Pure and substantially pure rebaudioside a

Abstract

The invention provides a high throughput, high purity, high yield system and method of isolating and purifying rebaudioside A (“Reb A”), with acceptable water solubility for all commercial uses, from commercially available Stevia rebaudiana starting material. The invention also provides a means of maximizing yields of 99+% purity Reb A based on the attributes of a given batch of Stevia starting material. The Reb A produced by the invention is water soluble, devoid of bitterness heretofore associated with rebaudioside sweeteners, non-caloric, and suitable for use as a reagent and as an ingredient in orally consumed products, e.g., as a sweetener, flavor enhancer, and flavor modifier.

1 . A method of determining the reflux solvent with which to purify rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 preparing a series of test mixtures comprising an alkanol mixed with water, the starting material, and optionally one or more denaturants, wherein the water content of the series of test mixtures is increased from a minimum water content that is ten percent or less to a maximum water content that is no more than fifteen percent,   refluxing each of the test mixtures,   collecting separately by filtration the precipitate of each reflux,   washing the precipitate of each reflux in an alkanol that optionally contains one or more   denaturants,   collecting separately by filtration the retentate of each wash,   optionally repeating one or more times the steps of washing and collecting retentate of each wash;   optionally drying the retentate of each wash;   determining the rebaudioside A yield, purity, or yield and purity of each retentate with reference to the water content of the text mixture from which the retentate was obtained, wherein the series of determinations corresponding to the series of test mixtures is called the water content-yield-purity relationship for the alkanol, starting material, and optional denaturants used in the test mixtures, and   examining the water content-yield-purity relationship and selecting the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from the starting material by interpolating between water contents used in the test mixtures, by extrapolating from the water contents used in the test mixtures, or by selecting a water content used in one of the test mixtures.   
     
     
         2 . A method of determining the reflux solvent with which to purify rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 preparing a series of test mixtures comprising an alkanol mixed with water, the starting material, and optionally one or more denaturants, wherein the water content of the series of test mixtures is increased from a minimum water content that is ten percent or less to a maximum water content that is no more than fifteen percent,   refluxing each of the test mixtures,   collecting separately by filtration the precipitate of each reflux,   washing the precipitate of each reflux in an alkanol that optionally contains one or more denaturants,   collecting separately by filtration the retentate of each wash,   optionally repeating one or more times the steps of washing and collecting retentate of each wash;   optionally drying the retentate of each wash;   determining the rebaudioside A yield, purity, or yield and purity of each retentate with reference to the water content of the text mixture from which the retentate was obtained, wherein the series of determinations corresponding to the series of test mixtures is called the water content-yield-purity relationship for the alkanol, starting material, and optional denaturants used in the test mixtures,   recording in at least one graph or table the water content-yield-purity relationship, and   selecting by reference to the at least one graph or table the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from the starting material by interpolating between water contents used in the test mixtures, by extrapolating from the water contents used in the test mixtures, or by selecting a water content used in one of the test mixtures.   
     
     
         3 . A method of determining the reflux solvent with which to purify rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 preparing a series of test mixtures comprising an alkanol mixed with water, the starting material, and optionally one or more denaturants, wherein the water content of the series of test mixtures is increased from a minimum water content that is ten percent or less to a maximum water content that is no more than fifteen percent,   refluxing each of the test mixtures,   collecting separately by filtration the precipitate of each reflux,   repeating one or more times the steps of refluxing and collecting each precipitate;   washing the precipitate of each reflux in an alkanol that optionally contains one or more denaturants,   collecting separately by filtration retentate of each wash,   optionally repeating one or more times the steps of washing and collecting retentate of each wash;   optionally drying the retentate of each wash;   determining the rebaudioside A yield, purity, or yield and purity of each retentate with reference to the water content of the text mixture from which the retentate was obtained, wherein the series of determinations corresponding to the series of test mixtures is called the water content-yield-purity relationship for the alkanol, starting material, and optional denaturants used in the test mixtures, and   examining the water content-yield-purity relationship and selecting the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from the starting material by interpolating between water contents used in the test mixtures, by extrapolating from the water contents used in the test mixtures, or by selecting a water content used in one of the test mixtures.   
     
