JP7432533B2 - High purity steviol glycosides - Google Patents

Description

The present invention relates to steviol glycosides and methods of making them, including highly purified steviol glycoside compositions.

BACKGROUND OF THE INVENTION High intensity sweeteners have sweetness levels many times higher than that of sucrose. They are essentially non-caloric and are commonly used in diet and reduced calorie products, including foods and beverages. High-intensity sweeteners do not cause a glycemic response, making them suitable for use in products intended for diabetics and other people interested in controlling their carbohydrate intake.

Steviol glycosides are a group of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae family (Compositae) that grows in certain regions of South America. It is. They are structurally characterized by a single base, steviol, which differs by the presence of carbohydrate residues at the C13 and C19 positions. They accumulate in Stevia leaves and constitute approximately 10% to 20% of the total dry weight. On a dry weight basis, the four major glycosides found in Stevia leaves are generally stevioside (9.1%), rebaudioside A (3.8%), and rebaudioside C (0.6-1.0%). , and dulcoside A (0.3%). Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.
Although methods for preparing steviol glycosides from Stevia rebaudiana are known, many of these methods are not suitable for commercial use.
Therefore, there remains a need for simple, efficient, and economical methods for preparing compositions containing steviol glycosides, including highly purified steviol glycoside compositions.

SUMMARY OF THE INVENTION The present invention provides for the production of target steviol glycosides by contacting a starting composition comprising an organic substrate with microbial cells and/or an enzyme preparation, thereby producing a composition comprising the target steviol glycosides. A method of preparing a composition comprising:
The starting composition can be any organic compound containing 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 glycosides are steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C , rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, or a synthetic steviol glycoside.
In one aspect, the target steviol glycoside is rebaudioside AM.
In some preferred embodiments, an enzyme preparation comprising one or more enzymes or a microbial cell comprising one or more enzymes capable of converting the starting composition to the target steviol glycoside. is used. Enzymes may be located on the surface and/or inside the cell. Enzyme preparations can be provided in the form of whole cell suspensions, crude lysates, or purified enzyme(s). Enzyme preparations can be in free form or immobilized on solid supports made of inorganic or organic materials.

In some embodiments, the microbial cell contains the necessary enzymes and genes encoding them to convert the starting composition to the target steviol glycoside. Accordingly, the present invention also provides for contacting a starting composition comprising an organic substrate with a microbial cell comprising at least one enzyme capable of converting the starting composition to a target steviol glycoside, thereby providing at least one Also provided is a method of preparing a composition comprising a target steviol glycoside by producing a medium comprising the target steviol glycoside.

Enzymes necessary to convert the starting composition to the target steviol glycosides include steviol biosynthetic enzymes, UDP-glucosyltransferases (UGTs), and/or UDP recycling enzymes.
In one aspect, the steviol biosynthetic enzymes include mevalonic acid (MVA) pathway enzymes.
In another aspect, the steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one aspect, the steviol biosynthetic enzymes include geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenate 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate Acid synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C- Methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, cleaved HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, and the like.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase that is capable of adding at least one glucose unit to a steviol and/or steviol glycoside substrate to provide a target steviol glycoside.
As used below, the term "SuSy_AT" refers to sucrose synthase having the amino acid sequence "SEQ ID NO: 1" as described in Example 1, unless specified otherwise.
As used below, the term "UGTS12" refers to a UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO: 2" as described in Example 1, unless specified otherwise.

As used below, the term "UGT76G1" refers to a UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO: 3" as described in Example 1, unless specified otherwise.
In one embodiment, steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in microbial cells. Microbial cells include, for example, E. Coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. It may be a genus or species (Yarrowia sp.) or the like. In another embodiment, UDP-glucosyltransferase is synthesized.

In one aspect, the UDP-glucosyltransferase is substantially (more than 85%, more than 86%, more than 87%, more than 88%, (more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) having amino acid sequence identity UGTs, as well as isolated nucleic acid molecules encoding these UGTs.

In one aspect, the steviol biosynthetic enzyme, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). Microorganisms include, for example, E. Coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. species (Yarrowia sp.).

