KR20200133251A - High-purity steviol glycoside - Google Patents

Abstract

Highly purified steviol glycosides, especially steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside B, rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudio Side E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a , Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside Baudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudio Side 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f , Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebau A method of preparing Dioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 is described. The method includes the use of enzyme preparations and recombinant microorganisms to convert various starting compositions to target steviol glycosides. Highly purified steviol glycosides are useful as non-calorie sweeteners, flavor enhancers, sweetness enhancers, and antifoam agents in edible and chewing compositions such as beverages, confectionery, bakery products, cookies, and chewing gum.

Description

High-purity steviol glycoside

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

High-intensity sweeteners have a sweetness level that is much greater than that of sucrose. High intensity sweeteners are essentially non-calorie and are commonly used in diet and reduced-calorie products, including foods and beverages. High-intensity sweeteners do not elicit a glycemic response and make them suitable for use in products that target diabetes and other conditions where restrictions on carbohydrate intake are important.

Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni of the group Asteraceae ( Compositae ), which are native to certain regions of South America. Steviol glycosides are structurally characterized by single base steviols that differ in the presence of carbohydrate residues at positions C13 and C19. Steviol glycosides accumulate in stevia leaves, which make up approximately 10%-20% of the total dry weight. On a dry weight basis, in stevia leaves, the four major glycosides found are typically Stevioside (9.1%), Rebaudioside A (3.8%), Rebaudioside C (0.6-1.0%) and Dulco Includes Side A (0.3%). Other known steviol glycosides include rebaudiosides B , C , D , E , F and M, steviolbiosides and rubusosides.

From Stevia rebaudiana While methods for preparing steviol glycosides are known, many of these methods are unsuitable for commercial use.

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

Summary of the invention

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

As used herein, "biocatalytic" or "biocatalytic" refers to a naturally or genetically engineered biocatalyst capable of single or multiple step chemical transformation of an organic compound, such as an enzyme, or a cell comprising one or more enzymes. Refers to the use of. Biocatalytic processes include fermentation, biosynthesis, bioconversion and bioconversion processes. Both isolated enzymes and whole-cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring or recombinant proteins.

As used herein, the term “steviol glycoside(s)” refers to, but is not limited to, naturally occurring steviol glycosides, such as steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, Rubusoside, Steviol Bioside A, Steviol Bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A , Rebaudioside E, Rebaudioside E 2 , Rebaudioside E 4 , Rebaudioside E 6 , Rebaudioside E 3 , Rebaudioside D, Rebaudioside I, Rebaudio Side A M , Rebaudioside D 7 , Rebaudioside M, Rebaudioside M 4 , Rebaudioside 1 a , Rebaudioside 1 b , Rebaudioside 1 c , Rebaudioside 1 d , Rebaudioside 1 e , Rebaudioside 1 f , Rebaudioside 1 g , Rebaudioside 1 h , Rebaudioside 1 i , Rebaudioside 1 j , Rebaudioside 1 k , Rebaudioside 1 l , Rebaudioside 1 m , Rebaudioside 1 n , Rebaudioside 1 o, Rebaudioside 1 p, Rebaudioside 1 q, Rebaudioside 1 r, Rebaudioside 1 s , Rebaudioside 1 t, Rebaudioside 2 a, Rebaudioside 2 b, Rebaudioside 2 c, Rebaudioside 2 d , Rebaudioside 2 e , Re Baudioside 2 f , Rebaudioside 2 g , Rebaudioside 2 h , Rebaudioside 2 i , Rebaudioside 2 j , Rebaudioside 2 k , Rebaudioside 2 l , Rebau Dioside 2m , Rebaudioside 2n, Rebaudioside 2o , Rebaudioside 2p , Rebaudioside 2q , Rebaudioside 2r , Rebaudioside 2s , Synthetic steviol Glycosides, such as enzymatically glucosylated steviol glycosides And a glycoside of steviol comprising a combination thereof.

The term "SvG7" as used herein refers to enzymatically glucosylated steviol glycosides and combinations thereof, particularly, but not limited to, reb 1a , reb 1b , reb 1c , reb 1d , reb 1e , reb 1f , reb 1g , reb 1h , reb 1i , reb 1j , reb 1k , reb 1l , reb 1m , reb 1n , reb 1o, reb 1p, reb 1q, reb 1r, reb 1s , reb 1t, reb 2a, reb 2b, reb 2c, reb 2d, reb 2e, reb 2f, reb 2g, reb 2h, reb 2i, reb 2j, reb 2k, reb 2l, reb 2m, reb 2n, reb 2o, reb 2p , reb 2q, reb 2r , and/or reb 2s . Any naturally occurring steviol glycoside or any synthetic steviol glycoside, including molecules comprising steviol having 7 covalently attached glucose residues, Refers to. SvG7 may refer to a single steviol glycoside with 7 glucose residues covalently attached or a mixture of steviol glycosides with 7 glucose residues covalently attached.

The present invention comprises 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 an enzyme preparation to prepare a composition comprising a target steviol glycoside. to provide.

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 D, rubusoside, steviolbioside A, steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4 , Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside Baudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudio Side 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q , Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside Baudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s, Synthetic steviol glycoside, e.g., enzymatically glucosylated ste It is a glycoside of steviol, including viol glycosides and combinations thereof.

In one embodiment, the target steviol glycoside is rebaudioside 1 a .

In one embodiment, the target steviol glycoside is rebaudioside 1 b .

In one embodiment, the target steviol glycoside is rebaudioside 1 c .

In one embodiment, the target steviol glycoside is rebaudioside 1 d .

In one embodiment, the target steviol glycoside is rebaudioside 1 e .

In one embodiment, the target steviol glycoside is rebaudioside 1 f .

In one embodiment, the target steviol glycoside is 1 g of rebaudioside.

In one embodiment, the target steviol glycoside is rebaudioside 1 h .

In one embodiment, the target steviol glycoside is rebaudioside 1 i .

In one embodiment, the target steviol glycoside is rebaudioside 1 j .

In one embodiment, the target steviol glycoside is rebaudioside 1 k .

In one embodiment, the target steviol glycoside is 1 l of rebaudioside.

In one embodiment, the target steviol glycoside is 1 m of rebaudioside.

In one embodiment, the target steviol glycoside is rebaudioside 1 n .

In one embodiment, the target steviol glycoside is rebaudioside 1 o .

In one embodiment, the target steviol glycoside is rebaudioside 1 p .

In one embodiment, the target steviol glycoside is rebaudioside 1 q .

In one embodiment, the target steviol glycoside is rebaudioside 1 r .

In one embodiment, the target steviol glycoside is rebaudioside 1 s .

In one embodiment, the target steviol glycoside is 1 t of rebaudioside.

In one embodiment, the target steviol glycoside is rebaudioside 2 a .

In one embodiment, the target steviol glycoside is rebaudioside 2 b .

In one embodiment, the target steviol glycoside is rebaudioside 2 c .

In one embodiment, the target steviol glycoside is rebaudioside 2 d .

In one embodiment, the target steviol glycoside is rebaudioside 2 e .

In one embodiment, the target steviol glycoside is rebaudioside 2 f .

In one embodiment, the target steviol glycoside is 2 g of rebaudioside.

In one embodiment, the target steviol glycoside is rebaudioside 2 h .

In one embodiment, the target steviol glycoside is rebaudioside 2 i .

In one embodiment, the target steviol glycoside is rebaudioside 2 j .

In one embodiment, the target steviol glycoside is rebaudioside 2 k .

In one embodiment, the target steviol glycoside is 2 l of rebaudioside.

In one embodiment, the target steviol glycoside is 2 m rebaudioside.

In one embodiment, the target steviol glycoside is rebaudioside 2 n .

In one embodiment, the target steviol glycoside is rebaudioside 2 o .

In one embodiment, the target steviol glycoside is rebaudioside 2 p .

In one embodiment, the target steviol glycoside is rebaudioside 2 q .

In one embodiment, the target steviol glycoside is rebaudioside 2 r .

In one embodiment, the target steviol glycoside is rebaudioside 2 s .

In one embodiment, the target steviol glycoside is rebaudioside M4 .

In one embodiment, the target steviol glycoside is SvG7.

In some preferred embodiments, one or more enzymes capable of converting the starting composition to a target steviol glycoside, or an enzyme preparation comprising microbial cells comprising one or more enzymes are used. Enzymes can be located on the cell surface and/or within. Enzyme preparations can be provided in the form of whole cell suspensions, crude lysates or as purified enzyme(s). The enzyme preparation can be immobilized in free form or on a solid support made from inorganic or organic materials.

In some embodiments, the microbial cell contains the necessary enzymes to convert its starting composition to a target steviol glycoside and a gene encoding it. Accordingly, the present invention provides a medium comprising at least one target steviol glycoside by contacting the starting composition comprising an organic substrate with a microbial cell comprising at least one enzyme convertible to the target steviol glycoside. It also provides a process for preparing a composition comprising the target steviol glycoside by preparing.

Enzymes required to convert the starting composition to the target steviol glycosides are steviol biosynthetic enzymes NDP-glucosyltransferase (NGTs), ADP-glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs), GDP -Glucosyltransferases (GGTs), TDP-glucosyltransferases (TDPs), UDP-glucosyltransferases (UGTs), and/or NDP-regeneration enzymes, ADP-regeneration enzymes, CDP-regeneration enzymes, GDP-regeneration Enzymes, TDP-regenerating enzymes and/or UDP-regenerating enzymes.

In one embodiment, the steviol biosynthetic enzyme comprises a mevalonate (MVA) pathway enzyme.

In another embodiment, the steviol biosynthetic enzyme comprises a non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the steviol biosynthetic enzyme is geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, cowene synthase, cowene oxidase, cowenic acid 13-hydroxylase (KAH), ste Viol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C- Methyl-D-erythritol synthase (CMS), 4-diphosphocytyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytyl-2-C-methyl-D -Erythritol 2,4-cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), l-hydroxy-2- Methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrolysis Phosphate decarboxylase, cytochrome P450 reductase, and the like.

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

In the following, the term "SuSy_AT" as used herein is equivalent to (>85% >86%) sucrose synthase or SEQ ID 1 polypeptide having the amino-acid sequence "SEQ ID 1" described in Example 1, unless otherwise specified. >87% >88% >89% >90% >91% >92% >93% >94% >95% >96% >97% >98% >99%) polypeptides with amino-acid sequence identity Refers to an isolated nucleic acid molecule that encodes a polypeptide.

In the following, the term ""UGTSl2" as used herein is equivalent to (>85%) a UDP-glucosyltransferase or SEQ ID 2 polypeptide having the amino-acid sequence "SEQ ID 2" described in Example 1, unless otherwise specified. >86% >87% >88% >89% >90% >91% >92% >93% >94% >95% >96% >97% >98% >99%) with amino-acid sequence identity Polypeptide also refers to an isolated nucleic acid molecule encoding such a polypeptide.

