HIGH-PURITY STEVIOL GLYCOSIDES

TECHNICAL FIELD

The present invention relates to a process for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.

BACKGROUND OF THE INVENTION

High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted to diabetics and others interested in controlling for their intake of carbohydrates.

Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves, composing approximately 10%-20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%). Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.

Although methods are known for preparing steviol glycosides from Stevia rebaudiana, many of these methods are unsuitable for use commercially.

Accordingly, there remains a need for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.

SUMMARY OF THE INVENTION

The present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or enzyme preparation, thereby producing a composition comprising a target steviol glycoside.

The starting composition can be any organic compound comprising at least one carbon atom. In one embodiment, the starting composition is selected from the group consisting of steviol glycosides, polyols or sugar alcohols, various carbohydrates.

The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside K4), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or a synthetic steviol glycoside.

In one embodiment, the target steviol glycoside is rebaudioside AM In some preferred embodiments enzyme preparation comprising one or more enzymes, or a microbial cell comprising one or more enzymes, capable of converting the starting composition to target steviol glycosides are used. The enzyme can be located on the surface and/or inside the cell. The enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate or as purified enzyme(s). The enzyme preparation can be in free form or immobilized to a solid support made from inorganic or organic materials.

In some embodiments, a microbial cell comprises the necessary enzymes and genes encoding thereof for converting the starting composition to target steviol glycosides. Accordingly, the present invention also provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell comprising at least one enzyme capable of converting the starting composition to target steviol glycosides, thereby producing a medium comprising at least one target steviol glycoside.

The enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes, UDP-glucosyltransferases (UGTs) and/or UDP-recycling enzyme.

In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.

In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one embodiment the steviol biosynthesis enzymes are selected from the group including 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-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), l-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 etc.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.

As used hereinafter, the term “SuSy_AT”, unless specified otherwise, refers to sucrose synthase having amino-acid sequence “SEQ ID 1” as described in Example 1.

As used hereinafter, the term “UGTS12”, unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence “SEQ ID 2” as described in Example 1.

As used hereinafter, the term “UGT76G1”, unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence “SEQ ID 3” as described in Example 1.

In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc. In another embodiment, the UDP-glucosyltransferases are synthesized.

In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS12, EUGT11 and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.

In one embodiment, steviol biosynthesis enzymes, UGTs and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing an —OH functional group at C13 to give a target steviol glycoside having an —O-glucose beta glucopyranoside glycosidic linkage at C13. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a —COOH functional group at C19 to give a target steviol glycoside having a —COO-glucose beta-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a UGT having >85% amino-acid sequence identity with UGT74G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1→2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1→2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside A (rebaudioside KA). In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to form rebaudioside E.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside C to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

Optionally, the method of the present invention further comprises using more than one UGT on a starting composition, to give a target steviol glycoside(s) having more than one glucose unit than the starting composition. In a particular embodiment, the UDP-glucosyltransferases are UGT74G1, UGT85C2, UGT76G1, UGTS12, EUGT11 and/or UGT91D2 or any UGT having >85% amino-acid sequence identity with UGT74G1, UGT85C2, UGT76G1, UGTS12, EUGT11 and/or UGT91D2 or any combination thereof, capable of adding more than one glucose unit to a starting composition to give a steviol glycoside(s) having more than one glucose unit than the starting composition.

In one embodiment, the UDP-glucosyltransferases are any UDP-glucosyltransferases capable of adding overall two glucose unit to stevioside to form rebaudioside AM. In a particular embodiment, the UDP-glucosyltransferases are selected from UGTS12, EUGT11, UGT91D2, UGT76G1 or any UGT having >85% amino-acid sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1 or any combination thereof. In another particular embodiment, the UDP-glucosyltransferases are UGTS12 and UGT76G1.

Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of steviol and/or the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.

In one embodiment, the recycling catalyst is sucrose synthase SuSy_At or a sucrose synthase having >85% amino-acid sequence identity with SuSy_At.

In one embodiment, the recycling substrate is sucrose.

Optionally, the method of the present invention further comprises the use of transglycosidases that use oligo- or poly-saccharides as the sugar donor to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosylinvertase, alkaline invertase, acid invertase, fructofuranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose are transferred to the recipient target steviol glycosides. In one embodiment, the recipient steviol glycoside is rebaudioside AM.

Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

In one embodiment, the target steviol glycoside can be produced within the microorganism. In another embodiment, the target steviol glycoside can be secreted out in the medium. In one another embodiment, the released steviol glycoside can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the completion of the conversion reaction.

In one embodiment, separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In particular embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside. In other embodiments, the composition comprises greater than about 99% by weight of the target steviol glycoside.

The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.

Purified target steviol glycosides can be used in consumable products as a sweetener, flavor modifier, flavor with modifying properties and/or foaming suppressor. Suitable consumable products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of rebaudioside AM.

FIG. 2 shows the pathways of producing rebaudioside AM and various steviol glycosides from steviol.

FIG. 3 shows the biocatalytic production of rebaudioside AM from stevioside using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.

FIG. 4 shows the biocatalytic production of rebausioside AM from rebaudioside E using the enzyme UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.

FIG. 5 shows the HPLC chromatogram of stevioside. The peak with retention time of 25.992 minutes corresponds to stevioside.

FIG. 6 shows the HPLC chromatogram of the product of the biocatalytic production of rebaudioside AM from stevioside. The peak with retention time of 10.636 minutes corresponds to rebaudioside AM.

FIG. 7 shows the HPLC chromatogram of rebaudioside E. The peak with retention time of 10.835 minutes corresponds to rebaudioside E.

FIG. 8 shows the HPLC chromatogram of the product of the biocatalytic production of rebaudioside AM from rebaudioside E. The peaks with retention time of 10.936 and 11.442 minutes correspond to rebaudioside E and rebaudioside AM respectively.