In one aspect, the UDP-glucosyltransferase adds at least one glucose unit to steviol or any starting steviol glycoside with an -OH functionality at C13 to target a target with an -O-glucose beta glucopyranoside glycosidic linkage at C13. Any UDP-glucosyltransferase that can give steviol glycosides. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to steviol or any starting steviol glycoside having a -COOH functional group at C19 to have a -COO-glucose beta glucopyranoside glycosidic linkage at C19. Any UDP-glucosyltransferase that can provide the target steviol glycoside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to provide at least one glucose unit in the newly generated glycosidic bond(s). Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having two beta 1→2 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside, in the glycosidic bond(s) of the newly created bond. Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having at least one beta 1→3 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to provide at least one glucose unit in the newly generated glycosidic bond(s). Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having two beta 1→2 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol to produce steviol monoside. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity with UGT85C2, or a UGT with greater than 85% amino acid sequence identity with UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol to produce steviol monoside A. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviol monoside A to produce steviol monoside B. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviol monoside A to produce steviolbioside A. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside A to produce rubusoside. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside to produce rubusoside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside to produce steviolbioside.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviolbioside B to produce stevioside B. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside B to produce stevioside C. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside A. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside C. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside B. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside A (rebaudioside KA). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside to produce stevioside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to produce rebaudioside E2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside C to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside to produce rebaudioside E2. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside to produce rebaudioside E. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to produce rebaudioside AM.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E2 to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

Optionally, the methods of the invention include using two or more UGTs on the starting composition to provide a target steviol glycoside(s) having two or more more glucose units than the starting composition. further including. In certain embodiments, the UDP-glucosyltransferase is 85% UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2, or 85% UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2. have extremely high amino acid sequence identity and any UGT or those that can add two or more glucose units to the starting composition to give a steviol glycoside(s) having two or more more glucose units than the starting composition. Any combination.

In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add a total of two glucose units to stevioside to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is selected from UGTS12, EUGT11, UGT91D2, UGT76G1, or any UGT with greater than 85% amino acid sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1, or any combination thereof. . In another particular aspect, the UDP-glucosyltransferases are UGTSl2 and UGT76G1.

Optionally, the method of the invention further comprises recycling the UDP to provide UDP-glucose. In one aspect, the method comprises using catalytic amounts of UDP-glucosyltransferase and UDP-glucose such that biotransformation of steviol and/or steviol glycoside substrates to target steviol glycosides occurs. including recycling UDP by providing a recycling catalyst and a recycling substrate.
In one aspect, the recycling catalyst is sucrose synthase SuSy_At, or a sucrose synthase having greater than 85% amino acid sequence identity with SuSy_At.
In one aspect, the recycling substrate is sucrose.

Optionally, the methods of the invention further include the use of a transglycosidase using an oligosaccharide or polysaccharide as a sugar donor to modify the recipient target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase , fructosylinevertase, alkaline invertase , acid invertase, and fructofuranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, are transferred to the recipient target steviol glycoside. Ru. In one aspect, the recipient steviol glycoside is rebaudioside AM.

Optionally, the method of the invention further comprises separating the target steviol glycoside from the culture medium to obtain a highly purified target steviol glycoside composition. The target steviol glycosides may be separated by at least one suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.
In one aspect, the target steviol glycoside can be produced within a microorganism. In another aspect, the target steviol glycoside can be secreted into the culture medium. In another aspect, the released steviol glycosides can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the conversion reaction is complete.

In one aspect, the separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on a dry basis, ie, a highly purified steviol glycoside composition. In another embodiment, the separation produces 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 other embodiments, the composition comprises greater than about 99% by weight of the target steviol glycoside.
The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrides, or combinations thereof.

The purified targeted steviol glycosides can be used in consumable products as sweeteners, flavor modifiers, flavors with modifying properties and/or antifoam agents. Suitable consumable products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional compositions, oral hygiene compositions, and cosmetic compositions.

Figure 1 shows the chemical structure of rebaudioside AM. Figure 2 shows the pathway for producing rebaudioside AM and various steviol glycosides from steviol. Figure 3 shows the biocatalytic production of rebaudioside AM from stevioside using the enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via the sucrose synthase SuSy_At. Figure 4 shows the biocatalytic production of rebaudioside AM from rebaudioside E using the enzyme UGT76G1 and the concomitant recycling of UDP to UDP-glucose via the sucrose synthase SuSy_At. Figure 5 shows the HPLC chromatogram of stevioside. The peak with a retention time of 25.992 minutes corresponds to stevioside. Figure 6 shows the HPLC chromatogram of the product of biocatalytic production of rebaudioside AM from stevioside. The peak with a retention time of 10.636 minutes corresponds to rebaudioside AM. Figure 7 shows the HPLC chromatogram of rebaudioside E. The peak with a retention time of 10.835 minutes corresponds to rebaudioside E. FIG. 8 shows the HPLC chromatogram of the product of biocatalytic production of rebaudioside AM from rebaudioside E. The peaks with retention times of 10.936 minutes and 11.442 minutes correspond to rebaudioside E and rebaudioside AM, respectively. Figure 9 shows the HPLC chromatogram of rebaudioside AM after purification by methanol crystallization. The peak with a retention time of 10.336 minutes corresponds to rebaudioside AM. Figure 10 shows the ¹ H NMR spectrum of rebaudioside AM (500 MHz, pyridine-d5). Figure 11 shows the HSQC spectrum of rebaudioside AM (500 MHz, pyridine-d5). Figure 12 shows the H,H COZY spectrum of rebaudioside AM (500 MHz, pyridine-d5). Figure 13 shows the HMBC spectrum of rebaudioside AM (500 MHz, pyridine-d5). Figure 14 shows the HSQC-TOCSY spectrum of rebaudioside AM (500 MHz, pyridine-d5). Figures 15a and 15b show the LC chromatogram and mass spectrum of rebaudioside AM, respectively. Figures 15a and 15b show the LC chromatogram and mass spectrum of rebaudioside AM, respectively. Figure 2 is a graph showing the flavor modifying effect of Reb AM in coconut water. FIG. 17 is a graph showing the flavor modification effect of rebaudioside AM in chocolate protein shake.

