 ° C to approximately 35 ° C. Seeding of the low purity steviolglucoside solution can generally be accomplished by adding substantially pure crystals of rebaudioside A to the solution of low purity steviolglucoside in an amount sufficient to promote the precipitation of rebaudioside A and other steviolglucosides. An amount sufficient to promote precipitation in general may comprise a composition of substantially pure rebaudioside A in an amount from about 0.0001% to about 1% by weight of the low purity steviolglucoside solution, from about 0.01% to about 1% by weight , or any amount between them.

In another exemplary embodiment, the method further comprises separating and washing the substantially pure steviolglucoside composition after crystallization. The substantially pure steviolglucoside composition can be separated from its supernatant (organic solvent and impurities) by a variety of solid-liquid separation techniques using centrifugal force, including, without limitation, vertical and horizontal perforated basket centrifuge, centrifuge solids bowl, decanter centrifuge, peeler type centrifuge, impeller type centrifuge, Heinkel type centrifuge, disk stack centrifuge and vortex separation. Additionally, separation can be improved by any pressure, vacuum, or gravity filtration method, including without limitation the use of belts, drums, nutsche type, foil, plate, Rosenmund type, flare type, and bag and filter filters press. The operation of the solid-liquid separation device can be continuous, semi-continuous or in batch mode. The substantially pure steviolglucoside composition can also be washed in the separation device using various organic solvents and mixtures thereof and can be partially or completely dried in the separation device using any number of gases, including, without limitation, nitrogen or argon, to evaporate the residual liquid solvent. The substantially pure steviolglucoside composition can be removed automatically or manually from the separation device using liquids, gases, or mechanical means either by dissolving the solid or maintaining the solid form.

In yet another exemplary embodiment, the method further comprises drying the composition of substantially pure steviolglucoside. Suitable methods for drying such compositions are known to those skilled in the art and include, but are not limited to, the use of a rotary vacuum drier, fluidized bed drier, continuous rotary drier, plate drier, tray drier, Nauta type dryer, spray dryer, instant dryer, micron dryer, tray dryer, high and low speed paddle dryer and microwave dryer.

In an exemplary embodiment, the substantially pure steviolglucoside composition is dried using a nitrogen or argon purge to remove the residual solvent at a temperature in a range of from about 4 0 ° C to about 60 ° C

<td colspan="2">for a period</td><td>time of about 5</td><td>hours</td><td>until</td>

<td>approximately</td><td> 5</td><td>days of about 1</td><td>day</td><td>until</td>

<td>approximately</td><td> 4</td><td>days of approximately 2</td><td>days</td><td>until</td>

<td>approximately</td><td> 3</td><td>days, or for any duration</td><td>of</td><td>weather</td>

between them.

If further purification is desired, the method for purifying the low purity steviolglucoside composition described herein can be repeated or the substantially pure steviolglucoside composition can be further purified using an alternative purification method, such as column chromatography.

Purity, when used herein with respect to the composition of steviolglucoside, represents the percentage by weight of the selected steviolglucosides (eg, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside

D, rebaudioside E, rebaudioside F, stevioside, dulcoside, rubusoside, and steviolbioside) in the composition of steviolglucoside. Purity, as used herein with respect to rebaudioside A, represents the weight percent of rebaudioside A in the low purity steviolglucoside composition or substantially pure steviolglucoside composition. Accordingly, the substantially pure steviolglucoside compositions provided herein comprise rebaudioside A in a particular purity and the combined total of selected steviolglucosides in a particular purity. The remainder of the composition may comprise impurities or other steviolglucosides present in amounts which are below detectable limits. Impurities and other steviolglucosides present in amounts less than about 0.1% of the total composition by weight are considered below the detectable limit.

The selected steviolglucosides generally comprise one or more of the steviolglucosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, stevioside, dulcoside A, rubusoside, and steviolbioside. For example, in one embodiment the total amount of the combined steviolglucosides is based on the combination of nine steviolglucosides - i.e., Rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, stevioside, dulcoside A, rubusoside, and steviolioside. In another embodiment the combined total of steviolglucosides is based on the combination of seven steviolglucosides - i.e., Rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside

D, rebaudioside F, stevioside, and steviolbioside. Those skilled in the art will appreciate that the steviolglucosides included within the combined total of steviolglucoside can be selected based on particular dietary regulations or recommendations established by various government institutions (eg, JECFA, United States Food and Drug Administration, etc.) .

The purity of the composition can be measured using methods known to those of ordinary skill in the art. Such a method includes high performance liquid chromatography (HPLC). Those of ordinary skill in the art should also appreciate that moisture in the sample can affect the accuracy of purity measurements. Accordingly, the composition should be dried substantially when the purity is measured. When used herein, a substantially dry composition and on a dry basis are used interchangeably and can comprise up to about 10% by weight moisture.

Polymorphic and Amorphous Forms of Rebaudioside A

As the Requesters have previously described in

US Patent Application Serial No. 11 / 751,627, the purification of the rebaudioside A compositions can result in the formation of various amorphous and polymorphic forms of rebaudioside A. The Applicants previously identified at least three different rebaudioside A polymorphs: Form 1: a rebaudioside hydrate A; Shape

2: an anhydrous rebaudioside A, and Form 3: a rebaudioside A solvate. In addition to the at least three polymorphic forms of rebaudioside A, Applicants have also identified an amorphous form of rebaudioside A, Form 4.

Polymorphism is defined as the ability of a substance to exist as two or more crystalline states that have different arrangements and / or conformations of the molecules in the crystal lattice. Approximately 30% of organic compounds are believed to exhibit polymorphism (Zell, et al., Tetrahedron 56 (36) 6603-16 (2000)). Polymorphism is important in the formulation of drugs, pigments and dyes, sweeteners, explosives, and agrochemicals. Polymorphism can cause physical properties such as density, melting point, and dissolution ratio in exchange.