FIG. 9 shows the HPLC chromatogram of rebaudioside AM after purification by methanol crystallization. The peak with retention time of 10.336 minutes corresponds to rebaudioside AM.

FIG. 10 shows the ¹H NMR spectrum of rebaudioside AM (500 MHz, pyridine-d5).

FIG. 11 shows the HSQC spectrum of rebaudioside AM (500 MHz, pyridine-d5).

FIG. 12 shows the H,H COSY spectrum of rebaudioside AM (500 MHz, pyridine-d5).

FIG. 13 shows the HMBC spectrum of rebaudioside AM (500 MHz, pyridine-d5).

FIG. 14 shows the HSQC-TOCSY spectrum of rebaudioside AM (500 MHz, pyridine-d5).

FIG. 15a and FIG. 15b show the LC chromatogram and mass spectrum of rebaudioside AM respectively.

DETAILED DESCRIPTION

One object of the invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or a synthetic steviol glycoside from various starting compositions.

As used herein, the abbreviation term “reb” refers to “rebaudioside”. Both terms have the same meaning and may be used interchangeably.

As used herein, “biocatalysis” or “biocatalytic” refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells including microorganisms, comprising one or more enzyme, capable of single or multiple step chemical transformations on organic compounds. Biocatalysis processes include fermentation, biosynthesis, bioconversion and biotransformation processes. Both isolated enzyme, and whole-cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

As used herein, the term “steviol glycoside(s)” refers to a glycoside of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

Starting Composition

As used herein, “starting composition” refers to any composition (generally an aqueous solution) containing one or more organic compound comprising at least one carbon atom.

In one embodiment, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols and various carbohydrates.

The starting composition steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 or other glycoside of steviol occurring in Stevia rebaudiana plant, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

In one embodiment, the starting composition is steviol.

In another embodiment, the starting composition steviol glycoside is steviolmonoside.

In yet another embodiment, the starting composition steviol glycoside is steviolmonoside A.

In still another embodiment, the starting composition steviol glycoside is rubusoside.

In yet another embodiment, the starting composition steviol glycoside is steviolbioside.

In yet another embodiment, the starting composition steviol glycoside is steviolbioside A.

In yet another embodiment, the starting composition steviol glycoside is steviolbioside B.

In still another embodiment, the starting composition steviol glycoside is stevioside.

In yet another embodiment, the starting composition steviol glycoside is stevioside A, also known as rebaudioside KA.

In still another embodiment, the starting composition steviol glycoside is stevioside B.

In still another embodiment, the starting composition steviol glycoside is stevioside C.

In another embodiment, the starting composition steviol glycoside is rebaudioside E.

In another embodiment, the starting composition steviol glycoside is rebaudioside E2.

In another embodiment, the starting composition steviol glycoside is rebaudioside E3.

The term “polyol” refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced.

The term “carbohydrate” refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH₂O)_n, wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivatives”, “substituted carbohydrate”, and “modified carbohydrates” are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.

The starting composition may be synthetic or purified (partially or entirely), commercially available or prepared.

In one embodiment, the starting composition is glycerol.

In another embodiment, the starting composition is glucose.

In still another embodiment, the starting composition is sucrose.

In yet another embodiment, the starting composition is starch.

In another embodiment, the starting composition is maltodextrin.

In yet another embodiment, the starting composition is cellulose.

In still another embodiment, the starting composition is amylose.

The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein.

Target Steviol Glycoside

The target steviol glycoside of the present method can be any steviol glycoside that can be prepared by the process disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or other glycoside of steviol occurring in Stevia rebaudiana plant, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

In one embodiment, the target steviol glycoside is steviolmonoside.

In another embodiment, the target steviol glycoside is steviolmonoside A.

In another embodiment, the target steviol glycoside is steviolbioside.

In another embodiment, the target steviol glycoside is steviolbioside A.

In another embodiment, the target steviol glycoside is steviolbioside B.

In another embodiment, the target steviol glycoside is rubusoside.

In another embodiment, the target steviol glycoside is stevioside.

In another embodiment, the target steviol glycoside is stevioside A (rebaudioside KA).

In another embodiment, the target steviol glycoside is stevioside B.

In another embodiment, the target steviol glycoside is stevioside C.

In another embodiment, the target steviol glycoside is rebaudioside E.

In another embodiment, the target steviol glycoside is rebaudioside E2.

In another embodiment, the target steviol glycoside is rebaudioside E3.

In another embodiment, the target steviol glycoside is rebaudioside AM The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.

In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside.

In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside A.

In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside.

In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside A.

In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside B.

In one embodiment, the present invention is a biocatalytic process for the production of rubusoside.

In one embodiment, the present invention is a biocatalytic process for the production of stevioside.

In one embodiment, the present invention is a biocatalytic process for the production of stevioside A (rebaudioside KA).

In one embodiment, the present invention is a biocatalytic process for the production of stevioside B.

In one embodiment, the present invention is a biocatalytic process for the production of stevioside C.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E2.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside AM.

In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside AM from a starting composition comprising stevioside and UDP-glucose.

In another particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside AM from a starting composition comprising rebaudioside E and UDP-glucose.

Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by any suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

In particular embodiments, the process described herein results in a highly purified target steviol glycoside composition. The term “highly purified”, as used herein, refers to a composition having greater than about 80% by weight of the target steviol glycoside on an anhydrous (dried) basis. In one embodiment, the highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous (dried) basis, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% target steviol glycoside content on a dried basis.

In one embodiment, when the target steviol glycoside is reb AM, the process described herein provides a composition having greater than about 90% reb AM content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is reb AM, the process described herein provides a composition comprising greater than about 95% reb AM content by weight on a dried basis.