In the following, the term ““UGT76G1” as used herein is equivalent to (>85%) a UDP-glucosyltransferase or SEQ ID 3 polypeptide having the amino-acid sequence “SEQ ID 3” described in Example 1, unless otherwise specified. >86% >87% >88% >89% >90% >91% >92% >93% >94% >95% >96% >97% >98% >99%) with amino-acid sequence identity Polypeptide also refers to an isolated nucleic acid molecule encoding such a polypeptide.

In one embodiment, steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in microbial cells. Microbial cell, for example, Escherichia coli (E. coli), saccharose in MRS (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, Yarrow subtotal (Yarrowia) species Etc. In another embodiment, UDP-glucosyltransferase is synthesized.

In one embodiment, UDP-glucosyltransferase is equivalent to these polypeptides (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGTs with amino-acid sequence identity, also It is selected from the group containing isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGTs and UDP-glucose regeneration system are present in one microorganism (microbial cell). Microorganism may be, for example, all E. coli (E. coli), saccharose in MRS (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, Yarrow subtotal (Yarrowia) .

In one embodiment, the UDP-glucosyltransferase is capable of adding at least one glucose unit at C13, to steviol bearing an -OH functional group, or to any starting steviol glycoside, so that at C13, O-glucose beta glucose. Copyranoside is any UDP-glucosyltransferase that produces a target steviol glycoside with a glycoside linkage. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is capable of adding at least one glucose unit at C19, to steviol bearing a -COOH functional group or to any starting steviol glycoside, so that at C19, -COO-glucose beta -Glucopyranoside Any UDP-glucosyltransferase that produces a target steviol glycoside with a glycoside linkage. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with >85% amino-acid sequence identity with UGT74G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→2 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed bound glycoside with at least one additional glucose. In binding(s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→3 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces the target steviol glycoside with at least one beta 1→4 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→6 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→2 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed bound glycoside with at least one additional glucose. In binding(s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→3 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces the target steviol glycoside with at least one beta 1→4 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→6 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a steviol mono side A to be added to at least one of the glucose units to steviol. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT74G1 or 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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a bio-steviol side D to be added to at least one glucose unit in steviol mono side A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a bio-steviol side A to be added to at least one glucose unit in steviol mono A side. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a steviol bio B side to be added to at least one glucose unit in steviol mono side A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 D to form rebaudioside G. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT74G1 or UGT74G1.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rubusoside to form stevioside . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 rebaudioside G. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or 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. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a stevioside B to be added to at least one glucose unit in the base fusu side. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form the at least one bio-glucose side A stevioside C to be added to the unit to steviol. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In one embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase can be added to the at least one side of rain bio B Ste the glucose unit forming the stevioside B. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT85C2 or UGT85C2 or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form at least one C stevioside, in addition to possible rain bio B side stacking a glucose unit. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 B to form rebaudioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a rebaudioside E to be added to at least one glucose unit in the stevioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 G to form rebaudioside E6 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a rebaudioside E to be added to at least one glucose unit on stevioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 A to form rebaudioside E4. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to form rebaudioside E6 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 steviolbioside C to form rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT85C2 or UGT85C2 or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 A to form rebaudioside I. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 E4 to form rebaudioside D. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 E6 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M4 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1a . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1b . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1c . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1d . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1e . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1f . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form 1 g of rebaudioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1h . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1i . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1j . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1k . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1l . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1o . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1p . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1r . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1s . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1t . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2b . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2c . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2d . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2e . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2f . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form 2g of rebaudioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 h . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2i . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2j . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2k . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2l . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2n . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2o . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2p . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2r . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2s . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is UGT76G1 or UGT76G1 with >85% amino-acid sequence identity with UGT. Optionally, the method of the present invention further comprises regenerating UDP to provide UDP-glucose. In one embodiment, the method is regenerated so that the bioconversion of steviol and/or steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose. And regenerating UDP by providing a catalyst and regeneration substrate.

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

In one embodiment, the regeneration substrate for the UDP-glucose regeneration catalyst is sucrose.

Optionally, the methods of the present invention further comprise the use of transglycosidases that use oligo- or poly-saccharides as sugar donors to modify the recipient target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, sacarase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, It includes fructosylvertase, alkaline invertase, acid invertase, and fructofuranosidase. In some embodiments, glucose including, but not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and sugar(s) other than glucose are transferred to the recipient target steviol glycoside. In one embodiment, the recipient steviol glycoside is Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebau Dioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2 r and/or Rebaudioside 2 s . In another embodiment, the recipient steviol glycoside is rebaudioside M4 . In another embodiment, the recipient steviol glycoside is SvG7.

Optionally, the method of the present invention further comprises isolating 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 crystallization, separation by membrane, centrifugation, extraction, chromatographic separation or a combination of such methods.

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

In one embodiment, the separation results in a composition comprising greater than about 80% target steviol glycosides by weight on an anhydride basis, ie, on a highly purified steviol glycoside composition. In another embodiment, separation results in a composition comprising greater than about 90% by weight of the target steviol glycoside. In certain embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside. In another embodiment, the composition comprises more than about 99% by weight of the target steviol glycoside.

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

The purified target steviol glycosides can be used in consumable products as sweetening agents, flavor modifiers, flavoring agents with modifying properties and/or antifoaming agents. Suitable consumer products include, but are not limited to, food, beverage, pharmaceutical compositions, tobacco products, functional compositions, oral care compositions, and cosmetic compositions.

1A to 1T show some of the SvG7 steviol glycosides rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, and rebau Dioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1 s and Rebaudioside 1 t shows the chemical structure of each.
Figures 2a to 2s show some SvG7 steviol glycosides rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, and rebau Dioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2 r and Rebaudioside 2 s shows the chemical structure of each.
3 shows the chemical structure of rebaudioside M4 .
Figures 4a to 4s are steviol and various intermediate steviol glycosides from Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside. 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1 s and Rebaudioside 1 t Rebaudioside 2a, Rebaudioside 2b, Rebaudioside Baudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudio Side 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2 r and Rebaudioside 2s, Rebaudioside M4 and the pathways for producing various steviol glycosides are shown.
Figures 5a-5t show rebaudioside 1a, rebaudioside 1b, rebaudioside 1a from rebaudioside A using simultaneous regeneration of enzymes UGTSl2 and UGT76G1 and UDP to UDP-glucose via sucrose synthase SuSy_At. Dioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1 g Rebaudioside 1 h Rebaudioside 1I, Rebaudioside 1 j Rebaudioside 1 k Rebaudioside 1 l Rebaudioside 1 m and Rebaudioside 1 n Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1 s and Rebaudioside 1 t shows the biocatalytic production of each.
Figures 6a-6t show from stevioside to rebaudioside 2a, rebaudioside 2b, rebaudioside 2c using the simultaneous regeneration of enzymes UGTSl2 and UGT76G1 and UDP to UDP-glucose via the sucrose synthase SuSy_At. , Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside Baudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and Rebaudioside 1 q represents the biocatalytic production of each.
Figure 7 shows the biocatalytic production of rebaudioside M4 from stevioside using the enzymes UGTSl2 and UGT76G1 and the concomitant regeneration of UDP to UDP-glucose via the sucrose synthase SuSy_At.
Figures 8a to 8t show rebaudioside 2a, rebaudioside 2b, rebaudioside 2b from rebaudioside A M using simultaneous regeneration of the enzymes UGTSl2 and UGT76G1 and UDP to UDP-glucose through the sucrose synthase SuSy_At. Baudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudio Side 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s And the biocatalytic production of each of rebaudioside 1q.
Figure 9 shows the biocatalytic production of rebaudioside M4 from rebaudioside A M using the enzymes UGTSl2 and UGT76G1 and the concomitant regeneration of UDP to UDP-glucose through the sucrose synthase SuSy_At.
Figures 10A-10T show rebaudioside 2a, rebaudioside 2b, rebaudio from rebaudioside M4 using simultaneous regeneration of enzymes UGTSl2 and UGT76G1 and UDP to UDP-glucose through sucrose synthase SuSy_At. Dioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and Rebaudioside 1q shows the biocatalytic production of each.
11A shows the HPLC chromatogram of stevioside. The peak with a retention time of 20.958 minutes corresponds to stevioside. The peak with a retention time of 20.725 min corresponds to Rebaudioside A. The peak at 32.925 min corresponds to Rebaudioside B. The peak at 33.930 min corresponds to steviol bioside.
11B shows the HPLC chromatogram of the product of biocatalytic production of SvG7 molecules from stevioside. The peak at 5.089 min corresponds to 2 m of rebaudioside. The peak at 6.459 minutes is equivalent to rebaudioside 2 a. The peak at 9.825 min corresponds to Rebaudioside AM . The peak at 13.845 min corresponds to Rebaudioside M. The peak at 32.974 min corresponds to Rebaudioside B. The peak at 33.979 min corresponds to steviol bioside.
11C shows the HPLC chromatogram of rebaudioside 2a after purification by HPLC. The peak with a retention time of 6.261 min corresponds to rebaudioside 2 a .
11D shows an HPLC chromatogram of 2m of rebaudioside after purification by HPLC. The peak with a retention time of 5.197 min corresponds to 2 m of rebaudioside.
Figure 12a shows the ¹ H NMR spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12b shows the HSQC spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12c shows the H,H COSY spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12d shows the HMBC spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12e shows the HSQC-TOCSY spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12f shows the 1D-NOESY spectrum of rebaudioside 2a (500 MHz, pyridine- d5 ).
Figure 12g shows the HPLC chromatogram of Rebaudioside 2a .
Figure 12h shows the mass spectrum of rebaudioside 2 a.
13A shows the 1H NMR spectrum of 2 m of rebaudioside (500 MHz pyridine- d5 ).
13B shows the HSQC spectrum of 2 m of rebaudioside (500 MHz pyridine- d5 ).
13C shows the H,H COSY spectrum of 2m of rebaudioside (500 MHz, pyridine- d5 ).
13D shows the HMBC spectrum of 2m of rebaudioside (500 MHz, pyridine- d5 ).
13E shows the HSQC-TOCSY spectrum of rebaudioside 2m (500 MHz, pyridine- d5 ).
13F shows the 1D-NOESY spectrum of rebaudioside 2m (500 MHz, pyridine- d5 ).
13G shows the HPLC chromatogram of Rebaudioside 2m .
13H shows the mass spectrum of 2m of rebaudioside.

The present invention comprises 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 an enzyme preparation to prepare a composition comprising a target steviol glycoside. to provide.

One object of the present invention is to target steviol glycosides from various starting compositions, particularly steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside Violbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebau Dioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebau Dioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 or to provide an efficient biocatalytic method for preparing synthetic steviol glycosides.