DETAILED DESCRIPTION The present invention provides methods for preparing target steviol glycosides by contacting a starting composition comprising an organic substrate with microbial cells and/or an enzyme preparation, thereby producing a composition comprising the target steviol glycosides. A method of preparing a composition comprising:

One object of the present invention is to target steviol glycosides, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside An object of the present invention is to provide an efficient biocatalytic method for preparing Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, Rebaudioside AM, or synthetic steviol glycosides from various starting compositions.
As used herein, the abbreviation "reb" refers to "rebaudioside." Both terms have the same meaning and may be used interchangeably.

As used herein, "biocatalyst" or "biocatalysis" refers to a natural or genetically modified biocatalyst, such as an enzyme, or one Refers to the use of cells containing species or microorganisms containing two or more enzymes. Biocatalytic processes include fermentation, biosynthesis, bioconversion, and biotransformation processes. Both isolated enzyme and whole cell biocatalytic methods are known in the art. A biocatalytic protein enzyme can be a naturally occurring protein or a recombinant protein.

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

Starting Composition As used herein, "starting composition" refers to any composition (generally an aqueous solution) that includes one or more organic compounds containing at least one carbon atom.
In one aspect, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols, and various carbohydrates.

The steviol glycosides of the starting composition are steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), steviolside B, stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, or other glycosides of steviol occurring in the Stevia rebaudiana plant, synthetic steviol glycosides, such as enzymatically glucosylated steviol glycosides, and combinations thereof. selected from the group consisting of.
In one aspect, the starting composition is steviol.
In another embodiment, the steviol glycoside of the starting composition is steviol monoside.
In yet another aspect, the steviol glycoside of the starting composition is steviol monoside A.

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

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

In another embodiment, the steviol glycoside of the starting composition is rebaudioside E.
In another embodiment, the steviol glycoside of the starting composition is rebaudioside E2.
In another embodiment, the steviol glycoside of the starting composition is rebaudioside E3.

The term "polyol" refers to a molecule containing two or more hydroxyl groups. Polyols may be diols, triols, or tetraols containing 2, 3, and 4 hydroxyl groups, respectively. Polyols may also contain more than four hydroxyl groups, such as pentaol, hexaol, heptaol, each containing 5, 6, or 7 hydroxyl groups. Additionally, the polyol may also be a sugar alcohol, a polyhydric alcohol, or a polyhydric alcohol that is a reduced form of a carbohydrate, where the carbonyl groups (aldehydes or ketones, reducing sugars) are reduced to primary or secondary hydroxyl groups. has been done. Examples of polyols are erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides. , reduced gentio-oligosaccharides, reduced maltitol syrups, reduced glucose syrups, hydrogenated starch hydrolysates, polyglycitols, and sugar alcohols, or any other reducible carbohydrate. Not done.

The term "carbohydrate" refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, and oligomers and polymers thereof, of the general formula (CH ₂ O) _n , where n is 3 to 30. Additionally, the carbohydrates of the invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrate includes unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivative,""substitutedcarbohydrate," and "modified carbohydrate" are synonymous. Modified carbohydrate means any carbohydrate in which at least one atom has been added, removed, or substituted, or in which combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate may optionally be deoxygenated and/or hydrogen, at any corresponding C position, provided that the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition. Halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, Can be substituted with one or more moieties such as carbalkoxy, carboxamide, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonate, or any other viable functional group. .

Examples of carbohydrates that may be used according to the invention are tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, Arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose , cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abequosose, galactosamine, sugar beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xylotriose, xylobiose, etc.), xylo-terminal oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose, etc.) ), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose , ribose, high fructose corn syrup, coupling sugars, and soybean oligosaccharides. Furthermore, the carbohydrates used herein may be in either the D or L configuration.