Microorganisms and Enzyme Preparations

In one embodiment of present invention, a microorganism (microbial cell) and/or enzyme preparation is contacted with a medium containing the starting composition to produce target steviol glycosides.

The enzyme can be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme or a combination thereof. In one embodiment, the biocatalyst is a purified enzyme capable of converting the starting composition to the target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising at least one enzyme capable of converting the starting composition to the target steviol glycoside. In still another embodiment, the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting the starting composition to the target steviol glycoside.

In another embodiment, the biocatalyst is one or more microbial cells comprising enzyme(s) capable of converting the starting composition to the target steviol glycoside. The enzyme can be located on the surface of the cell, inside the cell or located both on the surface of the cell and inside the cell.

Suitable enzymes for converting the starting composition to target steviol glycosides include, but are not limited to, the steviol biosynthesis enzymes and UDP-glucosyltransferases (UGTs). Optionally it may include UDP recycling enzyme(s).

In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.

In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one embodiment the steviol biosynthesis enzymes are selected from the group including 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-C-methyl-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 etc.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1-42 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to form rebaudioside E.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to starting composition steviol glycoside has >85% amino-acid sequence identity with UGTs selected from the following listing of GenInfo identifier numbers, preferably from the group presented in Table 1, and Table 2.

397567
30680413
115480946
147798902
218193594
225443294

454245
32816174
116310259
147811764
218193942
225444853

1359905
32816178
116310985
147827151
219885307
225449296

1685003
34393978
116788066
147836230
222615927
225449700

1685005
37993665
116788606
147839909
222619587
225454338

2191136
37993671
116789315
147846163
222623142
225454340

2501497
37993675
119394507
147855977
222625633
225454342

2911049
39104603
119640480
148905778
222625635
225454473

4218003
41469414
122209731
148905999
222636620
225454475

4314356
41469452
125526997
148906835
222636621
225458362

13492674
42566366
125534279
148907340
222636628
225461551

13492676
42570280
125534461
148908935
222636629
225461556

15217773
42572855
125540090
148909182
224053242
225461558

15217796
44890129
125541516
148909920
224053386
225469538

15223396
46806235
125545408
148910082
224055535
225469540

15223589
50284482
125547340
148910154
224056138
226316457

15227766
51090402
125547520
148910612
224056160
226492603

15230017
51090594
125554547
148910769
224067918
226494221

15231757
52839682
125557592
156138791
224072747
226495389

15234056
56550539
125557593
156138797
224080189
226495945

15234195
62734263
125557608
156138799
224091845
226502400

15234196
62857204
125559566
156138803
224094703
226507980

15238503
62857206
125563266
165972256
224100653
226531147

15239523
62857210
125571055
168016721
224100657
226532094

15239525
62857212
125579728
171674071
224101569
238477377

15239543
75265643
125588307
171906258
224103105
240254512

15239937
75285934
125589492
183013901
224103633
242032615

15240305
75288884
125599469
183013903
224103637
242032621

15240534
77550661
125601477
186478321
224109218
242038423

15982889
77556148
126635837
187373030
224114583
242043290

18086351
82791223
126635845
187373042
224116284
242044836

18418378
83778990
126635847
190692175
224120552
242051252

18418380
89953335
126635863
194701936
224121288
242056217

18418382
110741436
126635867
195620060
224121296
242056219

19743740
110743955
126635883
209954691
224121300
242056663

19911201
115438196
126635887
209954719
224130358
242059339

20149064
115438785
133874210
209954725
224140703
242059341

20260654
115441237
133874212
209954733
224143404
242060922

21435782
115454819
145358033
210063105
224143406
242067411

21553613
115456047
147772508
210063107
224144306
242067413

21593514
115457492
147776893
212275846
224285244
242076258

22759895
115459312
147776894
216296854
225431707
242076396

23955910
115464719
147776895
217074506
225435532
242084750

26452040
115471069
147786916
218185693
225436321
242091005

28393204
115471071
147798900
218187075
225440041
242095206

30679796
115474009
147798901
218189427
225441116
242345159

242345161
297724601
326492035
356523945
357140904
359486938

255536859
297725463
326493430
356523957
357165849
359487055

255538228
297728331
326500410
356523959
357165852
359488135

255541676
297738632
326506816
356523961
357168415
359488708

255547075
297745347
326507826
356523963
357437837
359493630

255552620
297745348
326508394
356524387
357442755
359493632

255552622
297795735
326509445
356524403
357442757
359493634

255555343
297796253
326511261
356527181
357445729
359493636

255555361
297796257
326511866
356533209
357445731
359493815

255555363
297796261
326512412
356533852
357445733
359495856

255555365
297797587
326517673
356534718
357446799
359495858

255555369
297798502
326518800
356535480
357446805
359495869

255555373
297799226
326521124
356542996
357452779
359495871

255555377
297805988
326525567
356543136
357452781
359497638

255556812
297807499
326525957
356543932
357452783
359807261

255556818
297809125
326526607
356549841
357452787
374256637

255563008
297809127
326527141
356549843
357452789
377655465

255564074
297811403
326530093
356554358
357452791
378405177

255564531
297820040
326534036
356554360
357452797
378829085

255572878
297821483
326534312
356558606
357452799
387135070

255577901
297825217
332071132
356560333
357470367
387135072

255583249
297832276
339715876
356560599
357472193
387135078

255583253
297832280
342306012
356560749
357472195
387135092

255583255
297832518
342306016
356566018
357474295
387135094

255585664
297832520
343457675
356566169
357474493
387135098

255585666