Starting composition

As used herein, “starting composition” refers to any composition (generally an aqueous solution) containing one or more organic compounds 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 steviol, steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside B, and lebau Dioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside Baudioside E6, Rebaudioside E3, Rebaudioside D Rebaudioside I, Rebaudioside AM, Rebaudioside D7 Rebaudioside M and/or Rebaudioside M4 or in plants Synthesis of other glycosides of steviol that occurs Steviol glycosides are selected from the group consisting of, for example, 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 another embodiment, the starting composition steviol glycoside is steviolbioside .

In still other embodiments, steviol glycoside starting composition is steviol bio D side.

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

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

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

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

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

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

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

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

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

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

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

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 E4 .

In another embodiment, the starting composition steviol glycoside is rebaudioside E6 .

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

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

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

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

In another embodiment, the starting composition steviol glycoside is rebaudioside D7 .

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

In another embodiment, the starting composition steviol glycoside is rebaudioside M4 .

The term “polyol” refers to a molecule containing more than one hydroxyl group. The polyol may be a diol triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. Polyols may also contain more than 4 hydroxyl groups, such as pentols, hexaols, heptaols, etc., each containing 5, 6, or 7 hydroxyl groups. Additionally, the polyol may also be a sugar alcohol, a polyhydric alcohol, or a polyalcohol in the reduced form of a 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, traitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosac Lides, reduced xylo-oligosaccharides, reduced genthio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzate, polyglycitol and sugar alcohols or any other reducible carbohydrate.

The term "carbohydrate" refers to the general formula (CH ₂ O) _n Aldehyde or ketone compounds substituted with multiple hydroxyl groups, where n refers to 3-30, also oligomers and polymers thereof. The carbohydrates of the present invention may also be substituted or deoxygenated at one or more positions. Carbohydrates used herein include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivative”, “substituted carbohydrate”, and “modified carbohydrate” are synonymous. Modified carbohydrates refer herein to any carbohydrate in which at least one valency is added, removed, or substituted, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Carbohydrate derivatives or substituted carbohydrates optionally at any corresponding C-position, deoxygenated, and/or one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative , Alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulphenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosph Phosphoryl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or a carbohydrate derivative or substituted carbohydrate function that improves the sweet taste of the sweetener composition. May be substituted with any other active functional group provided.

Examples of carbohydrates that can be used in accordance with the present invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose. Ross, Fructose, Threos, Arabinose, Xylose, Rixos, Allos, Altos, Mannos, Idos, Lactose, Maltose, Antiwar, Isotrehalose, Neotrehalose, Isomaltulose, Eritros, De Oxyribose, Gulose, Idos, Talos, Erythrulose, Xylulose, Psicose, Turanose, Cellobiose, Amylopectin, Glucosamine, Mannozamine, Fucose, Glucuronic Acid, Gluconic Acid, Glucono-lactone , Abecuose, galactozamine, sugar beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xylottriose, xylobiose, etc.) ), xylo-terminal oligosaccharide, genthio-oligosaccharide (genthiobiose, genthiotriose, genthiotetraose, etc.), sorbose, nigero-oligosaccharide, palatinose oligosaccharide, p Ructooligosaccharides (Kestose, Nistose, etc.), Maltotetraol, Maltotriol, Malto-oligosaccharides (Maltotriose, Maltotetraose, Maltopentaose, Maltohexaose, Maltoheptose, etc.), Starch , Inulin, inulo-oligosaccharide, lactulose, melibiose, rapinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, and soy oligosaccharides. Additionally, carbohydrates used herein may be of D- or L-configuration.

The starting composition can be synthetically 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 another embodiment, the starting composition is rhamnose.

In yet 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 yet another embodiment, the starting composition is amylose.

The organic compound(s) of the starting composition act as substrate(s) for the production of the target steviol glycoside(s) 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 steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4 , Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside Baudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudio Side 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q , Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside Baudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Synthesis of rebaudioside 2r, rebaudioside 2s, SvG7 or other glycosides of steviol occurring in plants Steviol glycosides from the group consisting of, for example, enzymatically glucosylated steviol glycosides and combinations thereof Is selected.

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 D.

In another embodiment, the target steviol glycoside is rubusoside.

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

In still other embodiments, the target steviol glycoside is steviol bio B side.

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

In another embodiment, the target steviol glycoside is stevioside .

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

In still other embodiments, the target steviol glycoside is stevioside A.

In still other embodiments, the target steviol glycoside is stevioside B.

In still other embodiments, the target steviol glycoside is stevioside C.

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

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 E4 .

In another embodiment, the target steviol glycoside is rebaudioside E6 .

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

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

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

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

In another embodiment, the target steviol glycoside is rebaudioside D7 .

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

In another embodiment, the target steviol glycoside is rebaudioside M4 .

In another embodiment, the target steviol glycoside is rebaudioside 1 a .

In another embodiment, the target steviol glycoside is rebaudioside 1 b .

In another embodiment, the target steviol glycoside is rebaudioside 1 c .

In another embodiment, the target steviol glycoside is rebaudioside 1 d .

In another embodiment, the target steviol glycoside is rebaudioside 1 e .

In another embodiment, the target steviol glycoside is rebaudioside 1 f .

In another embodiment, the target steviol glycoside is 1 g of rebaudioside.

In another embodiment, the target steviol glycoside is rebaudioside 1 h .

In another embodiment, the target steviol glycoside is rebaudioside 1 i.

In another embodiment, the target steviol glycoside is rebaudioside 1 j .

In another embodiment, the target steviol glycoside is rebaudioside 1 k .

In another embodiment, the target steviol glycoside is 1 l of rebaudioside.

In another embodiment, the target steviol glycoside is 1 m of rebaudioside.

In another embodiment, the target steviol glycoside is rebaudioside 1 n .

In another embodiment, the target steviol glycoside is rebaudioside 1 o .

In another embodiment, the target steviol glycoside is rebaudioside 1 p .

In another embodiment, the target steviol glycoside is rebaudioside 1 q .

In another embodiment, the target steviol glycoside is rebaudioside 1 r .

In another embodiment, the target steviol glycoside is rebaudioside 1 s .

In another embodiment, the target steviol glycoside is 1 t of rebaudioside.

In another embodiment, the target steviol glycoside is rebaudioside 2 a .

In another embodiment, the target steviol glycoside is rebaudioside 2 b .

In another embodiment, the target steviol glycoside is rebaudioside 2 c .

In another embodiment, the target steviol glycoside is rebaudioside 2 d .

In another embodiment, the target steviol glycoside is rebaudioside 2 e .

In another embodiment, the target steviol glycoside is rebaudioside 2 f .

In another embodiment, the target steviol glycoside is 2 g of rebaudioside.

In another embodiment, the target steviol glycoside is rebaudioside 2 h .

In another embodiment, the target steviol glycoside is rebaudioside 2 i .

In another embodiment, the target steviol glycoside is rebaudioside 2 j .

In another embodiment, the target steviol glycoside is rebaudioside 2 k. to be

In another embodiment, the target steviol glycoside is 2 l of rebaudioside.

In another embodiment, the target steviol glycoside is 2 m rebaudioside.

In another embodiment, the target steviol glycoside is rebaudioside 2 n .

In another embodiment, the target steviol glycoside is rebaudioside 2 o .

In another embodiment, the target steviol glycoside is rebaudioside 2 p .

In another embodiment, the target steviol glycoside is rebaudioside 2 q .

In another embodiment, the target steviol glycoside is rebaudioside 2 r .

In another embodiment, the target steviol glycoside is rebaudioside 2 s .

In another embodiment, the target steviol glycoside is SvG7.

The target steviol glycoside can be in any polymorph or amorphous form including hydrates, solvates, anhydrides 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 production catalytic process for the production of steviol mono side 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 D.

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 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 rebaudioside B.

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

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

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

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 A.

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 E4 .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E6 .

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 D.

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

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

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside D7 .

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 M.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside M4 .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 a .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 b .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 c .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 d .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 e .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 f .

In one embodiment, the present invention is a biocatalytic process for the production of 1 g of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of 1 h of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 i .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 j .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 k .

In one embodiment, the present invention is a biocatalytic process for the production of 1 l of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of 1 m of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 n .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 o .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 p .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 q .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 r .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1 s .

In one embodiment, the present invention is a biocatalytic process for the production of 1 t of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 a .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 b .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 c .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 d .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 e .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 f .

In one embodiment, the present invention is a biocatalytic process for the production of 2 g of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of 2 h of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 i .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 j .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 k .

In one embodiment, the present invention is a biocatalytic process for the production of 2 l of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of 2 m of rebaudioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 n .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 o .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 p .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 q .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 r .

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2 s .

In one embodiment, the present invention is a biocatalytic process for the production of SvG7.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 a from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 b from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 c from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 d from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 e from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the invention provides a biocatalytic process for the production of rebaudioside 1 f from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 g of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 h of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 i from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 j from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 k from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 liter of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 m of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 n from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 o from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 p from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 q from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 r from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 1 s from a starting composition comprising Rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 t of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 2 g of rebaudioside from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2 h from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2i from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the invention provides a biocatalytic process for the production of Rebaudioside 2L from a starting composition comprising Stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2m from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2n from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2p from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2q from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2s from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 2 g of rebaudioside from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 2 h of rebaudioside from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2L from a starting composition comprising Rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2L from a starting composition comprising Rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2m from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2n from a starting composition comprising Rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2q from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2s from a starting composition comprising Rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 2 g of rebaudioside from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2 h from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2i from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2L from a starting composition comprising Rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2m from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2n from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2q from a starting composition comprising Rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 2s from a starting composition comprising Rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 q from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 a from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 b from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 c from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 1 d from a starting composition comprising Rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 e from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 f from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 g of rebaudioside from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1h from a starting composition comprising rebaudioside M4 and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 i from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 j from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 k from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 liter of rebaudioside from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 m of rebaudioside from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 n from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 o from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 p from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 q from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1 r from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside 1 s from a starting composition comprising Rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of 1 t of rebaudioside from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising stevioside and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising Stevioside, Rebaudioside A and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside AM and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M and UDP-glucose.

In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M4 and UDP-glucose.

Optionally, the method of the present invention further comprises isolating 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 crystallization, separation by membrane, centrifugation, extraction, chromatographic separation or a combination of such methods.

In certain 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 (dry) basis. In one embodiment, the highly purified target steviol glycoside composition comprises greater than about 90% by weight of the target steviol glycoside on a dry basis, such as, for example, greater than about 91%, about 92 on a dry basis. %, 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.