Amorphous, when used herein, describes a non-crystalline solid material. The amorphous form of rebaudioside

A (Form 4) has an improved dissolution ratio compared to the polymorphic forms of rebaudioside A (Forms 1, 2, or 3). Those of ordinary skill in the art should appreciate that the dissolution ratio of a sweetening composition may be important in formulating solid and liquid sweetening compositions, non-limiting examples of which include chewing gum, baked goods, and beverages.

The material properties of the three polymorphs of rebaudioside A and the amorphous form of rebaudioside A are summarized in the following table:

Table 1: Polymorphic and Amorphous Forms of Rebaudioside A

<td></td><td>Form 1 Polymorphous</td><td>Form 2 Polymorphous</td><td>Form 3 Polymorphous</td><td>Form 4 Amorphous</td>

<td>Dissolution ratio</td><td>Very low &lt;002%</td><td>Intermediate</td><td>High (&lt;30% in</td><td>High (&lt;35% in</td>

<td>in H2O at 25 ° C</td><td>in 60 minutes)</td><td>(&lt;30% in 5 minutes)</td><td>5 minutes)</td><td>5 minutes)</td>

<td>Alcohol content</td><td> &lt; 0.5 %</td><td> &lt; 1 %</td><td> 1-3 %</td><td> &lt; 0.05 %</td>

<td>Moisture content</td><td> &gt;5%</td><td> &lt; 1 %</td><td> &lt;3 %</td><td> &lt;6%</td>

The properties of the materials described above are only illustrative of the particular embodiments of the amorphous and polymorphic forms of rebaudioside A. Those of ordinary skill in the art should appreciate that the anhydrous rebaudioside A polymorph (Form 2), polymorph of the rebaudioside solvate A, and amorphous rebaudioside A are hygroscopic and can absorb moisture in an amount of up to about 10% by weight on a dry basis. Without wishing to be bound by any theory, the methods described herein are believed to generally produce the anhydrous rebaudioside A polymorph (Form 2) and / or amorphous rebaudioside A composition (Form 4).

As illustrated in Fig. 1, the type of polymorphic or amorphous form produced may depend on factors such as the composition of the aqueous organic solution, the temperature of the crystallization step, and the temperature during the drying step. Without wishing to be bound by any theory, Form 1 and Form 3 are believed to form during the single crystallization stage although Form 2 is believed to form during the drying stage after conversion of Form 1 and

Form 3.

Low temperatures during the crystallization step, in the range of about 20 ° C to about 50 ° C, and a low ratio of water to organic solvent in aqueous organic solvent results in the formation of Form 3. High temperatures during the crystallization stage, in the range of about 50 "C to about 80 ° C, and a high ratio of water to organic solvent in aqueous organic solvent results in the formation of the

Form 1. Form 1 can be converted to Form 3 by suspending an anhydrous solvent at approximately room temperature for approximately 2 to approximately 16 hours or by suspending an anhydrous solvent at approximately reflux temperature for approximately 0.5 to approximately 3 hours. The

Form 3 can be converted to Form 1 by suspending the polymorph in water at approximately room temperature for approximately 16 hours or at approximately reflux temperature for approximately 2 to approximately 3 hours. Form 3 can become the

Form 2 during the drying process; however, increasing either the drying temperature above about 70 ° C or the drying time of a substantially pure Rebaudioside A composition may result in decomposition of Rebaudioside A and the remaining Rebaudioside B impurity. in the composition of substantially pure rebaudioside A. Form 2 can be converted to Form 1 with the addition of water.

Form 4 can be obtained during the initial purification of rebaudioside A or directly from any single polymorph or combination of polymorphs using methods well known to those of ordinary skill in the art. In addition, Form 4 can be obtained from a low purity rebaudioside A composition or a substantially pure rebaudioside A composition obtained through purification means other than those described herein above. Non-limiting examples of methods for preparing amorphous forms of rebaudioside A include ball milling, precipitation, lyophilization, freeze-grinding, and spray drying of a rebaudioside A composition.

The present invention is further illustrated by the following examples, which should not be construed in any way as limitations imposed on the scope thereof. On the contrary, it is clearly understood that the resource may have various other modalities, modifications, and equivalents thereof, after reading the description herein, may be suggested by themselves to those of skill in the art without departing from the spirit of the present invention and / or the scope of the appended claims. Unless otherwise specified, percentages (% s) are by weight.

EXAMPLES

The purity of the rebaudioside A compositions described in the examples hereinbelow were determined using HPLC. Methods for performing HPLC analysis are well known to those of ordinary skill in the art. Briefly described, HPLC analysis was performed using ZORBAX NH column₂(150 x 4.6 mm, 5 pm) at a temperature of 30 ° C. The mobile phase comprised a 20% buffer solution (0.0125% acetic acid and 0.0125% ammonium acetate) and 80% acetonitrile at a flow rate of 1.5 mL / min. 12 pL of each sample was injected in duplicate and the sample was analyzed using a UV detector at 210nm (4nm bandwidth) with a reference of 260nm (100nm bandwidth). HPLC analysis required a run time ranging from 40 to min.

A buffer solution of 0.0125% acetic acid and 0.0125% ammonium acetate was prepared by dissolving 0.125 g of ammonium acetate and 125 pL of glacial acetic acid in one liter of water. The retention time of rebaudioside B was adjusted by varying the ratio of ammonium acetate to acetic acid, maintaining a total of 0.025% of both combined. Increasing the amount of acetic acid decreased the retention time of rebaudioside B.

The mobile phase was prepared by mixing the buffer with acetonitrile to achieve a retention time of rebaudioside A of 7.0 ± 0.5 min. Initially, this was approximately 20% buffer (200 mL buffer and 800 mL acetonitrile). Increasing the amount of acetonitrile from 1 to 2% increased the retention time of rebaudioside A by approximately one minute.