297840825
343457677
356566173
357474497
387135100

255634688
297840827
350534960
356567761
357474499
387135134

255644801
297847402
356498085
356574704
357490035
387135136

255645821
297849372
356499771
356576401
357493567
387135174

255647456
300078590
356499777
356577660
357497139
387135176

255648275
300669727
356499779
357114993
357497581
387135184

260279126
302142947
356501328
357115447
357497671
387135186

260279128
302142948
356502523
357115451
357500579
387135188

261343326
302142950
356503180
357115453
357504663
387135190

283132367
302142951
356503184
357116080
357504691
387135192

283362112
302765302
356503295
357116928
357504699
387135194

289188052
302796334
356504436
357117461
357504707
387135282

295841350
302811470
356504523
357117463
357505859
387135284

296088529
302821107
356504765
357117829
357510851
387135294

296090415
302821679
356511113
357117839
357516975
387135298

296090524
319759260
356515120
357125059
359477003
387135300

296090526
319759266
356517088
357126015
359477998
387135302

297599503
320148814
356520732
357134488
359478043
387135304

297601531
326489963
356522586
357135657
359478286
387135312

297611791
326490273
356522588
357138503
359484299
387135314

297722841
326491131
356522590
357139683
359486936
387135316

387135318
449440433
460376293
460413408
462423864
475546199

387135320
449445896
460378310
460416351
470101924
475556485

387135322
449446454
460380744
462394387
470102280
475559699

387135324
449447657
460381726
462394433
470102858
475578293

387135326
449449002
460382093
462394557
470104211
475591753

387135328
449449004
460382095
462395646
470104264
475593742

388493506
449449006
460382754
462395678
470104266
475612072

388495496
449451379
460384935
462396388
470106317
475622476

388498446
449451589
460384937
462396389
470106357
475622507

388499220
449451591
460385076
462396419
470115448
475623787

388502176
449451593
460385872
462396542
470130404
482550481

388517521
449453712
460386018
462397507
470131550
482550499

388519407
449453714
460389217
462399998
470136482
482550740

388521413
449453716
460394872
462400798
470136484
482550999

388827901
449453732
460396139
462401217
470136488
482552352

388827903
449457075
460397862
462402118
470136492
482554970

388827907
449467555
460397864
462402237
470137933
482555336

388827909
449468742
460398541
462402284
470137937
482555478

388827913
449495638
460403139
462402416
470140422
482556454

393887637
449495736
460403141
462404228
470140426
482557289

393887646
449499880
460403143
462406358
470140908
482558462

393887649
449502786
460403145
462408262
470141232
482558508

393990627
449503471
460405998
462409325
470142008
482558547

397746860
449503473
460407578
462409359
470142010
482561055

397789318
449515857
460407590
462409777
470142012
482561555

413924864
449518643
460409128
462411467
470143607
482562795

414590349
449519559
460409134
462414311
470143939
482562850

414590661
449522783
460409136
462414416
470145404
482565074

414591157
449524530
460409459
462414476
473923244
482566269

414879558
449524591
460409461
462415526
474114354
482566296

414879559
449528823
460409463
462415603
474143634
482566307

414879560
449528825
460409465
462415731
474202268
482568689

414888074
449534021
460409467
462416307
474299266
482570049

431812559
460365546
460410124
462416920
474363119
482570572

449432064
460366882
460410126
462416922
474366157
482575121

449432066
460369823
460410128
462416923
474429346

449433069
460369829
460410130
462416924
475432777

449436944
460369831
460410132
462417401
475473002

449438665
460369833
460410134
462419769
475489790

449438667
460370755
460410213
462420317
475511330

449440431
460374714
460411200
462423366
475516200

TABLE 1

GI number
Accession
Origin

190692175
ACE87855.1

Stevia rebaudiana

41469452
AAS07253.1

Oryza saliva

62857204
BAD95881.1

Ipomoea nil

62857206
BAD95882.1

Ipomoea purperea

56550539
BAD77944.1

Bellis perennis

115454819
NP_001051010.1

Oryza sativa Japonica Group

115459312
NP_001053256.1

Oryza sativa Japonica Group

115471069
NP_001059133.1

Oryza saliva Japonica Group

115471071
NP_001059134.1

Oryza saliva Japonica Group

116310985
CAH67920.1

Oryza sativa Indica Group

116788066
ABK24743.1

Picea sitchensis

122209731
Q2V6J9.1

Fragaria × ananassa

125534461
EAY81009.1

Oryza sativa Indica Group

125559566
EAZ05102.1

Oryza sativa Indica Group

125588307
EAZ28971.1

Oryza sativa Japonica Group

148907340
ABR16806.1

Picea sitchensis

148910082
ABR18123.1

Picea sitchensis

148910612
ABR18376.1

Picea sitchensis

15234195
NP_194486.1

Arabidopsis thaliana

15239523
NP_200210.1

Arabidopsis thaliana

15239937
NP_196793.1

Arabidopsis thaliana

1685005
AAB36653.1

Nicotiana tabacum

183013903
ACC38471.1

Medicago truncatula

186478321
NP_172511.3

Arabidopsis thaliana

187373030
ACD03249.1

Avena strigosa

194701936
ACF85052.1

Zea mays

19743740
AAL92461.1

Solanum lycopersicum

212275846
NP_001131009.1

Zea mays

222619587
EEE55719.1

Oryza sativa Japonica Group

224055535
XP_002298527.1

Populus trichocarpa

224101569
XP_002334266.1

Populus trichocarpa

224120552
XP_002318358.1

Populus trichocarpa

224121288
XP_002330790.1

Populus trichocarpa

225444853
XP_002281094

Vitis vinifera

225454342
XP_002275850.1

Vitis vinifera

225454475
XP_002280923.1

Vitis vinifera

225461556
XP_002285222

Vitis vinifera

225469540
XP_002270294.1

Vitis vinifera

226495389
NP_001148083.1

Zea mays

226502400
NP_001147674.1

Zea mays

238477377
ACR43489.1

Triticum aestivum

240254512
NP_565540.4

Arabidopsis thaliana

2501497
Q43716.1

Petunia × hybrida

255555369
XP_002518721.1

Ricinus communis

26452040
BAC43110.1

Arabidopsis thaliana

296088529
CBI37520.3

Vitis vinifera

297611791
NP_001067852.2

Oryza sativa Japonica Group

297795735
XP_002865752.1

Arabidopsis lyrata subsp. lyrata

297798502
XP_002867135.1

Arabidopsis lyrata subsp. lyrata

297820040
XP_002877903.1

Arabidopsis lyrata subsp. lyrata

297832276
XP_002884020.1

Arabidopsis lyrata subsp. lyrata

302821107
XP_002992218.1

Selaginella moellendorffii

30680413
NP_179446.2

Arabidopsis thaliana

319759266
ADV71369.1

Pueraria montana var. lobata

326507826
BAJ86656.1

Hordeum vulgare subsp. Vulgare

343457675
AEM37036.1

Brassica rapa subsp. oleifera

350534960