In one embodiment, when the target steviol glycoside is rebaudioside M4 , the process described herein provides a composition having a rebaudioside M4 content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside M4 , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1a , the process described herein provides a composition having a rebaudioside 1a content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1a , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1b , the process described herein provides a composition having a rebaudioside 1b content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1b , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1c , the process described herein provides a composition having a rebaudioside 1c content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1c , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1d , the process described herein provides a composition having a rebaudioside 1d content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1d , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1e , the process described herein provides a composition having a rebaudioside 1e content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1e , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1f , the process described herein provides a composition having a rebaudioside 1f content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1f , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1 g of rebaudioside, the process described herein provides a composition having a 1 g content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 1 gram of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1 h of rebaudioside, the process described herein provides a composition having a 1 h content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 1 h of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1i , the process described herein provides a composition having a rebaudioside 1i content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1i , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1j , the process described herein provides a composition having a rebaudioside 1j content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1j , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1k rebaudioside, the process described herein provides a composition having a 1k content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1k , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1 liter of rebaudioside, the process described herein provides a composition having a 1 liter content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 1 liter of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1 m rebaudioside, the process described herein provides a composition having an 1 m content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 1 m of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1n , the process described herein provides a composition having a rebaudioside 1n content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1n , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1o , the process described herein provides a composition having a rebaudioside 1o content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1o , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is Rebaudioside 1p , the process described herein provides a composition having a rebaudioside 1p content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1p , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1q , the process described herein provides a composition having a rebaudioside 1q content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1q , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1r , the process described herein provides a composition having a rebaudioside 1r content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1r , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is Rebaudioside 1s , the process described herein provides a composition having a rebaudioside 1s content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is Rebaudioside 1s , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 1 t of rebaudioside, the process described herein provides a composition having a 1 t content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 1 t of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2a , the process described herein provides a composition having a content of rebaudioside 2a greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2a , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2b , the process described herein provides a composition having a rebaudioside 2b content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2b , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2c , the process described herein provides a composition having a rebaudioside 2c content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2c , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2d , the process described herein provides a composition having a rebaudioside 2d content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2d , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2e , the process described herein provides a composition having a rebaudioside 2e content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2e , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2f , the process described herein provides a composition having a rebaudioside 2f content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2f , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 2 g of rebaudioside, the process described herein provides a composition having a 2 g content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 2 grams of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 2 h of rebaudioside, the process described herein provides a composition having a 2 h content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 2 h of rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2i , the process described herein provides a composition having a rebaudioside 2i content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2i , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2j , the process described herein provides a composition having a rebaudioside 2j content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2j , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2k , the process described herein provides a composition having a rebaudioside 2k content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2k , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 2L rebaudioside, the process described herein provides a composition having a 2L content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 2L rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is 2m rebaudioside, the process described herein provides a composition having a 2m content of rebaudioside greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is 2m rebaudioside, the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2n , the process described herein provides a composition having a rebaudioside 2n content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2n , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2o , the process described herein provides a composition having a rebaudioside 2o content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2o , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2p , the process described herein provides a composition having a rebaudioside 2p content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2p , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2q , the process described herein provides a composition having a rebaudioside 2 q content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2q , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2r , the process described herein provides a composition having a rebaudioside 2r content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2r , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2s , the process described herein provides a composition having a rebaudioside 2s content greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is Rebaudioside 2s , the process described herein provides a composition comprising an amount greater than about 95% by weight on a dry basis.

In one embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition having an SvG7 content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition comprising a SvG7 content of greater than about 95% by weight on a dry basis.

Microbial and enzyme preparations

In one embodiment of the present invention, a target steviol glycoside is prepared by contacting a microorganism (microbial cell) and/or enzyme preparation with a medium containing the starting composition.

Enzymes can be provided in the form of whole cell suspensions, crude lysates, purified enzymes or combinations thereof. In one embodiment, the biocatalyst is an enzyme capable of converting the purified starting composition to the target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising an enzyme capable of converting at least one starting composition to a target steviol glycoside. In yet another embodiment, the biocatalyst is a total cell suspension comprising an enzyme capable of converting at least one starting composition to a target steviol glycoside.

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

Suitable enzymes for converting the starting composition to the target steviol glycoside include, but are not limited to, the steviol biosynthetic enzyme NDP-glucosyltransferase (NGTs), ADP-glucosyltransferase (AGTs), CDP-glucosyltransferase (CGTs). ), GDP-glucosyltransferases (GGTs), TDP-glucosyltransferases (TDPs), UDP-glucosyltransferases (UGTs). Optionally it will include NDP-regeneration enzyme(s) ADP-regeneration enzyme(s) CDP-regeneration enzyme(s) GDP-regeneration enzyme(s) TDP-regeneration enzyme(s) and/or UDP-regeneration enzyme(s). I can.

In one embodiment, the steviol biosynthetic enzyme comprises a mevalonate (MVA) pathway enzyme.

In another embodiment, the steviol biosynthetic enzyme comprises a non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the steviol biosynthetic enzyme is geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, cowene synthase, cowene oxidase, cowenic acid 13-hydroxylase (KAH), ste Viol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C- Methyl-D-erythritol synthase (CMS), 4-diphosphocytyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytyl-2-C-methyl-D -Erythritol 2,4-cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), l-hydroxy-2- Methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrolysis Phosphate decarboxylase, cytochrome P450 reductase, and the like.

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

In one embodiment, steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in microbial cells. Microbial cell, for example, Escherichia coli (E. coli), saccharose in MRS (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, Yarrow subtotal (Yarrowia) species Etc. In another embodiment, UDP-glucosyltransferase is synthesized.

In one embodiment, UDP-glucosyltransferase is equivalent to these polypeptides (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGTs with amino-acid sequence identity, also It is selected from the group containing isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGTs and UDP-glucose regeneration system are present in one microorganism (microbial cell). Microorganism may be, for example, all E. coli (E. coli), saccharose in MRS (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, Yarrow subtotal (Yarrowia) .

In one embodiment, the UDP-glucosyltransferase is capable of adding at least one glucose unit at C13, to steviol bearing an -OH functional group, or to any starting steviol glycoside, so that at C13, O-glucose beta glucose. Copyranoside is any UDP-glucosyltransferase that produces a target steviol glycoside with a glycoside linkage. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is capable of adding at least one glucose unit at C19, to steviol bearing a -COOH functional group or to any starting steviol glycoside, so that at C19, -COO-glucose beta -Glucopyranoside Any UDP-glucosyltransferase that produces a target steviol glycoside with a glycoside linkage. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with >85% amino-acid sequence identity with UGT74G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→2 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed bound glycoside with at least one additional glucose. In binding(s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→3 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces the target steviol glycoside with at least one beta 1→4 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C19 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→6 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→2 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed bound glycoside with at least one additional glucose. In binding(s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→3 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces the target steviol glycoside with at least one beta 1→4 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT with >85% amino-acid sequence identity with UGT76G1.

In another embodiment, UDP-glucosyltransferase is capable of adding at least one glucose unit on the C13 side of any starting steviol glycoside to the existing glucose so that a newly formed glycoside bond with at least one additional glucose In (s), it is any UDP-glucosyltransferase that produces a target steviol glycoside with at least one beta 1→6 glucopyranoside glycoside linkage(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT with >85% amino-acid sequence identity with UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a steviol mono side A to be added to at least one of the glucose units to steviol. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT74G1 or 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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a bio-steviol side D to be added to at least one glucose unit in steviol mono side A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a bio-steviol side A to be added to at least one glucose unit in steviol mono A side. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a steviol bio B side to be added to at least one glucose unit in steviol mono side A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 D to form rebaudioside G. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT74G1 or UGT74G1.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rubusoside to form stevioside . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 rebaudioside G. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or 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. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a stevioside B to be added to at least one glucose unit in the base fusu side. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form the at least one bio-glucose side A stevioside C to be added to the unit to steviol. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In one embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase can be added to the at least one side of rain bio B Ste the glucose unit forming the stevioside B. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT85C2 or UGT85C2 or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form at least one C stevioside, in addition to possible rain bio B side stacking a glucose unit. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 B to form rebaudioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a rebaudioside E to be added to at least one glucose unit in the stevioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 G to form rebaudioside E6 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, UDP- glucosyl transferase is any UDP- glucosyl transferase to form a rebaudioside E to be added to at least one glucose unit on stevioside A. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 A to form rebaudioside E4. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to form rebaudioside E6 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 steviolbioside C to form rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT85C2 or UGT85C2 or a UGT with >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 A to form rebaudioside I. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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 certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 E4 to form rebaudioside D. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 E6 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M4 . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1a . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1b . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1c . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1d . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1e . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1f . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form 1 g of rebaudioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1h . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1i . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1j . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1k . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1l . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M to form rebaudioside 1o . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1p . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1r . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1s . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1t . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2b . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2c . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2d . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2e . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2f . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form 2g of rebaudioside. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 h . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2i . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2j . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2k . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2l . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2m . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2n . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >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 M4 to form rebaudioside 2o . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2p . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2r . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2s . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1q . In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGTSl2 or UGTSl2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT with >85% amino-acid sequence identity with EUGT11. In yet another specific embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT with >85% amino-acid sequence identity with UGT91D2. In certain embodiments, the UDP-glucosyltransferase is a UGT with >85% amino-acid sequence identity with UGT76G1 or UGT76G1.

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

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

In one embodiment, the regeneration substrate for the UDP-glucose regeneration catalyst is sucrose.

Optionally, the methods of the present invention further comprise the use of transglycosidases that use oligo- or poly-saccharides as sugar donors to modify the recipient target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, sacarase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, It includes fructosylvertase, alkaline invertase, acid invertase, and fructofuranosidase. In some embodiments, glucose including, but not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and sugar(s) other than glucose are transferred to the recipient target steviol glycoside. In one embodiment, the recipient steviol glycoside is Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebau Dioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k, Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2 r and/or Rebaudioside 2 s . In another embodiment, the recipient steviol glycoside is rebaudioside M4 . In another embodiment, the recipient steviol glycoside is SvG7.

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

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

The microorganism may be any microorganism possessing the necessary enzyme(s) to convert the starting composition to the target steviol glycoside(s). These enzymes are encoded within the genome of the microorganism.

Suitable micro-organisms, but not limited to, Escherichia coli (E.coli), MRS with saccharide (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, Yarrow subtotal (Yarrowia) species such as Includes.

In one embodiment, there is no microorganism upon contact with the starting composition.

In another embodiment, the microorganism is immobilized upon contact with the starting composition. For example, microorganisms can be immobilized on a solid support made from an inorganic or organic material. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derivatized cellulose or glass, ceramics, metal oxides or membranes. Microorganisms can be immobilized on a solid support, for example by covalent attachment, adsorption, crosslinking, entrapment or encapsulation.

In still another embodiment, an enzyme capable of converting the starting composition to the target steviol glycoside is secreted from 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 accomplished by at least one suitable method that provides a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analysis), or a combination of such methods.

Usage

The highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, ste Violbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebau Dioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebau Dioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m , Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 "as is" or other sweetener flavoring food ingredients And combinations thereof.

Non-limiting examples of flavorings 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, colorants, extenders, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof. Include.

The highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, ste Violbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebau Dioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebau Dioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m , Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are, but are not limited to, hydrate solvate anhydride amorphous form. And it can be prepared in a variety of polymorphic forms including combinations thereof.

The highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, ste Violbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebau Dioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebau Dioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m , Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are food, beverage, pharmaceutical compositions, cosmetics , Chewing gum, tabletop products, cereals, dairy products, toothpaste and other oral compositions, etc., can be incorporated as a high strength natural sweetener.

In some embodiments, the highly purified target glycoside(s) of the present invention is from about 0.0001% to about 12% by weight, such as about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, About 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, About 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, Food, beverage, pharmaceutical composition, cosmetics, chewing gum, table in an amount of about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5% or about 12.0% by weight It is present in top products, cereals, dairy products, toothpaste and other oral compositions.

In certain embodiments, the sweetener is from about 0.0001% to about 8% by weight, such as from about 0.0001% to about 0.0005%, from about 0.0005% to about 0.001%, from about 0.001% to about 0.005% by weight. , About 0.005% to about 0.01%, about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1% by weight , About 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6% , From about 6% to about 7% and from about 7% to about 8% by weight.

The highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, ste Violbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebau Dioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebau Dioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m , Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s, SvG7 and/or combinations thereof can be used as sweetening compounds or Or it is at least one naturally occurring high intensity sweetener such as Dulcoside A, Dulcoside B, Dulcoside C, Dulcoside D, Rebaudioside A2, Rebaudioside A3, Rebaudioside A4, Rebaudioside B2. , Rebaudioside C, Rebaudioside C2, Rebaudioside C3, Rebaudioside C4, Rebaudioside C5, Rebaudioside C6, Rebaudioside D2, Rebaudioside D3, Rebaudioside Baudioside D4, Rebaudioside D5, Rebaudioside D6, Rebaudioside D8, Rebaudioside E5, Rebaudioside E7 , Rebaudioside F , Rebaudioside F2 , Rebaudio Side F3 , Rebaudioside H , Rebaudioside H2 , Rebaudioside H3 , Rebaudioside H4 , Rebaudioside H5 , Rebaudioside H6 , Rebaudioside I2 , Rebaudioside I3 , Rebaudioside J, Rebaudioside K, Rebaudioside K2, Rebaudioside KA, Rebaudioside L, Rebaudioside M2, Rebaudioside M3, Rebaudioside N, Re Baudioside N2, Rebaudioside N3, Rebaudioside N4, Rebaudioside N5, Rebaudioside O, Rebaudioside O2, Rebaudioside O3, Rebaudioside O4, Rebaudio Side Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebau Dioside Z2, Steviolbioside C, Steviolbioside E, Stevioside D, Stevioside E, Stevioside E2, Stevioside F, Stevioside G, Stevioside H, Mogroside, Brazzein, Neohesperidine, Dihydrochalcone, glycyrrhizic acid and salts thereof, taumatin, perillatin, fernandulcin, mucuroside, baunoside, flomisoside- I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, ferline Drin, Carnocifloside , Cyclocarioside, Pterocaryoside, Polypodoside A, Brasilin, Hernandulsin, Philodulsin, Glyciphilin, Florizine, Trilovatin, Dihydroflavonol, Dihydroquercetin-3-acetate, Neo Astilibin, trans -cinnamaldehyde, monatin, monatin salts, other indole derivative sweeteners, seligein A , hematoxylin, monelin, osladin, pterocaryoside A , pterocaryoside B , Marvinlin, pentadine, miraculin, curculin, neoculin, chlorogenic acid, cinarine, Luo Han Guo sweetener, mogroside V , cyanoside, syratose, and combinations thereof.

In certain embodiments, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6 , Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Re Baudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudio Side 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r , Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside Baudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are, Dulcoside A, Dulcoside B, Dulcoside C, Dulcoside D, Rebaudio Side A2, Rebaudioside A3, Rebaudioside A4, Rebaudioside B2, Rebaudioside C, Rebaudioside C2, Rebaudioside C3, Rebaudioside C4, Rebaudioside C5 , Rebaudioside C6, Rebaudio Oside D2, Rebaudioside D3, Rebaudioside D4, Rebaudioside D5, Rebaudioside D6, Rebaudioside D8, Rebaudioside E5, Rebaudioside E7 Rebaudioside F Rebaudioside F2 Rebaudioside F3 Rebaudioside H Rebaudioside H2 Rebaudioside H3 Rebaudioside H4 Rebaudioside H5 Rebaudioside H6 Rebaudioside I2 Rebaudioside I3 , Rebaudioside J, Rebaudioside K, Rebaudioside K2, Rebaudioside KA, Rebaudioside L, Rebaudioside M2, Rebaudioside M3, Rebaudioside N, Re Baudioside N2, Rebaudioside N3, Rebaudioside N4, Rebaudioside N5, Rebaudioside O, Rebaudioside O2, Rebaudioside O3, Rebaudioside O4, Rebaudio Side Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebau Dioside Z2, Steviolbioside C, Steviolbioside E, Stevioside D, Stevioside E, Stevioside E2, Stevioside F, Stevioside G, Stevioside H, NSF-02 Mogroside V siratose Luo Han Guo allulose allose D-tagatose may be used as a sweetener in a composition comprising a compound selected from the group consisting of erythritol and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, re Baudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebau Dioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebau Dioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n , Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are also synthetic high strength sweeteners such as sucralose, potassium acesulfame, aspartame, alitam , Saccharin, neohesperidine, dihydrochalcone, cyclamate, neotam, dulcin, suosan advantam, salts thereof, and combinations thereof.

Also highly purified target steviol glycoside(s), especially steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudio Side E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4 , Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside Baudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudio Side 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e , Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebau Dioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are natural sweetener inhibitors such as jimnemsan hodulsin zippin lactisol And may be used in combination with the like. Steviol Monoside, Steviol Monoside A, Steviol Bioside, Steviol Bioside D, Rubusoside, Steviol Bioside A, Steviol Bioside B, Rebaudioside B, Stevioside, Rebaudio Side G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3 , Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside Baudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudio Side 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s , Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside Baudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can also be combined with various umami enhancing agents. Steviol Monoside, Steviol Monoside A, Steviol Bioside, Steviol Bioside D, Rubusoside, Steviol Bioside A, Steviol Bioside B, Rebaudioside B, Stevioside, Rebaudio Side G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3 , Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside Baudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudio Side 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s , Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside Baudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are umami and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and It can be mixed with any combination thereof.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are carbohydrates, polyols, amino acids, and their corresponding salts, poly -Amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts and organic base salts including organic acid salts, inorganic salts, bitter compounds, flavoring and flavor components, bitter taste compounds, Proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof may be used in combination with one or more additives selected from the group consisting of.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can be combined with polyols or sugar alcohols. The term “polyol” refers to a molecule containing more than one hydroxyl group. The polyol may be a diol triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. Polyols may also contain more than 4 hydroxyl groups, such as pentols, hexaols, heptaols, etc., each containing 5, 6, or 7 hydroxyl groups. Additionally, the polyol may also be a sugar alcohol, a polyhydric alcohol, or a polyalcohol in the reduced form of a carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) is 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, traitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosac Ride, reduced xylo-oligosaccharide, reduced genthio-oligosaccharide, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzate, polyglycitol and sugar alcohol or sweetener composition that does not adversely affect the taste of the composition And any other carbohydrate that is reducible.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are reduced calorie sweeteners such as D-Tagatos L -Sugar, L-sorbose, L-arabinose and combinations thereof.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can also be combined with various carbohydrates. The term “carbohydrate” generally refers to an aldehyde or ketone compound having a number of substituted hydroxyl groups of the general formula (CH ₂ O) _n , wherein n is 3-30, also oligomers and polymers thereof. Carbohydrates of the invention may also be substituted or deoxygenated at one or more positions. Carbohydrates used herein include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivative”, “substituted carbohydrate”, and “modified carbohydrate” are synonymous. Modified carbohydrates refer herein to any carbohydrate in which at least one valency is added, removed, or substituted, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate is optionally deoxygenated at any corresponding C-position, and/or one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, Alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulphenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl , Phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any carbohydrate derivative or substituted carbohydrate function that improves the sweetness of the sweetener composition. May be substituted with other active functional groups of.

Examples of carbohydrates that can be used according to the present 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, lyxos, altose, mannose, idose, lactose, maltose, anti-sugar, isotrehalose, neotrehalose, isomaltulose , Erythrose, deoxyribose, gulose, idose, talos, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannozamine, fucose, glucuronic acid, glucose Conic acid, glucono-lactone, abecuose, galactozamine, sugar beet oligosaccharide, isomalto-oligosaccharide (isomaltose, isomalttriose, panose, etc.), xylo-oligosaccharide (xylot) Rios, xylobiose, etc.), xylo-terminated oligosaccharides, genthio-oligosaccharides (genthiobiose, genthiotriose, genthiotetraose, etc.), sorbose, nigero-oligosaccharide, palatino Oligosaccharides, fructo-oligosaccharides (kestos, nythose, etc.), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltose) Heptaose, etc.), starch, inulin, inulo-oligosaccharide, lactulose, melibiose, rapinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, and soy oligosaccharides. do. Additionally, carbohydrates used herein may be of D- or L-configuration.

The highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A , Steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebau Dioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudio Side 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are various physiologically active substances Or it may be used in combination with a functional ingredient. Functional ingredients generally include categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, functional foods, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols); Polyol; Prebiotics, live bacteria; Phytoestrogen; Soy protein; Sulfide/thiol; amino acid; protein; vitamin; And minerals. Functional ingredients can also be classified based on their health benefits, such as cardiovascular, cholesterol-reducing, and anti-inflammatory. Exemplary functional ingredients are provided in WO2013/096420, the contents of which are incorporated herein by reference.

The highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A , Steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebau Dioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudio Side 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are zero calories with improved taste characteristics , Reduced calorie or diabetic beverages and can be applied as high intensity sweeteners to prepare foods. It can also be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used. Also highly purified target steviol glycoside(s), especially steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudio Side E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4 , Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside Baudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudio Side 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e , Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebau Dioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are beverage foods and other products exclusively for human consumer products as well as enhanced features It can also be used as a sweetener in animal foods and feeds having.

Highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A , Steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebau Dioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudio Side 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are zero calories, reduced calories or It can be applied as a foam suppressant to prepare diabetes beverages and foods.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Examples of consumable products in which Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can be used as sweetening compounds are non- Alcoholic beverages such as vodka, wine, beer, liqueur, and sake, and the like; Natural juice; Soft drinks; carbonated drink; Diet drink; Zero-calorie drinks; Reduced calorie beverages and foods; Yogurt drink; Instant juice; Instant coffee; Powder type instant drinks; Can products; syrup; Fermented soybean paste; Soy sauce; vinegar; dressing; mayonnaise; ketchup; Curry; Soup; Instant bouillon; Powdered soy sauce; Powdered vinegar; Type of biscuit; Rice biscuits; cracker; bread; Chocolate; Caramel; candy; Chewing gum; jelly; Pudding; Preserved fruits and plants; Fresh cream; jam; marmalade; Flower paste; Powdered milk; Ice cream; Sherbet; Plants and bottled fruit; Cans and boiled beans; Meats and foods boiled in sweet sauces; Vegetable food for agriculture; Seafood; ham; sausage; Fish ham; Fish sausage; Fish paste; Deep fried fish products; Dry seafood products; Frozen food; Preserved seaweed; Preserved meat; tobacco; Medical products; And many others. In principle, the highly purified target steviol glycoside(s) may have non-limiting applications.

Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can be used as flavor modifiers or flavorings with modifying properties. Examples of consumable products include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liqueur, and sake, and the like; Natural juice; Soft drinks; carbonated drink; Diet drink; Zero-calorie drinks; Reduced calorie beverages and foods; Yogurt drink; Instant juice; Instant coffee; Powder type instant drinks; Can products; syrup; Fermented soybean paste; Soy sauce; vinegar; dressing; mayonnaise; ketchup; Curry; Soup; Instant bouillon; Powdered soy sauce; Powdered vinegar; Type of biscuit; Rice biscuits; cracker; bread; Chocolate; Caramel; candy; Chewing gum; jelly; Pudding; Preserved fruits and plants; Fresh cream; jam; marmalade; Flower paste; Powdered milk; Ice cream; Sherbet; Plants and bottled fruit; Cans and boiled beans; Meats and foods boiled in sweet sauces; Vegetable food for agriculture; Seafood; ham; sausage; Fish ham; Fish sausage; Fish paste; Deep fried fish products; Dry seafood products; Frozen food; Preserved seaweed; Preserved meat; tobacco; Medical products; And many others. In principle, the highly purified target steviol glycoside(s) may have non-limiting applications.

During the manufacture of products such as food, beverage, pharmaceutical, cosmetics, tabletop products, and chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, penetrating, filtering, sprinkling, spraying, infusion and other methods can be used.

In addition, the highly purified target steviol glycoside(s) obtained in the present invention, steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A , Steviolbioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebau Dioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudio Side 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 can be used in dry or liquid form. have.

The highly purified target steviol glycosides can be added before or after heat treatment of food products. Highly purified target steviol glycoside(s), in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B , Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebau Dioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudio The amount of Side 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 depends on the purpose of use. As discussed above, the highly purified target steviol glycoside(s) can be added alone or in combination with other compounds.

The present invention is also a sweetness increasing agent as steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudio Side E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a , Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside Baudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudio Side 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f , Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebau Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 to increase sweetness in beverages, wherein steviolmonoside, steviolmono Side A, Steviol Bioside, Steviol Bioside D, Rubusoside, Steviol Bioside A, Steviol Bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevio between De B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside Baudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudio Side 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l , Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside Baudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudio Side 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are present in concentrations below their respective sweetness perception thresholds.

As used herein, the term “sweetness increasing agent” refers to a compound capable of increasing or enhancing the perception of sweetness in a composition, such as a beverage. The term “sweetness enhancer” is synonymous with the terms “sweetness enhancer,” “sweetness enhancer,” “sweetness enhancer,” and “sweetness enhancer”.

The term "sweetness perception threshold concentration" as commonly used herein is the lowest known concentration of a sweetening compound perceivable by human taste, typically about 1.0% sucrose equivalent (1.0% SE). In general, the sweetness increasing agent, when present below the sweetness perception threshold concentration of a given sweetness increasing agent, is capable of increasing or enhancing the sweetness of a sweetener without providing a perceptible sweetness alone; However, the sweetness increasing agent itself can provide sweetness at concentrations above its sweetness perception threshold concentration. The sweetness perception threshold concentration is specific for a particular enhancer and can vary based on the beverage matrix. The sweetness perception threshold concentration can be readily determined by a taste test that increases the concentration of a given increasing agent until more than 1.0% sucrose equivalent is detected in a given beverage matrix. The concentration that provides about 1.0% sucrose equivalent is considered the sweetness perception threshold.

In some embodiments, the sweetener is about 0.0001% to about 12% by weight, such as about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1 wt%, about 0.5 wt%, about 1.0 wt%, about 1.5 wt%, about 2.0 wt%, about 2.5 wt%, about 3.0 wt%, about 3.5 wt%, about 4.0 wt%, about 4.5 wt%, about 5.0 wt%, about 5.5 wt%, about 6.0 wt%, about 6.5 wt%, about 7.0 wt%, about 7.5 wt%, about 8.0 wt%, about 8.5 wt%, about 9.0 wt%, about 9.5 wt%, about It is present in the beverage in an amount of 10.0%, about 10.5%, about 11.0%, about 11.5% or about 12.0% by weight.

In certain embodiments, the sweetener is from about 0.0001% to about 10% by weight, such as from about 0.0001% to about 0.0005%, from about 0.0005% to about 0.001%, from about 0.001% to about 0.005% by weight. , About 0.005% to about 0.01%, about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1% by weight , About 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6% , From about 6% to about 7%, from about 7% to about 8%, from about 8% to about 9% or from about 9% to about 10% by weight. In certain embodiments, the sweetener is present in the beverage in an amount from about 0.5% to about 10% by weight. In another specific 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 calorie sweetener. Suitable sweetening agents include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup and high fructose starch syrup.

In another embodiment, the sweetening agent 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 the sweetener may be used alone or in combination with other sweeteners.

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

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

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

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

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

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

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

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

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

The addition of a sweetness increasing agent at a concentration below its sweetness perception threshold increases the detected sucrose equivalent weight of a beverage comprising a sweetener and a sweetness increasing agent compared to a corresponding beverage without the sweetness increasing agent. In addition, the sweetness can be increased in an amount greater than the detectable sweetness of a solution containing the concentration of at least one sweetening agent in the absence of any sweetener.

Therefore, the present invention also provides a beverage containing a sweetening agent and steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bio Side B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebau Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebau Dioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebaudioside Adding a sweetness increasing agent selected from Baudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 or a combination thereof It provides a method of increasing the sweetness of a beverage comprising a sweetening agent comprising, wherein steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, Stevioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudio Side E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M , Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside Baudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudio Side 1p, Rebaudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d , Rebaudioside 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebau Dioside 2m, Rebaudioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 are below its sweetness perception threshold. Exists in concentration.

Steviol-monoside, steviol-monoside A, steviol-bioside, steviol-bioside D, rubusoside, steviol-bioside A, steviol-bioside in beverages containing a sweetener at a concentration below the sweetness recognition threshold B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudio Side E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4 , Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside Baudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudio Side 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudioside 2e , Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Rebau The addition of Dioside 2n, Rebaudioside 2o, Rebaudioside 2p, Rebaudioside 2q, Rebaudioside 2r, Rebaudioside 2s and/or SvG7 is a detection sucrose equivalent of about 1.0% to about 5.0 %, 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 are highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviol Bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside Baudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudio Side M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h , Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 1o, Rebaudioside 1p, Rebaudioside Baudioside 1q, Rebaudioside 1r, Rebaudioside 1s, Rebaudioside 1t, Rebaudioside 2a, Rebaudioside 2b, Rebaudioside 2c, Rebaudioside 2d, Rebaudio Side 2e, Rebaudioside 2f, Rebaudioside 2g, Rebaudioside 2h, Rebaudioside 2i, Rebaudioside 2j, Rebaudioside 2k Rebaudioside 2l, Rebaudioside 2m, Preferred embodiments of the present invention for the production of rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 Illustrate. It will be understood that the present invention is not limited to the materials, proportions, conditions and procedures specified in the illustrative examples only.

Example

Example 1

Protein sequence of engineered enzymes used in biocatalytic processes

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:

>UGTSl2 variant 0234 (engineered glucosyltransferase; source of WT gene: tomato (Solanum lycopersicum) )

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK

IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN

FSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSF

RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS

RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENS

TVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP

EGFLERIGERGRVLDKFAPQPRILNHPSTGG FISHCGWNSVMESIDFGVP

IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGE

TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3:

>UGT76G1 variant 0042 (engineered glucosyltransferase; source of WT gene: stevia rebaudiana )

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLVDSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWNSTLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

Example 2

Expression and formulation of the 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 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21 (DE3) cells.

Cells were cultured at 37° C. in ZYM505 medium (F. William Studier, Protein Expression and Purification (2005) 207-234) supplemented with kanamycin (50 mg/l). Expression of the gene was induced in the log phase by IPTG (0.2 mM) and performed at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220 xg, 20 min, 4° C.) and cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl ₂ , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL) With re-suspended to an optical density of 200 (measured at ₆₀₀ nm (OD ₆₀₀ )). The cells were then destroyed by sonication and the crude extract was separated from the cell fragments by centrifugation (18000 xg 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with distilled water to obtain an enzyme active agent.

For the enzymatic active formulation of SuSy_At, the activity in units is defined as follows: 1 mU of SuSy_At converts 1 nmol of sucrose to fructose in 1 minute. The reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose, 3 mM MgCl ₂ , and 15 mM uridine diphosphate (UDP) at t ₀ .

Example 3

Expression and formulation of the UGTSl2 variant of SEQ ID 2

The gene coding for the UGTSl2 variant of SEQ ID 2 (Example 1) was cloned into the expression vector pLE1A17 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21 (DE3) cells.

Cells were cultured at 37° C. in ZYM505 medium (F. William Studier, Protein Expression and Purification (2005) 207-234) supplemented with kanamycin (50 mg/l). Expression of the gene was induced in the log phase by IPTG (0.1 mM) and performed at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220 xg, 20 min, 4° C.) and cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl ₂ , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL) With re-suspended to an optical density of 200 (measured at ₆₀₀ nm (OD ₆₀₀ )). The cells were then destroyed by sonication and the crude extract was separated from the cell fragments by centrifugation (18000 xg 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 to obtain an enzyme active agent.

For the enzymatic active agent of UGTSl2, the unit-in activity is defined as follows: 1 mU of UGTSl2 converts 1 nmol of Rebaudioside A (Reb A ) to Rebaudioside D (Reb D ) in 1 minute. Let it. The reaction conditions for the assay are 30 ℃, 50 mM potassium phosphate buffer, pH 7.0, at _{t 0 10 mM Reb A, 500} mM sucrose, 3 mM MgCl _2, 0.25 mM uridine diphosphate (UDP), and 3 U / mL SuSy_At of.

Example 4

Expression and formulation of the 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 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21 (DE3) cells.

