High-Purity Steviol Glycosides

Abstract

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and M are described. The methods include utilizing recombinant microorganisms for converting various staring compositions to target steviol glycosides. In addition, novel steviol glycosides reb D2 and reb M2 are disclosed, as are methods of preparing the same. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

1 . A method for producing target steviol glycosides, comprising the steps of:
 a. providing a starting composition comprising an organic compound with at least one carbon atom;   b. providing a microorganism or a biocatalyst containing at least one enzyme selected from steviol biosynthesis enzymes, UDP-glycosyltransferases, and optionally providing UDP-glucose recycling enzymes;   c. contacting the microorganism with a medium containing the starting composition to produce a medium comprising at least one target steviol glycoside.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , further comprising the step of:
 d. purifying the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.   
     
     
         4 . The method of  claim 1 , wherein the organic compound is selected from the group consisting of polyols, carbohydrates, steviol glycosides and combinations thereof. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the biocatalyst is an enzyme, or a cell comprising one or more enzymes, capable of converting the organic compound to target steviol glycoside. 
     
     
         9 . The method of  claim 1 , wherein the target steviol glycoside is selected from the group consisting of stevioside, reb A, reb D, reb D2, reb E, reb M, reb M2 and mixtures thereof. 
     
     
         10 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-Cmethyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, UGT91D2, UGTSL2, UGT76G1, or UGT76G1 containing one or more point mutations selected from S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A and S456L. 
     
     
         11 . The method of  claim 3 , wherein the highly purified target steviol glycoside composition has a target steviol glycoside purity greater than about 95% by weight on a dry basis. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 11 , wherein the target steviol glycoside is reb M. 
     
     
         15 . The method of  claim 3 , wherein the target steviol glycoside is polymorphic. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . Isolated and highly purified reb D2 having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the method comprises:
 a. contacting a starting composition comprising reb A with an enzyme selected from the group of enzymes capable of transforming reb A to reb D2, UDP glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb D2; and   b. isolating, and optionally, purifying the composition comprising reb D2.   
     
     
         25 . The method of  claim 24 , wherein reb D2 has a purity greater than about 95% by weight on an anhydrous basis. 
     
     
         26 . Isolated and highly purified reb M2 having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         27 . A consumable product comprising isolated and highly purified reb M2 or highly purified reb D2. 
     
     
         28 . (canceled) 
     
     
         29 . The consumable product of  claim 27 , wherein the product is a beverage. 
     
     
         30 . The method of  claim 1 , wherein the method comprises:
 a. contacting a starting composition comprising reb D2 with an enzyme selected from the group consisting of enzymes capable of transforming reb D2 to reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb M2; and   b. isolating, and optionally, purifying the composition comprising reb M2.   
     
     
         31 . The method of  claim 30 , wherein reb M2 has a purity greater than about 95% by weight on an anhydrous basis. 
     
     
         32 . (canceled) 
     
     
         33 . The consumable product of  claim 27 , further comprising at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof. 
     
     
         34 . The consumable product of  claim 27 , further comprising a compound selected from the group consisting of stevia, stevia extract, steviolmonoside, steviolbioside, rubusoside, dulcoside A, dulcoside B, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside Nor rebaudioside 0, or other glycoside of steviol found in  Stevia rebaudiana , mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pemandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, camosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hemandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, mogroside VI, grosmomomside, siamenoside, NSF-02, NSF-03, NSF-04, allulose, allose, D-tagatose, erythritol and combinations thereof. 
     
     
         35 . The consumable product of  claim 27  wherein the product is a tabletop sweetener composition comprising isolated and purified reb D2 or isolated and purified reb M2. 
     
     
         36 . The consumable product of  claim 27 , further comprising at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof. 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . A method for enhancing the sweetness of a beverage comprising a sweetener comprising:
 a.) providing a beverage comprising a sweetener; and   b.) adding a sweetness enhancer selected from reb D2, reb M2 or a combination thereof, wherein reb D2 and/or reb M2 1s present m a concentration at or below the sweetness recognition threshold.