NP_001234680.1

Solanum lycopersicum

356501328
XP_003519477.1

Glycine max

356522586
XP_003529927.1

Glycine max

356535480
XP_003536273.1

Glycine max

357445733
XP_003593144.1

Medicago truncatula

357452783
XP_003596668.1

Medicago truncatula

357474493
XP_003607531.1

Medicago truncatula

357500579
XP_003620578.1

Medicago truncatula

357504691
XP_003622634.1

Medicago truncatula

359477998
XP_003632051.1

Vitis vinifera

359487055
XP_002271587

Vitis vinifera

359495869
XP_003635104.1

Vitis vinifera

387135134
AFJ52948.1

Linum usitatissimum

387135176
AFJ52969.1

Linum usitatissimum

387135192
AFJ52977.1

Linum usitatissimum

387135282
AFJ53022.1

Linum usitatissimum

387135302
AFJ53032.1

Linum usitatissimum

387135312
AFJ53037.1

Linum usitatissimum

388519407
AFK47765.1

Medicago truncatula

393887646
AFN26668.1

Barbarea vulgaris subsp. arcuata

414888074
DAA64088.1

Zea mays

42572855
NP_974524.1

Arabidopsis thaliana

449440433
XP_004137989.1

Cucumis sativus

449446454
XP_004140986.1

Cucumis sativus

449449004
XP_004142255.1

Cucumis sativus

449451593
XP_004143546.1

Cucumis sativus

449515857
XP_004164964.1

Cucumis sativus

460382095
XP_004236775.1

Solanum lycopersicum

460409128
XP_004249992.1

Solanum lycopersicum

460409461
XP_004250157.1

Solanum lycopersicum

460409465
XP_004250159.1

Solanum lycopersicum

462396388
EMJ02187.1

Prunus persica

462402118
EMJ07675.1

Prunus persica

462409359
EMJ14693.1

Prunus persica

462416923
EMJ21660.1

Prunus persica

46806235
BAD17459.1

Oryza saliva Japonica Group

470104266
XP_004288529.1

Fragaria vesca subsp. vesca

470142008
XP_004306714.1

Fragaria vesca subsp. vesca

475432777
EMT01232.1

Aegilops tauschii

51090402
BAD35324.1

Oryza sativa Japonica Group

TABLE 2

Internal

GI number
Accession
Origin
reference

460409128
XP.004249992.1

Solanum lycopersicum

UGTSl

460386018
XP.004238697.1

Solanum lycopersicum

—

460409134
XP.004249995.1

Solanum lycopersicum

—

460410132
XP.004250485.1

Solanum lycopersicum

UGTSl2

460410130
XP.004250484.1

Solanum lycopersicum

—

460410128
XP.004250483.1

Solanum lycopersicum

—

460378310
XP.004234916.1

Solanum lycopersicum

—

209954733
BAG80557.1

Lycium barbarum

UGTLB

209954725
BAG80553.1

Lycium barbarum

—

One embodiment of the present invention is a microbial cell comprising an enzyme, i.e. an enzyme capable of converting the starting composition to the target steviol glycoside. Accordingly, some embodiments of the present method include contacting a microorganism with a medium containing the starting composition to provide a medium comprising at least one target steviol glycoside.

The microorganism can be any microorganism possessing the necessary enzyme(s) for converting the starting composition to target steviol glycoside(s). These enzymes are encoded within the microorganism's genome.

Suitable microoganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc.

In one embodiment, the microorganism is free when contacted with the starting composition.

In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, the microorganism may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the microorganism include derivatized cellulose or glass, ceramics, metal oxides or membranes. The microorganism may be immobilized to the solid support, for example, by covalent attachment, adsorption, cross-linking, entrapment or encapsulation.

In still another embodiment, the enzyme capable of converting the starting composition to the target steviol glycoside is secreted out of the microorganism and into the reaction medium.

The target steviol glycoside is optionally purified. Purification of the target steviol glycoside from the reaction medium can be achieved by at least one suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.

Uses

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this invention can be used “as-is” or in combination with other sweeteners, flavors, food ingredients and combinations thereof.

Non-limiting examples of flavors include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla and combinations thereof.

Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this invention can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous, amorphous forms and combinations thereof.

Highly purified target glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this invention may be employed as a sweetening compound as the sole sweetener, or it may be used together with at least one naturally occurring high intensity sweeteners such as rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside rebaudioside M2, rebaudioside N, rebaudioside O, rebaudioside O2, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2, stevioside F, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, 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, siamenoside and combinations thereof.

In a particular embodiment, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in a sweetener composition comprising a compound selected from the group consisting of rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside O, rebaudioside O2, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2, stevioside F, NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside K4), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be used in combination with synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.

Moreover, highly purified target steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be combined with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be mixed with umami tasting and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and combinations thereof.

Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be combined with polyols or sugar alcohols. The term “polyol” refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be combined with various carbohydrates. The term “carbohydrate” generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH₂O)_n, wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivatives”, “substituted carbohydrate”, and “modified carbohydrates” are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used as a flavor modifier or flavor with modifying properties include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.