A diluent solution was prepared by mixing 750 mL of acetonitrile and 250 mL of the buffer solution. Rebaudioside A standards were prepared by diluting 20.0 ± 0.5 mg (closest to 0.1 mg) of the rebaudioside A standard with 4 mL of the diluent solution to make a standard solution of approximately 5000 mg / L. Rebaudioside A standard solution was injected at 10.8, 11.4,

12.6 and 13.2 pL. The moisture content was measured by Karl Fischer analysis each time a standard was prepared and corrections were made based on the purity of the solvent according to the certificate of analysis. Alternatively, rebaudioside A standards were prepared by diluting individual samples of 18, 19, 21, and 22 (each ± ​​0.2) mg of rebaudioside A standard with 4 mL of the diluent solution (correcting humidity and purity). The individually prepared samples were injected at the same level as the samples (12 pL).

Stevioside standards were prepared by diluting 12.5 + 20 0.5 mg (recorded closest to 0.1 mg) of the stevioside standard with 5 mL of the diluent solution to make a standard solution of approximately 2500 mg / L (stock A) (correcting humidity and purity). The stevioside standard was then diluted using one mL of stock A to ten mL of diluent to produce a standard of 250 mg / L (stock B), and stock standards were diluted to final concentrations ranging from 2.5 to 50 mg / L.

Samples of the rebaudioside A compositions were prepared by diluting 125 ± 2 mg (recorded closest to 0.1 mg) of the rebaudioside A composition with 25 mL of the diluent solution to make a sample solution of approximately 5000 mg / L (correcting the humidity). If the samples were not analyzed immediately, they were stored without vacuum, under nitrogen, and dried.

The following table provides a guide to the retention times (RT) of rebaudioside A and other steviolglucosides. However, those of ordinary skill in the art should appreciate that retention times can be modified as necessary.

Table 2: HPLC Retention Guidelines

<td>Compound</td><td>RT (min)</td>

<td>Stevioside</td><td> 4.53</td>

<td>Rebaudioside C</td><td> 5.21</td>

<td>Rebaudioside F</td><td> 5.62</td>

<td>Rebaudioside A</td><td> 7.07</td>

<td>Rebaudioside D</td><td> 15.79</td>

<td>Steviolbioside</td><td> 18.35</td>

<td>Rebaudioside B</td><td> 35.83</td>

Low purity rebaudioside * compositions (having 41-62% by weight of rebaudioside A) were purified using a single crystallization step to obtain a substantially pure steviolglucoside composition having high purity of rebaudioside A and high yield.

Five different low purity rebaudioside A compositions having purities of rebaudioside A of from about 41% to about 62% by weight and total steviolglucoside content from about 88% to 92% by weight (based on analytical HPLC analysis) were used. The total amount of steviolglucosides in these examples was based on the combined amounts of rebaudioside A, rebaudioside

B, Rebaudioside C, Rebaudioside D, Rebaudioside F, Stevioside, and Steviolbioside.

Two of the low purity rebaudioside A compositions were obtained from Shenzhen (Shenzhen NII Natural Food

Ingredients C, Ltd., China). Two of the low purity rebaudioside A compositions were obtained from Shandong (Shandong Huaxian Stevia Limited-Liability Company, China). In addition, another of the Hailin low purity rebaudioside A compositions was obtained (Hailin Farm, China). The amount of rebaudioside A and total steviolglucoside content in each of these compositions is set forth in Table 3 below.

Table 3: Summary of Rebaudioside A Compositions of

Low purity

<td>Provider</td><td>Amount of Rebaudioside A (% in weigh)</td><td>Amount of Seven Steviolglucosides (% in weigh)</td><td>Number of Nine Steviolglucosides (% in weigh)</td>

<td>Shenzhen (41%)</td><td> 41 %</td><td> 90%</td><td> 93 %</td>

<td>Shenzhen (62%)</td><td> 62%</td><td> 89%</td><td> 92%</td>

<td>Shandong (42%)</td><td> 42%</td><td> 88%</td><td> 92%</td>

<td>Shandong (51%)</td><td> 51 %</td><td> 92%</td><td> 94%</td>

<td>Hailin</td><td> 56%</td><td> 88%</td><td> 91 %</td>

Set A of Examples

The experimental crystallization conditions are summarized in Tables 4 and 5 and the results are summarized in

Table 6.

Low purity rebaudioside A compositions (50

g) combined with an organic solvent during stirring at a mixing temperature for a desired period of time to obtain a substantially clear solution. The solution was allowed to cool to room temperature (22 ° C) and optionally seeded with substantially pure rebaudioside A (&gt; 99.0%). In general, precipitation was almost instantaneous after cooling to room temperature. The precipitate was filtered and washed with either room temperature or cold ethanol (95%, 22 ° C or 4 ° C) and dried in a vacuum drying oven at 50-60 ° C for 2 to 4 days.

The amount of the resulting precipitate was determined and the precipitate was subjected to analytical HPLC (Agilent 1100) using the universal CAD (charged aerosol) detector to obtain a quantitative estimate of both the amount of rebaudioside A and the total amount of seven steviolglucosides (including rebaudioside A, rebaudioside B, rebaudioside C, stevioside, dulcoside A, rubusoside and

Steviolbioside) and nine steviolglucosides (including rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside

F, stevioside, dulcoside A, rubusoside and Steviolbioside). The total mass yield and yield of total rebaudioside A (both as a whole and relative to the total amount of rebaudioside A present in the low purity rebaudioside A compositions) were then determined. Examples numbers 6, 7, 10, 11, 12, 15, 16, 25, 26, 27, 31, 32,

36, and 37 are described in more detail below.