     
         4 . A method of determining the reflux solvent with which to purify rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 preparing a series of test mixtures comprising an alkanol mixed with water, the starting material, and optionally one or more denaturants, wherein the water content of the series of test mixtures is increased from a minimum water content that is ten percent or less to a maximum water content that is no more than fifteen percent,   refluxing each of the test mixtures,   collecting separately by filtration the precipitate of each reflux,   repeating one or more times the steps of refluxing and collecting the precipitate of each reflux;   washing the precipitate of each reflux in an alkanol that optionally contains one or more denaturants,   collecting separately by filtration retentate of each wash,   optionally repeating one or more times the steps of washing and collecting retentate of each wash;   optionally drying the retentate of each wash;   determining the rebaudioside A yield, purity, or yield and purity of each retentate with reference to the water content of the text mixture from which the retentate was obtained, wherein the series of determinations corresponding to the series of test mixtures is called the water content-yield-purity relationship for the alkanol, starting material, and optional denaturants used in the test mixtures,   recording in at least one graph or table the water content-yield-purity relationship, and   selecting by reference to the at least one graph or table the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from the starting material by interpolating between water contents used in the test mixtures, by extrapolating from the water contents used in the test mixtures, or by selecting a water content used in one of the test mixtures.   
     
     
         5 . Performing the method of  claim 1 ,  2 ,  3 , or  4  two or more times, each time with a starting material of a different rebaudioside A content, but with the same alkanol and optional one or more denaturants for each series of test mixtures containing a given starting material, further comprising:
 examining the water content-yield-purity relationships of each of the different starting materials, 
 deriving an approximation of reflux solvent water content to use in purifying rebaudioside A from starting materials as a function of rebaudioside A content of a starting material, wherein the alkanol, and optional denaturants, to be used in purifying rebaudioside A are those used in the test mixtures, and 
 selecting, based on the labelled, asserted, or actual rebaudioside A content of a starting material to be processed and by reference to the approximation, the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from a starting material to be processed. 
 
     
     
         6 . Performing the method of  claim 1 ,  2 ,  3 , or  4  two or more times, each time with a starting material of a different rebaudioside A content, but with the same alkanol and optional one or more denaturants for each series of test mixtures containing a given starting material, further comprising:
 consolidating in at least one graph or table the water content-yield-purity relationships of each of the starting materials, 
 deriving by reference to the at least one graph or table of the water content-yield-purity relationships of the different starting materials an approximation of reflux solvent water content to use in purifying rebaudioside A from starting materials as a function of rebaudioside A content of a starting material, wherein the alkanol, and optional denaturants, to be used in purifying rebaudioside A are those used in the test mixtures, and 
 selecting, based on the labelled, asserted, or actual rebaudioside A content of a starting material to be processed and by reference to the approximation, the water content of the reflux solvent with which to produce a desired yield or purity of rebaudioside A from a starting material to be processed. 
 
     
     
         7 . A method of purifying rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 mixing the selected starting material of  claim 1 ,  2 ,  3 , or  4  with a reflux solvent with water content selected by the method of  claim 1 ,  2 ,  3 , or  4 , wherein the mixture contains the alkanol and optional one or more denaturants used in the one or more test mixtures of  claim 1 ,  2 ,  3 , or  4 ,   refluxing the mixture,   collecting by filtration the precipitate of the reflux,   optionally repeating one or more times the steps of refluxing and collecting the precipitate of each reflux;   washing the collected precipitate in an alkanol that optionally contains one or more denaturants,   collecting by filtration the retentate of the wash,   optionally repeating one or more times the steps of washing and collecting retentate; and   drying final product.   
     
     
         8 . A method of purifying rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 mixing the starting material with a reflux solvent with water content selected by the method of  claim 5 , wherein the mixture contains the alkanol and optional one or more denaturants used in  claim 5 ,   refluxing the mixture,   collecting by filtration the precipitate of the reflux,   optionally repeating one or more times the steps of refluxing and collecting the precipitate of each reflux;   washing the collected precipitate in an alkanol that optionally contains one or more denaturants,   collecting by filtration the retentate of the wash,   optionally repeating one or more times the steps of washing and collecting retentate; and   drying final product.   
     
     
         9 . A method of purifying rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 mixing the starting material with a reflux solvent with water content selected by the method of  claim 6 , wherein the mixture contains the alkanol and optional one or more denaturants used in  claim 6 ,   refluxing the mixture,   collecting by filtration the precipitate of the reflux,   optionally repeating one or more times the steps of refluxing and collecting the precipitate of each reflux;   washing the collected precipitate in an alkanol that optionally contains one or more denaturants,   collecting by filtration the retentate of the wash,   optionally repeating one or more times the steps of washing and collecting retentate; and   drying final product.   
     