During the manufacturing of products such as foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, and chewing gum, the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.

Moreover, the highly purified target steviol glycoside(s) steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained in this invention may be used in dry or liquid forms.

The highly purified target steviol glycoside can be added before or after heat treatment of food products. The amount of the highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM depends on the purpose of usage. As discussed above, it can be added alone or in combination with other compounds.

The present invention is also directed to sweetness enhancement in beverages using steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. Accordingly, the present invention provides a beverage comprising a sweetener and steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM as a sweetness enhancer, wherein steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM is present in a concentration at or below their respective sweetness recognition thresholds.

As used herein, the term “sweetness enhancer” refers to a compound capable of enhancing or intensifying the perception of sweet taste in a composition, such as a beverage. The term “sweetness enhancer” is synonymous with the terms “sweet taste potentiator,” “sweetness potentiator,” “sweetness amplifier,” and “sweetness intensifier.”

The term “sweetness recognition threshold concentration,” as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE). Generally, the sweetness enhancers may enhance or potentiate the sweet taste of sweeteners without providing any noticeable sweet taste by themselves when present at or below the sweetness recognition threshold concentration of a given sweetness enhancer; however, the sweetness enhancers may themselves provide sweet taste at concentrations above their sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific for a particular enhancer and can vary based on the beverage matrix. The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.

In some embodiments, sweetener is present in the beverage in an amount from about 0.5% to about 12% by weight, such as, for example, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight, about 11.5% by weight or about 12.0% by weight.

In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.5% of about 10%, such as for example, from about 2% to about 8%, from about 3% to about 7% or from about 4% to about 6% by weight. In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.5% to about 8% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount from about 2% to about 8% by weight.

In one embodiment, the sweetener is a traditional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup and high fructose starch syrup.

In another embodiment, the sweetener is erythritol.

In still another embodiment, the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-psicose, D-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose, D-turanose, D-leucrose and combinations thereof.

It is contemplated that a sweetener can be used alone, or in combination with other sweeteners.

In one embodiment, the rare sugar is D-allose. In a more particular embodiment, D-allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In another embodiment, the rare sugar is D-psicose. In a more particular embodiment, D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In still another embodiment, the rare sugar is D-ribose. In a more particular embodiment, D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In yet another embodiment, the rare sugar is D-tagatose. In a more particular embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In a further embodiment, the rare sugar is L-glucose. In a more particular embodiment, L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In one embodiment, the rare sugar is L-fucose. In a more particular embodiment, L-fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In another embodiment, the rare sugar is L-arabinose. In a more particular embodiment, L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In yet another embodiment, the rare sugar is D-turanose. In a more particular embodiment, D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

In yet another embodiment, the rare sugar is D-leucrose. In a more particular embodiment, D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

The addition of the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer. Moreover, sweetness can be increased by an amount more than the detectable sweetness of a solution containing the same concentration of the at least one sweetness enhancer in the absence of any sweetener.

Accordingly, the present invention also provides a method for enhancing the sweetness of a beverage comprising a sweetener comprising providing a beverage comprising a sweetener and adding a sweetness enhancer selected from steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM or a combination thereof, wherein steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM are present in a concentration at or below their sweetness recognition thresholds.

Addition of steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM in a concentration at or below the sweetness recognition threshold to a beverage containing a sweetener may increase the detected sucrose equivalence from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.

The following examples illustrate preferred embodiments of the invention for the preparation of highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are only illustrative.

EXAMPLES
Example 1

Protein Sequences of Engineered Enzymes Used in the Biocatalytic Process

SEQ ID 1:

>SuSy_At, variant PM1-54-2-E05 (engineered

sucrose synthase; source of WT gene:

Arabidopsis thaliana)

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII

AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL

RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT

LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKI

QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL

DMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD

TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCG

ERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVEL

SKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDI

YWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHT

AFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEI

EELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL

RELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMD

RVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPA

EIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIE

EKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQ

AVPLAQDD

SEQ ID 2:

>UGTS12 variant 0234 (engineered

glucosyltransferase; source of WT gene:

Solanum lycopersicum)

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK

IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN

FSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSF

RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS

RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENS

TVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP

EGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVP

IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGE

TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3:

>UGT76G1 variant 0042 (engineered

glucosyltransferase; source of WT gene:

Stevia rebaudiana)

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNF

NKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADE

LRRELELLMLASEEDEEVSCLITDALWYFAQDVADSLNLRRLVLMTSSLF

NFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIG

KEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHL

TASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLV

DSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAI

GAFWTHSGWNSTLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLEN

GWERGEVVNAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLES

LVSYISSL

Example 2

Expression and Formulation of SuSy_At Variant of SEQ ID 1

The gene coding for the SuSy_At variant of SEQ ID 1 (EXAMPLE 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37° C. Expression of the genes was induced at logarithmic phase by IPTG (0.2 mM) and carried out at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220×g, 20 min, 4° C.) and re-suspended to an optical density of 200 (measured at 600 nm (OD₆₀₀)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl₂, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000×g 40 min, 4° C.). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with distilled water, resulting in an enzymatic active preparation.

For enzymatic active preparations of SuSy_At, activity in Units is defined as follows: 1 mU of SuSy_At turns over 1 nmol of sucrose into fructose in 1 minute. Reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose at t₀, 3 mM MgCl₂, and 15 mM uridine diphosphate (UDP).