Table 4: Summary of Experimental Conditions

<td>Exp. No.</td><td>System Solvent*</td><td>Solvent (mL)</td><td>Mix Temperature (° C)</td><td>Mixing Time (min.)</td>

<td> 1</td><td>TO</td><td> 250</td><td> 55</td><td> 10</td>

<td> 2</td><td>B</td><td> 250</td><td> 50</td><td> 10</td>

<td> 3</td><td>C</td><td> 400</td><td> 50</td><td> 10</td>

<td> 4</td><td>D</td><td> 400</td><td> 55-59</td><td> 8</td>

<td> 5</td><td>AND</td><td> 300</td><td> 52-56</td><td> 8</td>

<td> 6</td><td>D</td><td> 300</td><td> 52</td><td> 6</td>

<td> 7</td><td>D</td><td> 200</td><td> 57</td><td> 13</td>

<td> 8</td><td>F</td><td> 300</td><td> 52</td><td> 10</td>

<td> 9</td><td>D</td><td> 200</td><td> 35-40</td><td> 30</td>

<td> 10</td><td>G *</td><td> 210</td><td> 35.40</td><td> 30</td>

<td> 11</td><td>G *</td><td> 210</td><td> 85</td><td> 5-6</td>

<td> 12</td><td>TO</td><td> 250</td><td> 50</td><td> 10</td>

<td> 13</td><td>B</td><td> 250</td><td> 50</td><td> 10</td>

<td> 14</td><td>C</td><td> 400</td><td> 50</td><td> 10</td>

<td> 15</td><td>D</td><td> 400</td><td> 53</td><td> 21</td>

<td> 16</td><td>D</td><td> 250</td><td> 22</td><td> 60</td>

<td> 17</td><td>TO</td><td> 250</td><td> 50</td><td> 10</td>

<td> 18</td><td>B</td><td> 250</td><td> 50</td><td> 10</td>

<td> 19</td><td>C</td><td> 400</td><td> 50</td><td> 10</td>

<td> 20</td><td>D</td><td> 400</td><td> 53-57</td><td> 16</td>

<td> 21</td><td>AND</td><td> 300</td><td> 52</td><td> 7</td>

<td> 22</td><td>D</td><td> 300</td><td> 53</td><td> 15</td>

<td> 23</td><td>D</td><td> 200</td><td> 57</td><td> 10</td>

<td> 24</td><td>F</td><td> 300</td><td> 53</td><td> 10</td>

<td> 25</td><td>D</td><td> 200</td><td> 35-40</td><td> 30</td>

<td> 26</td><td>G *</td><td> 210</td><td> 35-40</td><td> 30</td>

<td> 27</td><td>G *</td><td> 210</td><td> 85</td><td> 5-6</td>

<td> 28</td><td>TO</td><td> 250</td><td> 50</td><td> 10</td>

<td> 29</td><td>B</td><td> 250</td><td> 50</td><td> 10</td>

<td> 30</td><td>C</td><td> 500</td><td> 50</td><td> 15</td>

<td> 31</td><td>D</td><td> 400</td><td> 56</td><td> 8</td>

<td> 32</td><td>G</td><td> 210</td><td> 78</td><td> 5-6</td>

<td> 33</td><td>TO</td><td> 250</td><td> 50</td><td> 10</td>

<td> 34</td><td>B</td><td> 250</td><td> 50</td><td> 10</td>

<td> 35</td><td>C</td><td> 400</td><td> 50</td><td> 10</td>

<td> 36</td><td>D</td><td> 400</td><td> 56</td><td> 7.5</td>

<td> 37</td><td>H</td><td> 265</td><td> 22</td><td> 30</td>

* Denotes rebaudioside A methods in which the pure low purity (&gt; 99.0%) was seeded with the rebaudioside in a quantity of

A from about 0.0001% to about 0.001% by weight of the low purity rebaudioside A solution.

Table 5: Summary of Organic Solvent Systems

<td>Method</td><td>Solvent System</td>

<td>TO</td><td>4 parts ethanol (95%): 1 part methanol</td>

<td>B</td><td>1.5 parts ethanol (95%): 1 part methanol</td>

<td>C</td><td>methanol (100%)</td>

<td>D</td><td>ethanol (95%)</td>

<td>AND</td><td>methanol (95%)</td>

<td>F</td><td>extraction of ethyl acetate + ethanol (95%)</td>

<td>G</td><td>20 parts ethanol (95%): 1 part water</td>

<td>H</td><td>50 parts ethanol (95%): 3 parts water</td>

Table 6: Summary of Experimental Results

<td>Exp. No.</td><td>performance Total (mass%)</td><td>performance Total Reb A (% by mass)</td><td>performance from Reb A on Relation to Reb raw (mass%)</td><td>Total 7 Steviolglucosides (% by mass)</td><td>Total 9 Steviolglucosides (% by mass)</td><td>Reb A (mass%)</td>

<td> 1</td><td> 75.30</td><td> 31.63</td><td> 77</td><td> 91</td><td> 94</td><td> 42</td>

<td> 2</td><td> 80.20</td><td> 34.49</td><td> 84</td><td> 93</td><td> 95</td><td> 43</td>

<td> 3</td><td> 56.70</td><td> 25.52</td><td> 62</td><td> 95</td><td> 97</td><td> 45</td>

<td> 4</td><td> 24.60</td><td> 18.45</td><td> 45</td><td> 95</td><td> 97</td><td> 75</td>

<td> 5</td><td> 73.85</td><td> 32.49</td><td> 79</td><td> 92</td><td> 95</td><td> 44</td>

<td> 6</td><td> 34.90</td><td> 28.27</td><td> 69</td><td> 93</td><td> 95</td><td> 81</td>

<td> 7</td><td> 36.34</td><td> 28.71</td><td> 70</td><td> 94</td><td> 96</td><td> 79</td>

<td> 8</td><td> 35.91</td><td> 25.86</td><td> 63</td><td> 92</td><td> 95</td><td> 72</td>

<td> 9</td><td> 34.52</td><td> 27.27</td><td> 67</td><td> 93</td><td> 96</td><td> 79</td>

<td> 10</td><td> 24.60</td><td> 23.86</td><td> 58</td><td> 96</td><td> 98</td><td> 97</td>

<td> 11</td><td> 25.94</td><td> 23.35</td><td> 57</td><td> 96</td><td> 98</td><td> 90</td>