The starting composition may be synthetic or purified (partially or completely), commercially available or prepared.
In one aspect, the starting composition is glycerol.
In another embodiment, the starting composition is glucose.
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 serve as substrate(s) for the production of the target steviol glycoside(s), as described herein.

Target Steviol Glycoside The target steviol glycoside of the present method can be any steviol glycoside that can be prepared by the methods disclosed herein. In one embodiment, the target steviol glycosides are steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C , rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or other glycosides of steviol occurring in the Stevia rebaudiana plant, synthetic steviol glycosides, such as enzymatically glucosylated steviol glycosides and combinations thereof. selected from the group consisting of:

In one aspect, the target steviol glycoside is steviol monoside.
In another aspect, the target steviol glycoside is steviol monoside A.
In another aspect, the target steviol glycoside is steviolbioside.
In another aspect, the target steviol glycoside is steviolbioside A.
In another aspect, the target steviol glycoside is steviolbioside B.
In another aspect, the target steviol glycoside is rubusoside.
In another aspect, the target steviol glycoside is stevioside.
In one aspect, the target steviol glycoside is stevioside A (rebaudioside KA).
In another aspect, the target steviol glycoside is stevioside B.

In another aspect, the target steviol glycoside is stevioside C.
In another aspect, the target steviol glycoside is rebaudioside E.
In another aspect, the target steviol glycoside is rebaudioside E2.
In another aspect, the target steviol glycoside is rebaudioside E3.
In another aspect, the target steviol glycoside is rebaudioside AM.

The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrides, or combinations thereof.
In one aspect, the invention is a biocatalytic process for the production of steviol monoside.
In one aspect, the invention is a biocatalytic process for the production of steviol monoside A.
In one aspect, the invention is a biocatalytic process for the production of steviolbioside.
In one aspect, the invention is a biocatalytic process for the production of steviolbioside A.

In one aspect, the invention is a biocatalytic process for the production of steviolbioside B.
In one aspect, the invention is a biocatalytic process for the production of rubusoside.
In one aspect, the invention is a biocatalytic process for the production of stevioside.
In one aspect, the invention is a biocatalytic process for the production of Stevioside A (Rebaudioside KA).
In one aspect, the invention is a biocatalytic process for the production of Stevioside B.

In one aspect, the invention is a biocatalytic process for the production of Stevioside C.
In one aspect, the invention is a biocatalytic process for the production of rebaudioside E.
In one aspect, the invention is a biocatalytic process for the production of rebaudioside E2.
In one aspect, the invention is a biocatalytic process for the production of rebaudioside E3.

In one aspect, the invention is a biocatalytic process for the production of rebaudioside AM.
In certain embodiments, the invention provides a biocatalytic process for producing rebaudioside AM from a starting composition comprising stevioside and UDP-glucose.
In another specific aspect, the invention provides a biocatalytic process for producing rebaudioside AM from a starting composition comprising rebaudioside E and UDP-glucose.
Optionally, the method of the invention further comprises separating the target steviol glycoside from the culture medium to obtain a highly purified target steviol glycoside composition. The target steviol glycosides may be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

In certain embodiments, the methods described herein result in highly purified target steviol glycoside compositions. 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 aspect, the highly purified target steviol glycoside composition comprises greater than about 90% by weight of target steviol glycoside on an anhydrous (dry) basis, e.g., greater than about 91% on a dry weight basis, about Target steviol concentration of greater than 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%. Contains glycoside content.

In one aspect, when the target steviol glycoside is reb AM, the methods described herein result in a composition having a reb AM content of greater than about 90% by weight on a dry weight basis. In another particular aspect, when the target steviol glycoside is reb AM, the methods described herein result in a composition comprising greater than about 95% reb AM content by weight on a dry weight basis. .

Microorganisms and Enzyme Preparations In one aspect of the invention, microorganisms (microbial cells) and/or enzyme preparations are contacted with a medium containing a starting composition to produce target steviol glycosides.
Enzymes can be provided in the form of whole cell suspensions, crude lysates, purified enzymes, or combinations thereof. In one aspect, the biocatalyst is a purified enzyme capable of converting the starting composition to the target steviol glycoside. In another aspect, the biocatalyst is a crude lysate containing at least one enzyme capable of converting the starting composition to the target steviol glycoside. In yet another embodiment, the biocatalyst is a whole cell suspension containing at least one enzyme capable of converting the starting composition to the target steviol glycoside.