Example 3

Expression and Formulation of UGTS12 Variant of SEQ ID 2

The gene coding for the UGTS12 variant of SEQ 1D 2 (EXAMPLE 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37° C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220×g, 20 min, 4° C.) and re-suspended to an optical density of 200 (measured at 600 nm (OD₆₀₀)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl₂, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000×g 40 min, 4° C.). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

For enzymatic active preparations of UGTS12, activity in Units is defined as follows: 1 mU of UGTS12 turns over 1 nmol of rebaudioside A (RebA) into rebaudioside D (Reb D) in 1 minute. Reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at t₀, 500 mM sucrose, 3 mM MgCl₂, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.

Example 4

Expression and Formulation of UGT76G1 Variant of SEQ ID 3

The gene coding for the UGT76G1 variant of SEQ ID 3 (EXAMPLE 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/I) at 37° C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220×g, 20 min, 4° C.) and re-suspended to an optical density of 200 (measured at 600 nm (OD₆₀₀)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl₂, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000×g 40 min, 4° C.). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

For enzymatic active preparations of UGT76G1, activity in Units is defined as follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D) into rebaudioside M (Reb M) in 1 minute. Reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at t₀, 500 mM sucrose, 3 mM MgCl₂, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.

Example 5

Synthesis of Rebaudioside AM from Stevioside in a One-Pot Reaction, Adding UGTS12, SuSy_At and UGT76G1 at the Same Time

Rebaudioside AM (reb AM) was synthesized directly from stevioside in a one-pot reaction (FIG. 3), utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4): UGTS12 (variant of SEQ ID 2), SuSy_At-(variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 105 U/L UGTS12, 405 U/L SuSy_At, 3 U/L UGT76G1, 5 mM stevioside, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl₂and potassium phosphate buffer (pH 6.6). First, 207 mL of distilled water were mixed with 0.24 g MgCl₂.6H₂O, 103 g sucrose, 9.9 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 15 g stevioside. After dissolving the components, the temperature was adjusted to 45° C. and UGTS12, SuSy_At, UGT76G1 and 39 mg UDP were added. The reaction mixture was incubated at 45° C. shaker for 24 hrs. Additional 39 mg UDP was added at 8 hrs and 18 hours. The content of reb AM, reb E, stevioside, reb M, reb B, steviolbioside and reb/at several time points was analyzed by HPLC.

For analysis, biotransformation samples were inactivated by adjusting the reaction mixture to pH5.5 using 17% H₃PO₄and then boiled for 10 minutes. Resulting samples were filtered, the filtrates were diluted 10 times and used as samples for HPLC analysis. HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a pump, a column thermostat, an auto sampler, a UV detector capable of background correction and a data acquisition system. Analytes were separated using Agilent Poroshell 120 SB— C18, 4.6 mm×150 mm, 2.7 μm at 40° C. The mobile phase consisted of two premixes:

- premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile, and
- premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32% acetonitrile.

Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5 minute, changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes. The column temperature was maintained at 40° C. The injection volume was 5 μL. Rebaudioside species were detected by UV at 210 nm.

Table 3 shows for each time point the conversion of stevioside into identified rebaudioside species (area percentage). The chromatograms of stevioside and the reaction mixture at 24 hours are shown in FIG. 5 and FIG. 6, respectively. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.

TABLE 3

Biotransformation of stevioside to reb AM

Time,
% conversion from stevioside

hrs
Reb E
Reb AM
Reb M
Reb I
Stevioside
Reb B
Steviolbioside

0
0
0
0
0
100
0
0

6
1.9
35.9
1.3
1.7
58.7
0.0
0.4

18
0.9
96.7
1.3
0.6
0.0
0.0
0.4

24
0.3
96.4
2.1
0.7
0.0
0.2
0.4

Example 6

Synthesis of Rebaudioside AM from Rebaudioside E in a One-Pot Reaction, SuSy_At and UGT76G1 at the Same Time

Rebaudioside AM (reb AM) was synthesized directly from rebaudioside E (reb E) in a one-pot reaction (FIG. 4), utilizing the two enzymes (see EXAMPLES 1, 2 and 4): SuSy_At-(variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 405 U/L SuSy_At, 3 U/L UGT76G1, 5 mM reb E, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl₂.6H₂O and potassium phosphate buffer (pH 6.6). First, 37 mL of distilled water were mixed with 40.3 mg MgCl₂, 17.12 g sucrose, 1.65 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 5.04 g reb E. After dissolving the components, the temperature was adjusted to 45° C. and SuSy_At, UGT76G1 and 6.5 mg UDP were added. The reaction mixture was incubated at 45° C. shaker for 24 hrs. Additional 6.5 mg UDP was added at 8 hrs and 18 hours. The content of reb AM, reb E, stevioside, reb A, reb M, reb B, and steviolbioside at several time points was analyzed by HPLC.

- premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile, and
- premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32% acetonitrile.

Table 4 shows for each time point the conversion of reb E into identified rebaudioside species (area percentage). The chromatograms of reb E and the reaction mixture at 24 hours are shown in FIG. 7 and FIG. 8, respectively. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.

TABLE 4

Biotransformation of reb E to reb AM

% conversion from Reb E

Time,
Reb
Reb
Reb
Reb

Reb
Steviol-

hrs
E
AM
M
A
Stevioside
B
bioside

0
99.46
0
0
0.54
0
0
0

4
40.75
57.92
0
0.59
0
0.73
0

7
24.79
73.92
0
0.58
0
0.71
0

24
4.38
94.33
0
0.59
0
0.70
0

Example 7

Purification of Rebaudioside AM

The reaction mixture of EXAMPLE 5, after 24 hrs, was inactivated by adjusting the pH to pH 5.5 with H₃PO₄and then boiled for 10 minutes. After boiling the reaction mixture was filtered and diluted with RO water to 5% solids content. The diluted solution was passed through 1 L column packed with YWD03 macroporous adsorption resin (Cangzhou Yuanwei, China). Adsorbed steviol glycosides were eluted with 5 L 70% ethanol. The obtained eluate was evaporated until dryness to yield 16 g of dry powder which was dissolved in 80 mL of 70% methanol. The solution was crystallized at 20° C. for 3 days. The crystals were separated by filtration and dried in vacuum oven at 80° C. for 18 hours to yield 10.4 g of pure reb AM crystals with 95.92% purity, determined by HPLC assay. The chromatogram of reb AM is shown in FIG. 9. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.