<td> 12</td><td> 59.17</td><td> 47.93</td><td> 77</td><td> 93</td><td> 97</td><td> 81</td>

<td> 13</td><td> 73.50</td><td> 49.25</td><td> 79</td><td> 93</td><td> 96</td><td> 67</td>

<td> 14</td><td> 66.20</td><td> 47.66</td><td> 77</td><td> 93</td><td> 96</td><td> 72</td>

<td> 15</td><td> 53.87</td><td> 47.94</td><td> 77</td><td> 94</td><td> 97</td><td> 89</td>

<td> 16</td><td> 53.20</td><td> 49.48</td><td> 80</td><td> 95</td><td> 98</td><td> 93</td>

<td> 17</td><td> 68.80</td><td> 31.65</td><td> 75</td><td> 91</td><td> 95</td><td> 46</td>

<td> 18</td><td> 73.60</td><td> 33.12</td><td> 79</td><td> 92</td><td> 95</td><td> 45</td>

<td> 19</td><td> 59.60</td><td> 27.42</td><td> 65</td><td> 98</td><td> 98</td><td> 46</td>

<td> 20</td><td> 31.67</td><td> 22.49</td><td> 54</td><td> 91</td><td> 95</td><td> 71</td>

<td> 21</td><td> 67.47</td><td> 25.14</td><td> 60</td><td> 86</td><td> 91</td><td> 39</td>

<td> 22</td><td> 40.04</td><td> 26.83</td><td> 64</td><td> 89</td><td> 94</td><td> 67</td>

<td> 23</td><td> 42.73</td><td> 27.77</td><td> 66</td><td> 90</td><td> 94</td><td> 65</td>

<td> 24</td><td> 37.67</td><td> 27.88</td><td> 66</td><td> 90</td><td> 94</td><td> 74</td>

<td> 25</td><td> 47.00</td><td> 28.67</td><td> 68</td><td> 89</td><td> 94</td><td> 61</td>

<td> 26</td><td> 28.60</td><td> 23.74</td><td> 57</td><td> 92</td><td> 96</td><td> 83</td>

<td> 27</td><td> 23.12</td><td> 21.04</td><td> 50</td><td> 92</td><td> 97</td><td> 91</td>

<td> 28</td><td> 69.66</td><td> 38.31</td><td> 75</td><td> 94</td><td> 97</td><td> 55</td>

<td> 29</td><td> 77.10</td><td> 41.63</td><td> 82</td><td> 95</td><td> 97</td><td> 54</td>

<td> 30</td><td> 52.50</td><td> 28.88</td><td> 57</td><td> 98</td><td> 98</td><td> 55</td>

<td> 31</td><td> 53.18</td><td> 42.01</td><td> 82</td><td> 95</td><td> 97</td><td> 79</td>

<td> 32</td><td> 35.82</td><td> 34.39</td><td> 67</td><td> 97</td><td> 99</td><td> 96</td>

<td> 33</td><td> 64.60</td><td> 28.42</td><td> 62</td><td> 87</td><td> 91</td><td> 44</td>

<td> 34</td><td> 71.20</td><td> 32.75</td><td> 71</td><td> 88</td><td> 92</td><td> 46</td>

<td> 35</td><td> 67.60</td><td> 34.48</td><td> 75</td><td> 93</td><td> 96</td><td> 51</td>

<td> 36</td><td> 37.28</td><td> 28.71</td><td> 62</td><td> 92</td><td> 96</td><td> 77</td>

<td> 37</td><td> 26.40</td><td> 23.76</td><td> 52</td><td> 93</td><td> 96</td><td> 90</td>

Example 6

The composition of low purity rebaudioside A (50 g,

41%) was combined with 300 mL of ethanol (95%) and heated at 52 ° C for 6 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) for three days while stirring at the same time. The precipitate was filtered, washed with ethanol (5-10 mL 95%, 22 ° C), and dried for two days in a vacuum-desiccator oven (50 ° C) to obtain 17.45 g of a steviolglucoside composition ( 34.90% yield) having 81% rebaudioside A and 95% steviolglucosides in total.

Example 7

The composition of low purity rebaudioside A (50 g,

41%) was combined with 200 mL of ethanol (95%) and heated at 57 ° C for 13 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) for four days while stirring at the same time. The precipitate was filtered, washed with ethanol (5-10 mL 95%, 22 ° C), and dried for two days in a vacuum-desiccator oven (50 ° C) to obtain 18.17 g of a steviolglucoside composition ( 36.34% yield) having 79% rebaudioside A and 96% steviolglucosides in total.

Example 10

The low purity rebaudioside A composition (50 g, 41%) was combined with 200 mL of ethanol (95%) and 10 mL of water and heated at 35-40 ° C for 30 minutes while stirring at the same time. The solution was cooled and then seeded with substantially pure rebaudioside A (+ 99.0%) at room temperature (22 ° C) and allowed to settle one day at room temperature, for one day at 4 ° C, and again one day at room temperature. The precipitate was filtered, washed with ethanol (3 x 10 mL 95%, 4 ° C), and dried two days in a vacuum-desiccator oven (50 ° C) to obtain 12.3 g of a steviolglucoside composition (24.6 Yield%) having 97% rebaudioside A and 98% steviolglucosides in total.

Example 11

The composition of low purity rebaudioside A (50 g,

41%) was combined with 200 mL of ethanol (95%) and 10 mL of water, conditioned with a cold water reflux condenser, and heated in a water bath at 85 ° C for 5-6 minutes with stirring. weather. The solution was cooled and then seeded with substantially pure rebaudioside A (+ 99.0%) at room temperature (22 ° C) and left at room temperature for three days while stirring at the same time. The precipitate was filtered and washed with ethanol (3x10 mL 95%, 4 ° C) on the fourth day and dried for two days in a vacuum-desiccator oven (60 ° C) to obtain 12.97 g of a steviolglucoside composition ( 25.94% yield) having 90% rebaudioside A and 98% steviolglucosides in total.