Cells were cultured at 37° C. in ZYM505 medium (F. William Studier, Protein Expression and Purification (2005) 207-234) supplemented with kanamycin (50 mg/l). Expression of the gene was induced in the log phase by IPTG (0.1 mM) and performed at 30° C. and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220 xg, 20 min, 4° C.) and cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl ₂ , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL) With re-suspended to an optical density of 200 (measured at ₆₀₀ nm (OD ₆₀₀ )). The cells were then destroyed by sonication and the crude extract was separated from the cell fragments by centrifugation (18000 xg 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 to obtain an enzyme active agent.

For the enzymatic active formulation of UGT76G1, the unit-in activity is defined as follows: 1 mU of UGT76G1 converts 1 nmol of Rebaudioside D (Reb D ) to Rebaudioside M (Reb M ) in 1 min. Let it. The reaction conditions for the assay were 30° C., 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb D at t ₀ , 500 mM sucrose, 3 mM MgCl ₂ , 0.25 mM uridine diphosphate (UDP) and 3 U/mL. SuSy_At of.

Example 5

Synthesis of SvG7 in a one-pot reaction by adding UGTSl2, SuSy_At and UGT76G1 at the same time.

Various SvG7 The molecule was synthesized directly from stevioside in a one-pot reaction using the following three enzymes (see Examples 1, 2, 3 and 4) (see Fig. 11A): UGTSl2 (variant of SEQ ID 2) SuSy_At (SEQ ID 1) and UGT76G1 (variant of SEQ ID 3).

The final reaction solution was 348 U/L UGTSl2,1341 U/L SuSy_At, 10 U/L UGT76G1, 47 mM stevioside, 0.32 mM uridine diphosphate (UDP), 0.99 M sucrose, 3.9 mM MgCl ₂ and potassium phosphate buffer. (pH 6.6). First, 206 mL of distilled water was mixed with 0.24 g MgCl ₂ ㅇ6H ₂ O, 102 g sucrose, 9.8 mL of 1.5 M potassium phosphate buffer (pH 6.6), and 15 g stevioside. The final volume of the reaction mixture was adjusted to 300 mL.

After dissolution, the temperature was adjusted to 45° C. and UGTSl2, SuSy_At, UGT76G1 and 39 mg UDP were added. The reaction mixture was incubated for 24 hrs on a 45° C. shaker. Additional 39 mg UDP was added at 12 hours, 24 hours and 36 hours. The contents of reb 2a , reb 2m and various SvG7 at the end of the reaction (48 hours) were analyzed by HPLC.

Example 6

HPLC analysis

For analysis, bioconversion samples were inactivated by adjusting the reaction mixture to pH 5.5 with 17% H ₃ PO ₄ and then boiled for 10 minutes. The obtained sample was filtered, and the filtrate was diluted 10 times and used as a sample for HPLC analysis. HPLC assays were performed on an Agilent HP 1200 HPLC system consisting of a pump, column thermostat, auto sampler, UV detector with background correction and data acquisition system. The analyte was separated at 40° C. using an Agilent Poroshell 120 SB-C18, 4.6 mm x 150 mm, 2.7 μm. 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.

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

Table 3 shows the conversion of stevioside to identified rebaudioside species (area percent) for each time point. Chromatograms of stevioside and reaction mixture at 48 hours are shown in FIGS. 11A and 11B, respectively. One skilled in the art will understand that changes in solvent and/or equipment can often cause residence times to vary.

Example 7

Le Bout audio purification of the side 2a, 2m and various SvG7

300 mL of the reaction mixture (after 48 hrs) was inactivated by adjusting the pH to pH 5.5 with H3PO4 and then boiled for 10 minutes. The filtrate was loaded into a column containing 500 mL YWD03 (Cangzhou Yuanwei China) resin pre-equilibrated with water. The resin was washed with 2.5 L water and the water effluent from this step was discarded.

Steviol glycoside was eluted from the YWD03 resin column by elution with 2.5 L 70% v/v ethanol/water. The effluent from this step was collected and dried under vacuum at 60 ^o C to give 20 g of dry solid product. This sample was dissolved in water and subjected to further fractionation and separation by HPLC using the conditions listed in Table 4 below.

HPLC fractions corresponding to individual compounds from multiple runs were combined depending on the retention time. Fractions were freeze-dried.

The purity of the obtained fractions was evaluated by the analytical HPLC method described in Example 6. The purified rebaudioside 2a chromatogram is shown in FIG. 11C. The chromatogram of purified rebaudioside 2m is shown in FIG. 11D.

Example 8

Investigation of the structure of Rebaudioside 2a

Using samples dissolved in pyridine- d5 , NMR experiments were performed on a Bruker 500 MHz spectrometer. Along with the signal from the sample, the signal from pyridine- d5 at 123.5, 135.5, 149.9 ppm _C and 7.19, 7.55, 8.71 ppm _H was observed.

Of rebaudioside 2a recorded in pyridine- d ₅ ^{The 1} H-NMR spectrum confirmed the excellent quality of the sample (see Fig. 12A). HSQC (see Figure 12b) shows the presence of exo-methylene groups in the sugar region with long-range coupling to C-15 observable in H H-COSY (Figure 12c). Other deep-field signals of quaternary carbons (C-13 C-16 and C-19) are detected by HMBC (Fig. 12D). The correlation of HSQC HMBC and H H-COSY signals indicates the presence of glycosides of steviol with the following aglycone structure:

The correlation between HSQC and HMBC indicates the presence of 7 anomer signals indicated by 1i, 1ii, 1iii, 1iv, 1v, 1vi and 1vii. The coupling constant of the anomer proton of about 8 Hz, the broad signal of its sugar linkage and the NOE-correlation of the anomer proton allows the identification of these 7 sugars as β-D-glucopyranosides.

Combination data from HSQC and HMBC shows sugar-sugar linkages and sugar-aglycon linkages. The assignment of sugar sequences was confirmed using a combination of NOESY-TOCSY (Fig. 12E) and NOESY (Fig. 12F).

All together, the results from the above NMR experiments were used to assign the chemical shift of the carbon of the structure of proton and rebaudioside 2a (see Table 5).

Table 5 (continued)

Table 5 (continued)

Correlation of all NMR results shows rebaudioside 2 a with 7 β-D-glucose attached to steviol aglycone as shown in the following chemical structure:

Rebaudioside 2a LCMS (FIG. 12g and 12h) Analysis _{C 62 H 100 O 38 ([} C 62 H 99 O 38] - calculated for the single isotope _ion: 1451.6) in good agreement with expression molecule expected, at m / z 1451.6 [ MH] ^- represents an ion. MS data show that rebaudioside 2a is C ₆₂ H ₁₀₀ O ₃₈ Confirm that it has a molecular formula. LCMS analysis was performed under the conditions listed in Table 6.

Example 9

Rebaudioside 2m Structure of

Using samples dissolved in pyridine- d5 , NMR experiments were performed on a Bruker 500 MHz spectrometer. Along with the signal from the sample, the signal from pyridine- d5 at 123.5, 135.5, 149.9 ppm _C and 7.19, 7.55, 8.71 ppm _H was observed.

Of the rebaudioside 2m recorded in pyridine- d ₅ ^{The 1} H-NMR spectrum confirmed the excellent quality of the sample (see Fig. 13A). HSQC (see Figure 13b) shows the presence of exo-methylene groups in the sugar region with long-range coupling to C-15 observable in H H-COSY (Figure 13c). Other deep-field signals of quaternary carbons (C-13 C-16 and C-19) are detected by HMBC (Fig. 13D). The correlation of HSQC HMBC and H H-COSY signals indicates the presence of glycosides of steviol with the following aglycone structure:

The correlation between HSQC and HMBC indicates the presence of 7 anomer signals indicated by 1i, 1ii, 1iii, 1iv, 1v, 1vi and 1vii. The coupling constant of the anomer proton of about 8 Hz, the broad signal of its sugar linkage and the NOE-correlation of the anomer proton allows the identification of these 7 sugars as β-D-glucopyranosides.

Combination data from HSQC and HMBC shows sugar-sugar linkages and sugar-aglycon linkages. The assignment of sugar sequences was confirmed using a combination of NOESY-TOCSY (Fig. 13E) and NOESY (Fig. 13F).

All together, the results from the above NMR experiments were used to assign the chemical shift of the carbon of the structure of proton and rebaudioside 2m (see Table 7).

Table 7 (continued)

Table 7 (continued)

◆ Correlation of all NMR results shows rebaudioside 2m with 7 β-D-glucose attached to steviol aglycone as shown in the following chemical structure:

Rebaudioside 2m LCMS (FIG. 13g and 13h) Analysis _{C 62 H 100 O 38 ([} C 62 H 99 O 38] - for a single isotope ions _Calculation: The very good agreement with expression of the molecule expected 1451.6), [MH] at m / z 1451.6 ^- represents an ion. MS data show that rebaudioside 2m is C ₆₂ H ₁₀₀ O ₃₈ Confirm that it has a molecular formula. LCMS analysis was performed under the conditions listed in Table 6.

While the present invention and its advantages have been described in detail, it is to be understood that various changes, substitutions and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. In addition, the scope of this application is not intended to be limited to the specific embodiments of the invention described in this specification. As one of ordinary skill in the art will readily understand from this disclosure, currently existing or future developed compositions, processes, and methods that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein. And steps can be used in accordance with the present invention.

SEQUENCE LISTING <110> PureCircle USA Inc. <120> HIGH-PURITY STEVIOL GLYCOSIDES <130> 39227-77WO <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 808 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu 1 5 10 15 Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu 20 25 30 Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln 35 40 45 Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu 50 55 60 Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile 65 70 75 80 Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu 100 105 110 Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val 115 120 125 Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155 160 Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser 165 170 175 Leu Leu Pro Leu Leu Asp Phe Leu Arg Leu His Ser His Gln Gly Lys 180 185 190 Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His 195 200 205 Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr 210 215 220 Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg 225 230 235 240 Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu 245 250 255 Leu Asp Leu Leu Glu Ala Pro Asp Pro Ser Thr Leu Glu Thr Phe Leu 260 265 270 Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280 285 Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln 290 295 300 Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu 305 310 315 320 Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile 325 330 335 Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg 340 345 350 Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro 355 360 365 Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu 370 375 380 Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu 385 390 395 400 Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser 405 410 415 Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr 420 425 430 Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser 435 440 445 Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln 450 455 460 Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr 465 470 475 480 Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr 485 490 495 Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His 500 505 510 Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala 515 520 525 Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr 530 535 540 Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn 545 550 555 560 Asp Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe 565 570 575 Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu 580 585 590 Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Val Asn Leu Val Val 595 600 605 Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala 610 615 620 Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly 625 630 635 640 Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu 645 650 655 Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala 660 665 670 Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly 675 680 685 Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val 690 695 700 His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala 705 710 715 720 Ala Asp Leu Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser 725 730 735 His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys 740 745 750 Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val 755 760 765 Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu His Arg 770 775 780 Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln 785 790 795 800 Ala Val Pro Leu Ala Gln Asp Asp 805 <210> 2 <211> 442 <212> PRT <213> Solanum lycopersicum <400> 2 Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly 1 5 10 15 His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly 20 25 30 Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile 35 40 45 Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu 50 55 60 Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn 65 70 75 80 Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met 85 90 95 Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu 100 105 110 Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu 115 120 125 Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe 130 135 140 Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe 145 150 155 160 Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile 165 170 175 Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys 180 185 190 Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile 195 200 205 Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro 210 215 220 Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile 225 230 235 240 Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe 245 250 255 Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe 260 265 270 Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro 275 280 285 Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Glu Gly Phe Leu 290 295 300 Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln 305 310 315 320 Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys 325 330 335 Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile 340 345 350 Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val 355 360 365 Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile 370 375 380 His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu 385 390 395 400 Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu 405 410 415 Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile 420 425 430 Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys 435 440 <210> 3 <211> 458 <212> PRT <213> Stevia rebaudiana <400> 3 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ala Ile Thr Ile Leu His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Asp Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Gly Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Glu Trp Leu Asp Gln Gln Ala 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Gly Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Lys Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Pro Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Ser Phe Gly Gly Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Arg Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Val Val Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455