Example 8

Structure Elucidation of Rebaudioside AM

NMR experiments were performed on a Bruker 500 MHz spectrometer, with the sample dissolved in pyridine-d5. Along with signals from the sample, signals from pyridine-d5 at δ_C123.5, 135.5, 149.9 ppm and δ_H7.19, 7.55, 8.71 ppm were observed.

¹H-NMR-spectrum of rebaudioside AM in pyridine-d₅reveal the excellent quality of the sample (see FIG. 10). The HSQC (see FIG. 11) shows the presence of an exo-methylene group in the sugar region with a long-range coupling to C-15, observable in the H,H—COSY (FIG. 12). Other deep-fielded signals of the quaternary carbons (C-13, C-16 and C-19) are detected by the HMBC (FIG. 13). Correlation of the signals in the HSQC, HMBC and H,H—COSY reveal the presence of steviol glycoside with the following aglycone structure:

embedded image

Correlation of HSQC and HMBC signals reveal five anomeric signals. The coupling constant of the anomeric protons of about 8 Hz and the broad signals of their sugar linkage allows the identification of these five sugars as β-D-glucopyranosides.

The observation of the anomeric protons in combination with HSQC and HMBC reveal the sugar linkage and the correlation to the aglycone. The assignment of the sugar sequence was confirmed by using the combination of HSQC-TOCSY (FIG. 14) and HSQC.

The NMR experiments above were applied to assign the chemical shifts of the protons and carbons, main coupling constants and main HMBC correlations (see Table 5).

TABLE 5

Chemical shifts of rebaudioside AM

Position
δ_C[ppm]
δ_H[ppm]
J [Hz]
HMBC (H → C)

Aglycone moiety

1
39.9
t
0.68
m

1.64
m

2
19.4
t
1.39
m

2.08
m

3
37.4
t
1.05
m

2.80
m

4
44.2
s
—

5
57.3
d
0.95
m

6
21.7
t
1.90
m

2.12
m

7
41.0
t
1.26
m

1.38
m

8
41.9
s
—

9
53.3
d
0.85
m

10
39.2
s
—

11
20.1
t
1.59
m

1.61
m

12
36.9
t
1.65
m

1.92
m

13
85.9
s
—

14
43.8
t
1.78
d
11.0

2.52
d
11.0

15
47.4
t
2.00
d
16.0
7, 8, 9, 14

2.06
d
16.0

16
154.6
s
—

17
104.3
t
5.03
br s

13, 15, 16

5.71
br s

18
28.5
q
1.40
s

3, 4, 5, 19

19
175.2
s
—

20
16.2
q
1.06
s

1, 5, 9, 10

Sugar moiety

Sugar I: β-D-Glucopyranoside

1ⁱ
97.5
d
5.13
d
7.7
13

2ⁱ
84.0
d
4.14
m

3ⁱ
77.6
d
4.20
m

4ⁱ
71.3
d
4.19
m

5ⁱ
77.6
d
3.70
m

6ⁱ
62.0
t
4.23
m

4.32
m

Sugar II: β-D-Glucopyranoside

1ⁱⁱ
106.3
d
5.26
d
8.0
2*

2ⁱⁱ
76.8
d
4.13
m

3ⁱⁱ
77.3
d
4.21
m

4ⁱⁱ
71.6
d
4.18
m

5ⁱⁱ
77.9
d
3.91
m

6ⁱⁱ
62.4
t
4.29
m

4.41
m

Sugar III: β-D-Glucopyranoside

1ⁱⁱⁱ
92.9
d
6.20
d
8.1
19

2ⁱⁱⁱ
77.0
d
4.46
m

3ⁱⁱⁱ
88.1
d
4.24
m

4ⁱⁱⁱ
69.0
d
4.12
m

5ⁱⁱⁱ
78.4
d
3.82
m

6ⁱⁱⁱ
61.3
t
4.20
m

4.33
m

Sugar IV: β-D-Glucopyranoside

1^iv
103.4
d
5.73
d
7.7
2ⁱⁱⁱ

2^iv
75.4
d
3.98
m

3^iv
78.1
d
4.09
m

4^iv
72.6
d
4.08
m

5^iv
77.4
d
3.92
m

6^iv
62.9
t
4.32
m

4.51
m

Sugar V: β-D-Glucopyranoside

1^v
104.4
d
5.29
d
8.1
3ⁱⁱⁱ

2^v
75.1
d
4.00
m

3^v
78.2
d
4.24
m

4^v
71.4
d
4.27
m

5^v
78.2
d
3.99
m

6^v
61.9
t
4.27
m

4.48
m

Correlation of all NMR data indicates rebaudioside AM having five β-D-glucopyranoses attached to a steviol aglycone, as depicted with the following chemical structure:

embedded image

The chemical formula of rebaudioside AM is C₅₀H₈₀O₂₈, which corresponds to a calculated monoisotopic molecular mass of 1128.5. For LCMS analysis, rebaudioside AM was dissolved in methanol and analyzed using Shimadzu Nexera 2020 UFLC LCMS instrument on a Cortecs UPLC C18 1.6 μm, 50×2.1 mm column. The observed LCMS (negative ESI mode) result of 1127.3 (see FIG. 15a and FIG. 15b respectively) is consistent with rebaudioside AM and corresponds to the ion (M-H)⁻.

	Number	Date	Country
	62644065	Mar 2018	US
	62644407	Mar 2018	US

HIGH-PURITY STEVIOL GLYCOSIDES

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