Example 12

The composition of low purity rebaudioside A (50 g,

62%) was combined with 200 mL of ethanol (95%) and 50 mL of methanol (100%) and heated at 50 ° C for 10 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) while stirring overnight. The precipitate was filtered, washed with ethanol (2x30 mL 95%, 22 ° C) and dried for two days in a vacuum-desiccator oven (50 ° C) to obtain 29.58 g of a steviolglucoside composition (59.17% of yield) having 81% rebaudioside A and 97% steviolglucosides in total.

Example 15

The low purity rebaudioside A composition (50 g, 62%) was combined with 400 mL of ethanol (95%) and heated at 53 ° C for 21 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) while stirring overnight. The precipitate was filtered, washed with ethanol (10-15 mL 95%, 22 ° C) and dried two days in a vacuum-desiccator oven (50 ° C) to obtain 26.93 g of a steviolglucoside composition (53.87% yield) having 89% rebaudioside A and 97% steviolglucosides in total.

Example 16

The low purity rebaudioside A composition (50 g, 62%) was combined with 250 mL of ethanol (95%) at room temperature (22 ° C) for 60 minutes while stirring at the same time. The solution was left at room temperature while stirring overnight. The precipitate was filtered, washed with ethanol (10-15 mL 95%, 22 ° C), and dried two days in a vacuum-desiccator oven (60 ° C) to obtain 26.6 g of a steviolglucoside composition (53.2 Yield%) having 93% rebaudioside A and 98% steviolglucosides in total.

Example 24

The composition of low purity rebaudioside A (50 g,

42%) was combined with 150 mL of ethyl acetate at room temperature (22 ° C) for 15 minutes while stirring at the same time. The precipitate was filtered and then re-suspended in 300 mL of ethanol (95%) and heated at 52 ° C for 10 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) for four days while stirring at the same time.

The precipitate was filtered, washed with ethanol (5-10 mL 95%, ° C), and dried two days in a vacuum-desiccator oven (50 ° C) to obtain 18.83 g of a steviolglucoside composition (37.67% yield) having 74% rebaudioside A and 94% steviolglucosides in total.

Example 26

The low purity rebaudioside A composition (50 g, 42%) was combined with 200 mL of ethanoi (95%) and 10 mL of water and heated at 35-40 ° C for 30 minutes while stirring at the same time. The solution was cooled and then seeded with substantially pure rebaudioside A (+ 99.0%) at room temperature (22 ° C) and left intact at room temperature for one day and at 4 ° C for one day. The precipitate was filtered, washed with ethanol (3x10 mL 95%, 4 ° C), and dried two days in a vacuum-desiccator oven (50 ° C) to obtain 14.3 g of a steviolglucoside composition (28.6% of yield) having 97% rebaudioside A and 98% steviolglucosides in total.

Example 27

The composition of low purity rebaudioside A (50 g,

42%) was combined with 200 mL of ethanol (95%) and 10 mL of water, conditioned with a cold water reflux condenser, and heated in a water bath at 85 ° C for 5-6 minutes, stirring therein. weather. The solution was cooled and then seeded with substantially pure rebaudioside A (+ 99.0%) at room temperature (22 ° C) and left at room temperature for three days while stirring at the same time. The precipitate was filtered and washed with ethanol (3x10 mL 95%, 4 ° C) on the fourth day and dried for two days in a vacuum-desiccator (60 ° C) to obtain 11.56 g of a steviolglucoside composition ( 23.12% yield) having 91% rebaudioside A and 97% steviolglucosides in total.

Example 31

The composition of low purity rebaudioside A (50 g,

51%) was combined with 400 mL of ethanol (95%) and heated at 56 ° C for 8 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) overnight stirring at the same time. The precipitate was filtered, washed with ethanol (10-15 mL 95%, 22 ° C), and dried for two days in a vacuum-desiccator oven (50 ° C) to obtain 26.59 g of a steviolglucoside composition ( 53.18% yield) having 79% rebaudioside A and 97% steviolglucosides in total.

Example 32

The composition of low purity rebaudioside A (50 g,

51%) was combined with 200 mL of ethanol (95%) and 10 mL of water, conditioned with a cold water reflux condenser, and heated in a water bath at 78 ° C for 6-8 minutes with stirring. weather. The solution was cooled and left at room temperature (22 ° C) overnight stirring at the same time. The precipitate was filtered and washed with ethanol (3x10 mL

95%, 4 ° C) and dried four days in a vacuum-desiccator oven (60 ° C) to obtain 17.91 g of a steviolglucoside composition (35.82% yield) having 96% rebaudioside A and 99% of steviolglucosides in total.

Example 36

The composition of low purity rebaudioside A (50 g,

46%) was combined with 400 mL of ethanol (95%) and heated at 56 ° C for 7.5 minutes while stirring at the same time. The solution was cooled and left at room temperature (22 ° C) for five days while stirring at the same time. The precipitate was filtered, washed with ethanol (10-15 mL 95%, 22 ° C), and dried two days in a vacuum-desiccator oven (50 ° C) to obtain 18.64 g of a steviolglucoside composition (37.28 Yield%) having 77% rebaudioside A and 96% steviolglucosides in total.

Example 37

The composition of low purity rebaudioside A (50 g,

6%) was combined with 250 mL of ethanol (95%) and 15 mL of water at room temperature (22 ° C) for 60 minutes while stirring at the same time. The solution was left at room temperature overnight stirring at the same time. The precipitate was filtered, washed with ethanol (10-15 mL 95%, 22 ° C), and dried four days in a vacuum-desiccator oven (60 ° C) to obtain 13.2 g of a steviolglucoside composition (26.40 Yield%) having 90% rebaudioside A and 96% steviolglucosides in total.