Claims (17)

Steviol Glycoside I-XL of the formula:

Wherein the R1 and R2 sugar chains are defined in the following table;

A method for preparing at least one steviol glycoside of claim 1 comprising the following steps:
a. Providing a starting composition comprising an organic compound having at least one carbon atom;
b. Providing an enzyme preparation or microorganism containing at least one enzyme selected from steviol biosynthetic enzyme NDP-glucosyltransferase and optionally NDP-glucose regeneration enzyme;
c. A step of preparing a medium comprising at least one steviol glycoside of claim 1 by contacting the enzyme preparation or microorganism with a medium containing the starting composition. A method for preparing at least one steviol glycoside of claim 1 comprising the following steps:
a. Providing a starting composition comprising an organic compound having at least one carbon atom;
b. Providing a biosynthetic enzyme containing at least one enzyme selected from steviol biosynthetic enzyme NDP-glucosyltransferase and optionally NDP-glucose regeneration enzyme;
c. A step of preparing a medium comprising at least one steviol glycoside of claim 1 by contacting a biosynthetic enzyme with a medium containing the starting composition. The method of claim 2 or 3, further comprising the following steps:
From the badge of paragraph 1 Separating at least one steviol glycoside of claim 1 At least one Providing a highly purified composition of steviol glycoside. According to claim 2, 3 or 4, the starting composition is steviol, steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol Bioside B, Rebaudioside B, Stevioside, Rebaudioside G, Stevioside A, Stevioside B, Stevioside C, Rebaudioside A, Rebaudioside E, Rebaudioside E2, Rebaudioside Baudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4 , other steviol glycosides, polyols, carbohydrates and combinations thereof. The method of claim 2, wherein the microorganism is Escherichia coli (E.coli), Saccharomyces in MRS (saccharomyces) species, Aspergillus (Aspergillus) species, Pichia (Pichia) species, Bacillus (Bacillus) species, and Yarrow subtotal (Yarrowia) Method selected from the group consisting of species. 4. The method of claim 3, wherein the biocatalyst is an enzyme capable of converting the starting composition to at least one steviol glycoside of claim 1 or a cell comprising one or more enzymes. The method of claim 2, wherein the enzyme is selected from the group consisting of: steviol biosynthetic enzyme, UDP glucosyltransferase, UDP glucose regeneration enzyme, mevalonate (MVA) pathway enzyme, 2-C-methyl-D- Erythritol-4-phosphate pathway (MEP/DOXP) enzyme, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, cowene synthase, cowene oxidase, cowenic 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-diphosphocytyl-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), l-hydr Roxy-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, UGT74G1, UGT85C2, UGT91D2, EUGT11, UGTSl2, UGT76G1, UGlyT91C1 or >85% amino-acid sequence identity, >86% amino-acid sequence identity, >87% amino -Acid sequence identity, >88% amino-acid sequence identity, >89% amino-acid sequence identity, >90% amino-acid sequence identity, >91% amino-acid sequence identity, >92% amino-acid sequence identity, >93% amino-acid sequence identity, >94% amino-acid sequence identity, >95% amino-acid sequence identity, >96% amino-acid sequence identity, >97% amino-acid sequence identity, >98% amino- Mutant variants thereof with acid sequence identity, >99% amino-acid sequence identity; And combinations thereof. The method of claim 4, wherein the content of at least one steviol glycoside in the highly purified composition of at least one steviol glycoside of claim 1 is greater than about 95% by weight on a dry basis. A consumable product comprising at least one steviol glycoside of claim 1, wherein the product is selected from the group consisting of food, beverage, pharmaceutical compositions, tobacco products, functional compositions, oral care compositions, and cosmetic compositions. 11. The consumable product of claim 10, wherein the product is selected from the group consisting of: beverage; Natural juice; Soft drinks; carbonated drink; Diet drink; Zero-calorie drinks; Reduced calorie beverages and foods; Yogurt drink; Instant juice; Instant coffee; Powder type instant drinks; Can products; syrup; Fermented soybean paste; Soy sauce; vinegar; dressing; mayonnaise; ketchup; Curry; Soup; Instant bouillon; Powdered soy sauce; Powdered vinegar; Type of biscuit; Rice biscuits; cracker; bread; Chocolate; Caramel; candy; Chewing gum; jelly; Pudding; Preserved fruits and plants; Fresh cream; jam; marmalade; Flower paste; Powdered milk; Ice cream; Sherbet; Plants and bottled fruit; Cans and boiled beans; Boiled meats and foods in sweet sauces; Vegetable food for agriculture; Seafood; ham; sausage; Fish ham; Fish sausage; Fish paste; Deep fried fish products; Dry seafood products; Frozen food; Preserved seaweed; Preserved meat; Tobacco and medical products. The organic acid salt and organic salt of claim 10, including carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts. Add at least one additive selected from the group consisting of base salts, inorganic salts, bitter compounds, caffeine, flavoring and flavor ingredients, astringent compounds, protein or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof Consumable products containing as. The method of claim 10, wherein saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, active bacteria, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long-chain primary aliphatic acids A consumable product further comprising at least one functional ingredient selected from the group consisting of saturated alcohols, phytosterols and combinations thereof. The consumable product according to claim 8, further comprising a compound selected from the group consisting of: steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, Steviolbioside C , Steviolbioside D , Steviolbioside E , Rubusoside, Dulcoside A, Dulcoside B , Dulcoside C , Dulcoside D , Stevioside, Stevioside A, Stevioside B, Stevio Side C , Stevioside D , Stevioside E , Stevioside E2, Stevioside F , Stevioside G , Stevioside H , Rebaudioside A, Rebaudioside A2, Rebaudioside A3, Rebaudioside A4 , Rebaudioside B, Rebaudioside B2, Rebaudioside C, Rebaudioside C2, Rebaudioside C3, Rebaudioside C4, Rebaudioside C5, Rebaudioside C6, Rebaudioside Baudioside D, Rebaudioside D2, Rebaudioside D3, Rebaudioside D4, Rebaudioside D5, Rebaudioside D6, Rebaudioside D7, Rebaudioside D8, Rebaudio Side E, Rebaudioside E2, Rebaudioside E3, Rebaudioside E4, Rebaudioside E5, Rebaudioside E6, Rebaudioside E7, Rebaudioside F, Rebaudioside F2 , Rebaudioside F3, Rebaudioside G, Rebaudioside H, Rebaudioside H2, Rebaudioside H3, Rebaudioside H4, Rebaudioside H5, Rebaudioside H6, Rebaudioside H6 Baudioside I, Rebaudioside I2, Rebaudioside I3, Rebaudioside J, Rebaudioside K, Rebaudioside K2, Rebaudioside KA, Rebaudioside L, Rebaudio Side M, Rebaudioside M2, Rebaudioside M3, Rebaudioside N, Rebaudioside N2, Rebaudioside N3, Rebaudioside N4, Rebaudioside N5, Rebaudioside O, Rebaudioside O2, Rebaudioside O3, Rebaudioside O4, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebau Rebaudioside S, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Rebaudioside AM, SvG7 NSF-02 Mogroside V , Syratos, Luo Han Guo, allulose, D-allose, D-tagatose, erythritol, brazein, neohesperidine dihydrochalcone, glycyrrhizic acid and its salts, taumatin, ◀ perillatin, fernandulsin, mucurgio Side, baunoside, flomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, perindrin, carnosyfloside, cyclocarioside, pterocaryoside, polypodoside A, Brasilin, Hernandulsin, Phyllodulsin, Glyciphilin, Florizine, Trilovatin, Dihydroflavonol, Dihydroquercetin-3-acetate, Neoastilibin, Trans-cinnamaldehyde, Monatin and its salts , Seligein A, Hematoxylin, Monelin, Osladin, Pterocaryoside A, Pterocaryoside B, Marvinlin, Pentadine, Miraculin, Curculin, Neoculin, Chlorogenic Acid, Cina Lin, ▶Cyamineside◀, ▶Sucralose, potassium acesulfame, aspartame, alitam, saccharin, cyclamate, neotam, dulsin, suosan advantam, ◀ Jimnemic acid, Hodulsin, Zippin, lactisol, glutamate , Aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, traitol, galactitol, hydrogenated isomaltulose , Reduced isomalto-oligosaccharide, reduced xylo-oligosaccharide, reduced genthio-oligosaccharide, ring Wanted maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzate, polyglycitol, sugar alcohol, L-sugar, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic Oligosaccharides, various maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altose, mannose, idose, lactose, maltose, anti-sugar, isotrehalose, neotrehalose, iso Maltulose, erythrose, deoxyribose, gulose, talos, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannozamine, glucuronic acid, gluconic acid, glucono-lactone, abecu Ose, galactozamine, sugar beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xilotriose, xylobiose, etc.), xyl Rho-terminal oligosaccharide, genthio-oligosaccharide (genthiobiose, genthiotriose, genthiotetraose, etc.), nigero-oligosaccharide, palatinose oligosaccharide, fructooligosaccharide (Kes Toss, nythose, etc.), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulo- Oligosaccharides, lactulose, melibiose, rapinose, isomerized liquid sugars such as high fructose corn syrup, coupling sugar, soybean oligosaccharide, ◀D-psicose, D-ribose, L-glucose, L- Fucose, D-Turanose, D-Lucrose. A method of increasing the sweetness of a beverage or food comprising a sweetener comprising:
a. Providing a beverage or food containing a sweetener; And
b. Adding a sweetness increasing agent comprising at least one steviol glycoside of claim 1, wherein the at least one steviol glycoside of claim 1 is present at a concentration below or below the sweetness perception threshold. A method for modifying the flavor of a beverage or food, including:
a. Providing a beverage or food, and
b. Adding a composition comprising at least one steviol glycoside of claim 1. How to suppress foaming in beverages or foods, including:
a. Providing a beverage or food, and
b. Adding a foam suppressant comprising at least one steviol glycoside of claim 1.

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