     
         10 . The method of  claim 7 , wherein drying final product is performed by spray-drying. 
     
     
         11 . The method of  claim 8 , wherein drying final product is performed by spray-drying. 
     
     
         12 . The method of  claim 9 , wherein drying final product is performed by spray-drying. 
     
     
         13 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the minimum water content of the test mixtures is selected from the group consisting of zero percent, less than two percent, less than four percent, less than six percent, and less than eight percent. 
     
     
         14 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein filtration is selected from the group consisting of surface, rotary, centrifugal, and membrane. 
     
     
         15 . The method of  claim 7 , wherein filtration is selected from the group consisting of surface, rotary, centrifugal, and membrane. 
     
     
         16 . The method of  claim 8 , wherein filtration is selected from the group consisting of surface, rotary, centrifugal, and membrane. 
     
     
         17 . The method of  claim 9 , wherein filtration is selected from the group consisting of surface, rotary, centrifugal, and membrane. 
     
     
         18 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein collection of wash retentate is by evaporation of the alkanol. 
     
     
         19 . The method of  claim 7 , wherein collection of wash retentate is by evaporation of the alkanol. 
     
     
         20 . The method of  claim 8 , wherein collection of wash retentate is by evaporation of the alkanol. 
     
     
         21 . The method of  claim 9 , wherein collection of wash retentate is by evaporation of the alkanol. 
     
     
         22 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the alkanol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol. 
     
     
         23 . The method of  claim 7 , wherein the alkanol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol. 
     
     
         24 . The method of  claim 8 , wherein the alkanol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol. 
     
     
         25 . The method of  claim 9 , wherein the alkanol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, and butanol. 
     
     
         26 . The method of  claim 1 ,  2 ,  3 ,  4 , wherein the alkanol is ethanol and a denaturant is selected from the group consisting of methanol, propanol, isopropanol, butanol, ethyl acetate, toluene, 4-methyl-2-pentanone, methyl isobutyl-ketone, and kerosene. 
     
     
         27 . The method of  claim 7 , wherein the alkanol is ethanol and a denaturant is selected from the group consisting of methanol, propanol, isopropanol, butanol, ethyl acetate, toluene, 4-methyl-2-pentanone, methyl isobutyl-ketone, and kerosene. 
     
     
         28 . The method of  claim 8 , wherein the alkanol is ethanol and a denaturant is selected from the group consisting of methanol, propanol, isopropanol, butanol, ethyl acetate, toluene, 4-methyl-2-pentanone, methyl isobutyl-ketone, and kerosene. 
     
     
         29 . The method of  claim 9 , wherein the alkanol is ethanol and a denaturant is selected from the group consisting of methanol, propanol, isopropanol, butanol, ethyl acetate, toluene, 4-methyl-2-pentanone, methyl isobutyl-ketone, and kerosene. 
     
     
         30 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the water content is uniformly incremented in the series of test mixtures. 
     
     
         31 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the reflux temperature is selected from the group consisting of room temperature, between room temperature and 79° C., 79° C., and more than 79° C. 
     
     
         32 . The method of  claim 7 , wherein the reflux temperature is selected from the group consisting of room temperature, between room temperature and 79° C., 79° C., and more than 79° C. 
     
     
         33 . The method of  claim 8 , wherein the reflux temperature is selected from the group consisting of room temperature, between room temperature and 79° C., 79° C., and more than 79° C. 
     
     
         34 . The method of  claim 9 , wherein the reflux temperature is selected from the group consisting of room temperature, between room temperature and 79° C., 79° C., and more than 79° C. 
     
     
         35 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the ratio of the mass in grams of starting material to the volume of reflux solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         36 . The method of  claim 7 , wherein the ratio of the mass in grams of starting material to the volume of reflux solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         37 . The method of  claim 8 , wherein the ratio of the mass in grams of starting material to the volume of reflux solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         38 . The method of  claim 9 , wherein the ratio of the mass in grams of starting material to the volume of reflux solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         39 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the ratio of the mass in grams of retentate to the volume of wash solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         40 . The method of  claim 7 , wherein the ratio of the mass in grams of retentate to the volume of wash solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         41 . The method of  claim 8 , wherein the ratio of the mass in grams of retentate to the volume of wash solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         42 . The method of  claim 9 , wherein the ratio of the mass in grams of retentate to the volume of wash solvent in milliliters is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, and 1:8. 
     