Set B of Examples

A single stage crystallization of a low purity rebaudioside A composition was performed using various organic solvents and experimental conditions. The amount of the resulting precipitate was determined for each crystallization and the precipitate was subjected to analytical HPLC (Agilent 1100) using the universal CAD (charged aerosol) detector to obtain a quantitative estimate of rebaudioside A in the resulting composition. The total mass yield and total yield of rebaudioside A (both in total and in relation to the total amount of rebaudioside

A present in the low purity rebaudioside A compositions) were then determined. The results are summarized in Table 7 and the experimental conditions are described in detail below.

Table 7: Summary of Experimental Results

<td>Exp. No.</td><td>Mass Initial (g)</td><td>Purity of Reb A Initial (mass%)</td><td>Mass Final g)</td><td>performance Total (mass%)</td><td>Purity of Reb A Final (mass%)</td><td>Performance Reb A in Relationship to Reb A raw (% by mass)</td>

<td> 1</td><td> 5.0</td><td> 62</td><td> 2.15</td><td> 43.00</td><td> 92.5</td><td> 64.15</td>

<td> 2</td><td> 5.0</td><td> 62</td><td> 2.20</td><td> 44.00</td><td> 94.96</td><td> 67.39</td>

<td> 3</td><td> 5.0</td><td> 62</td><td> 2.55</td><td> 51.00</td><td> 96.3</td><td> 79.21</td>

<td> 4</td><td> 5.0</td><td> 62</td><td> 3.25</td><td> 65.00</td><td> 87</td><td> 91.21</td>

<td> 5</td><td> 5.0</td><td> 62</td><td> 3.32</td><td> 66.40</td><td> 77.6</td><td> 83.11</td>

<td> 6</td><td> 5.0</td><td> 41</td><td> 1.87</td><td> 37.40</td><td> 81</td><td> 73.89</td>

<td> 7</td><td> 5.0</td><td> 41</td><td> 1.65</td><td> 33.00</td><td> 81.62</td><td> 65.69</td>

<td> 8</td><td> 10.0</td><td> 62</td><td> 5.85</td><td> 58.50</td><td> 90.6</td><td> 85.49</td>

<td> 9</td><td> 10.0</td><td> 46</td><td> 4.40</td><td> 44.00</td><td> 73</td><td> 69.83</td>

<td> 10</td><td> 5.0</td><td> 51</td><td> 2.60</td><td> 52.00</td><td> 84.72</td><td> 86.38</td>

<td> 11</td><td> 5.0</td><td> 46</td><td> 2.24</td><td> 44.80</td><td> 78</td><td> 75.96</td>

<td> 12</td><td> 5.0</td><td> 62</td><td> 2.30</td><td> 46.00</td><td> 83.46</td><td> 61.92</td>

<td> 13</td><td> 5.0</td><td> 41</td><td> 1.85</td><td> 37.00</td><td> 80</td><td> 72.20</td>

<td> 14</td><td> 5.0</td><td> 54</td><td> 1.85</td><td> 37.00</td><td> 88</td><td> 60.30</td>

<td> 15</td><td> 5.0</td><td> 43</td><td> 1.36</td><td> 27.20</td><td> 85.82</td><td> 54.29</td>

Example 1

A composition of low purity rebaudioside A (5 g,

62%) was combined with 40 mL of ethanol (90%) and heated from 45 ° C to 58 ° C for 20 minutes. The solution was filtered while hot to remove traces of insoluble material. The filtrate was cooled and left at room temperature (22 ° C) for 44 hours while stirring at the same time. The precipitate was filtered, washed with absolute ethanol (22 ° C), and dried for hours to obtain 2.15 g of a steviolglucoside composition (43% yield) having 92.5% rebaudioside A.

Example 2

A composition of low purity rebaudioside A (5 g,

62%) was combined with 20 mL of ethanol (90%) and heated from 45 ° C to 58 ° C for 20 minutes. The solution was filtered while hot to remove traces of insoluble material. The filtrate was cooled and left at room temperature (22 ° C) for 44 hours while stirring at the same time. The precipitate was filtered, washed with absolute ethanol (70%, -5 ° C), and dried for 24 hours at 65 ° C under vacuum overnight to obtain 2.2 g of a steviolglucoside composition (44% yield ) which has 94.96% rebaudioside A.

Example 3

A low purity rebaudioside A composition (5 g, 62%) was combined with 20 mL of ethanol (90%) and heated from 45 ° C to 50 ° C for 20 minutes. The solution was filtered while hot to remove traces of insoluble material. The filtrate was cooled and left at room temperature (22 ° C) for 44 hours and at 5 ° C for 1 while stirring at the same time.

<td>The precipitate leaked,</td><td>He washed</td><td colspan="2">with ethanol</td><td>absolute (0 ° C),</td><td>and</td>

<td>dried for 24 hours</td><td>at 65 ° C</td><td>low</td><td>empty</td><td>during teda</td><td>the</td>

<td>night to get 2</td><td>.55 g</td><td>of</td><td>a</td><td>composition</td><td>of</td>

<td colspan="3">steviolglucoside (51% yield)</td><td>than</td><td>has 96.3%</td><td>of</td>

<td>rebaudioside A.</td><td></td><td></td><td></td><td></td><td></td>

<td>Example 4</td><td></td><td></td><td></td><td></td><td></td>

<td>A composition of</td><td colspan="2">rebaudioside A</td><td colspan="2">low purity (5</td><td>g,</td>

62%) was combined with 20 mL of ethanol (95%) and 0.35 mL of water and stirred at room temperature (22 ° C) for 2 days. The solution was cooled to 0 to 5 ° C and left at this temperature for four hours while stirring at the same time. The precipitate was filtered, washed with aqueous ethanol (95%, 0 ° C), and dried for 24 hours at 60 ° C - under vacuum to obtain 3.25 g of a steviolglucoside composition (65% yield) having 87 % of rebaudioside A.

Example 5

A low purity rebaudioside A composition (5 g, 62%) was combined with 20 mL of absolute ethanol and 1.0 mL of methanol and stirred at room temperature (22 ° C) for 2 days. The solution was cooled to 0 to 5 ° C and left at this temperature for four hours while stirring at the same time. The precipitate was filtered, washed with absolute ethanol (0 ° C), and dried for 20 hours at 60 ° C under vacuum to obtain 3.32 g of a steviolglucoside composition (66.4% yield) having 77.6% rebaudioside A .

Example 6

A low purity rebaudioside A composition (5 g, 41%) was combined with 20 mL of aqueous ethanol (95%, 22 ° C). The solution was seeded with substantially pure (&gt; 99.0%) rebaudioside A at room temperature (22 ° C) for 17 hours while shaking. The solution was cooled to 0 to 5 ° C and left at this temperature for four hours while stirring at the same time. The precipitate was filtered, washed with aqueous ethanol (95%, 0 ° C), and dried for 48 hours at 60 ° C under vacuum to obtain 1.87 g of a steviolglucoside composition (37.2% yield) having 81.0% from rebaudioside A.

Example 7

A composition of low purity rebaudioside A (5 g,

41%) was combined with 20 mL of aqueous ethanol (95%, 22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 72 hours while shaking. The precipitate was filtered and dried for 24 hours at 60 ° C under vacuum to obtain 1.65 g of a steviolglucoside composition (33% yield) having 81.62% rebaudioside A.

Example 8

A low purity rebaudioside A composition (10 g, 62%) was combined with 59.4 mL of aqueous ethanol (95%, 22 ° C) and 0.6 mL at room temperature (22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 2 days while shaking. The precipitate was filtered, washed with aqueous ethanol (95%, 4 ° C), and dried for 24 hours at 60 ° C under vacuum to obtain 5.82 g of a steviolgiucoside composition (58.2% yield) having 90.6% from rebaudioside A.

Example 9

A low purity rebaudioside A composition (10.0 g, 46%) was combined with 30 mL of aqueous ethanol (95%, 22 ° C) at room temperature (22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 2 days while shaking. The precipitate was filtered, washed with aqueous ethanol (95%, 5 ° C), and dried for 24 hours at 60 ° C under vacuum to obtain 4.4 g of a steviolgiucoside composition (44% yield) having 73% from rebaudioside A.

Example 10

A composition of low purity rebaudioside A (5.0 g,

51%) was combined with 12.5 mL of aqueous ethanol (90%, 22 ° C) at room temperature (22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 18 hours while shaking. The solution was cooled to 0 to 5 ° C and left at this temperature for three hours while stirring at the same time. The precipitate was filtered and dried for 20 hours at 60 ° C under vacuum to obtain 2.6 g of a steviolglucoside composition (52% yield) having 84.72% rebaudioside A.

Example 11

A low purity rebaudioside A composition (5.0 g, 10 46%) was combined with 12.5 mL of aqueous ethanol (90%, 22 ° C) at room temperature (22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 24 hours while shaking. The solution was cooled to 0 to 5 ° C and left at this temperature for four hours while stirring at the same time. The precipitate was filtered and dried for 24 hours at 60 ° C under vacuum to obtain 2.24 g of a steviolglucoside composition (45% yield) having 78% rebaudioside A.

Example 12

A composition of low purity rebaudioside A (5.0 g,

62%) was combined with 25 mL of aqueous ethanol (95%, 22 ° C) at room temperature (22 ° C) for 15 minutes. The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 24 hours while shaking. The precipitate was filtered, washed with aqueous ethanol, and dried for 24 hours at 60 ° C under vacuum to obtain 2.3 g of a steviolglucoside composition (46% yield) having 83.46% rebaudioside A.

Example 13

A low purity rebaudioside A composition (5.0 g, 41%) was combined with 25 mL of aqueous ethanol (95%, 22 ° C) at room temperature (22 ° C). The solution was seeded with substantially pure rebaudioside A (&gt; 99.0%) at room temperature (22 ° C) for 48 hours while shaking. The precipitate was filtered, washed with absolute ethanol (4 ° C), and dried for 24 hours at 60 ° C under vacuum to obtain 1.85 g of a steviolglucoside composition (37% yield) having 80.0% rebaudioside A .

Example 14

A composition of low purity rebaudioside A (5.0 g,

<td>51%) combined</td><td>with 20 mL</td><td>aqueous ethanol</td><td> (95%,</td><td>22 ° C) and</td><td> 0.1</td>

<td>mL of water a</td><td>temperature</td><td>ambient (22 ° C)</td><td>. The</td><td>solution</td><td>I know</td>

<td colspan="2">sowed with rebaudioside A</td><td>substantially</td><td>pure</td><td> (&gt; 99.0%</td><td>) to</td>

rebaudioside A.

room temperature (22 ° C) for 24 hours while stirring at the same time. The precipitate was filtered and dried for 24 hours at

60 ° C under vacuum to obtain 1.85 g of a steviolglucoside composition (37% yield) having 88.0% of

Example 15

A composition of low purity rebaudioside A (5.0 g,

<td>43%)</td><td>combined</td><td>with 20 mL</td><td>aqueous ethanol</td><td> (95%,</td><td> 22</td><td>° C) and</td><td> 0.1</td>

<td>mL of</td><td>water to</td><td>temperature</td><td>ambient (22 ° C)</td><td>. The</td><td colspan="2">solution</td><td>I know</td>

<td>5 sowed</td><td colspan="2">with rebaudioside A</td><td>substantially</td><td>pure</td><td> (&gt;</td><td> 99.0%</td><td>) to</td>

room temperature (22 ° C) for 72 hours while stirring at the same time. The precipitate was filtered, washed with aqueous ethanol (95%), and dried for 24 hours at 60 ° C under vacuum to obtain 1.36 g of a steviolglucoside composition (27% yield) having 85.82% rebaudioside A.

Although the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated that those skilled in the art, upon obtaining an understanding of the above, can readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present invention should be evaluated in the same way as that of the appended claims and any equivalent thereof.