     
         43 . The method of  claim 1 ,  2 ,  3 , or  4 , wherein the alkanol a washing step is different from the alkanol in a refluxing step. 
     
     
         44 . The method of  claim 7 , wherein the alkanol in a washing step is different from the alkanol in a refluxing step. 
     
     
         45 . The method of  claim 8 , wherein the alkanol in a washing step is different from the alkanol in a refluxing step. 
     
     
         46 . The method of  claim 9 , wherein the alkanol in a washing step is different from the alkanol in a refluxing step. 
     
     
         47 . Rebaudioside A produced using the method set forth in  claim 7 . 
     
     
         48 . Rebaudioside A produced using the method set forth in  claim 7 , and used in beverages, broths, and beverage preparations selected from the group comprising carbonated, non-carbonated, frozen, semi-frozen, non-frozen, ready-to-drink, concentrated, dairy, non-dairy, herbal, non-herbal, caffeinated, non-caffeinated, alcoholic, non-alcoholic, flavored, non-flavored, vegetable-based, fruit-based, root/tuber/corm-based, nut-based, other plant-based, cola-based, chocolate-based, meat-based, seafood-based, other animal-based, algae-based, calorie enhanced, calorie-reduced, and calorie-free products, optionally dispensed in open containers, cans, bottles or other packaging. 
     
     
         49 . Rebaudioside A produced using the method set forth in  claim 7 , and used in foods and food preparations selected from the group comprising dairy-based, cereal-based, baked, vegetable-based, fruit-based, root/tuber/corm-based, nut-based, other plant-based, egg-based, meat-based, seafood-based, other animal-based, algae-based, processed, preserved, and synthesized products. 
     
     
         50 . Rebaudioside A produced using the method set forth in  claim 7 , and used in candies, confections, desserts, and snacks selected from the group comprising dairy-based, cereal-based, baked, vegetable-based, fruit-based, root/tuber/corm-based, nut-based, gum-based, other plant-based, egg-based, meat-based, seafood-based, other animal-based, algae-based, processed, preserved, and synthesized products. 
     
     
         51 . Rebaudioside A produced using the method set forth in  claim 7 , and used in prescription and over-the-counter pharmaceuticals, assays, diagnostic kits, and therapies selected from the group comprising weight control, nutritional supplement, vitamins, infant diet, diabetic diet, athlete diet, geriatric diet, low carbohydrate diet, low fat diet, low protein diet, high carbohydrate diet, high fat diet, high protein diet, low calorie diet, non-caloric diet, oral hygiene products, personal care products, professional dentistry products, medical, veterinarian, and surgical products, and pharmaceutical compounding fillers, syrups, capsules, gels, and coating products. 
     
     
         52 . Rebaudioside A produced using the method set forth in  claim 7 , and used in consumer goods packaging materials and containers selected from the group comprising plastic film, thermoset and thermoplastic resin, gum, foil, paper, bottle, box, ink, paint, adhesive, and packaging coating products. 
     
     
         53 . Rebaudioside A produced using the method set forth in  claim 7 , and used in goods selected from the group comprising sweeteners, co-sweeteners, coated sweetener sticks, frozen confection sticks, medicine spoons (human and veterinary uses), dental instruments, pre-sweetened disposable tableware and utensils, sachets, edible sachets, pot pourris, edible pot pourris, hotch potches, edible hotch potches, artificial flowers, edible artificial flowers, clothing, edible clothing, massage oils, and edible massage oils. 
     
     
         54 . A method of purifying rebaudioside A from a starting material selected from the group consisting of  Stevia  starting material, a crude extract of  Stevia  plant material, and a crude extract of  Stevia  plant material which extract has at least 40% rebaudioside A purity, comprising:
 identifying approximate rebaudioside A purity in the starting material;   formulating an aqueous alkanol solvent, wherein said formulating comprises selecting a water to alkanol balance in the alkanol solvent so that the water content is substantially inversely proportional to the rebaudioside A purity of the starting material;   refluxing the starting material in the alkanol solvent; and   removing alkanol and water to obtain purified rebaudioside A.