In another aspect, the biocatalyst is one or more microbial cells containing enzyme(s) capable of converting the starting composition to the target steviol glycoside. Enzymes can be located on the surface of the cell, inside the cell, or both on the surface of the cell and inside the cell.
Suitable enzymes for converting the starting composition to the target steviol glycoside include, but are not limited to, steviol biosynthetic enzymes and UDP-glucosyltransferases (UGTs). Optionally, it may include UDP recycling enzyme(s).
In one aspect, the steviol biosynthetic enzymes include mevalonic acid (MVA) pathway enzymes.
In another aspect, the steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one aspect, the steviol biosynthetic enzymes include geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenate 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate Acid synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C- Methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, cleaved HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, and the like.
The UDP-glucosyltransferase can be any UDP-glucosyltransferase that is 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 cells include, for example, E. Coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. It may be a genus or species (Yarrowia sp.) or the like. In another embodiment, UDP-glucosyltransferase is synthesized.

In one aspect, the UDP-glucosyltransferase is substantially (more than 85%, more than 86%, more than 87%, more than 88%, (more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) having amino acid sequence identity UGTs, as well as isolated nucleic acid molecules encoding these UGTs.

In one aspect, the steviol biosynthetic enzyme, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). Microorganisms include, for example, E. Coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. species (Yarrowia sp.).

In one aspect, the UDP-glucosyltransferase adds at least one glucose unit to steviol or any starting steviol glycoside with an -OH functionality at C13 to target a target with an -O-glucose beta glucopyranoside glycosidic linkage at C13. Any UDP-glucosyltransferase that can give steviol glycosides. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to steviol or any starting steviol glycoside having a -COOH functional group at C19 to have a -COO-glucose beta glucopyranoside glycosidic linkage at C19. Any UDP-glucosyltransferase that can provide the target steviol glycoside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to provide at least one glucose unit in the newly generated glycosidic bond(s). Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having two beta 1→2 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside, in the glycosidic bond(s) of the newly created bond. Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having at least one beta 1→3 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase adds at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to provide at least one glucose unit in the newly generated glycosidic bond(s). Any UDP-glucosyltransferase capable of providing a target steviol glycoside with at least one additional glucose having two beta 1→2 glucopyranoside glycosidic bond(s). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol to produce steviol monoside. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity with UGT85C2, or a UGT with greater than 85% amino acid sequence identity with UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol to produce steviol monoside A. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviol monoside A to produce steviol monoside B. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviol monoside A to produce steviolbioside A. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside A to produce rubusoside. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside to produce rubusoside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviol monoside to produce steviolbioside.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to steviolbioside B to produce stevioside B. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside B to produce stevioside C. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside A. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside C. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside B. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside A (rebaudioside KA). In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to produce stevioside.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside to produce stevioside. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, or a UGT with greater than 85% amino acid sequence identity to UGT74G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside B to produce rebaudioside E2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside C to produce rebaudioside E3. In certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with greater than 85% amino acid sequence identity to UGT85C2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside to produce rebaudioside E2. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to stevioside to produce rebaudioside E. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.
In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that is capable of adding at least one glucose unit to rebaudioside E3 to produce rebaudioside AM.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E2 to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is UGTSl2, or a UGT having greater than 85% amino acid sequence identity with UGTSl2. In another particular aspect, the UDP-glucosyltransferase is EUGT11, or a UGT with greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2, or a UGT with greater than 85% amino acid sequence identity to UGT91D2.

In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is UGT76G1, or a UGT with greater than 85% amino acid sequence identity to UGT76G1.

Optionally, the method of the invention uses two or more UGTs relative to the starting composition to provide a target steviol glycoside(s) having two or more more glucose units than the starting composition. It further includes: In certain embodiments, the UDP-glucosyltransferase is 85% UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2, or 85% UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2. have extremely high amino acid sequence identity and any UGT or those that can add two or more glucose units to the starting composition to give a steviol glycoside(s) having two or more more glucose units than the starting composition. Any combination.

In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add a total of two glucose units to stevioside to produce rebaudioside AM. In certain embodiments, the UDP-glucosyltransferase is selected from UGTS12, EUGT11, UGT91D2, UGT76G1, or any UGT with greater than 85% amino acid sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1, or any combination thereof. . In another particular aspect, the UDP-glucosyltransferases are UGTSl2 and UGT76G1.

Optionally, the method of the invention further comprises recycling the UDP to provide UDP-glucose. In one aspect, the method comprises using catalytic amounts of UDP-glucosyltransferase and UDP-glucose such that biotransformation of steviol and/or steviol glycoside substrates to target steviol glycosides occurs. including recycling UDP by providing a recycling catalyst and a recycling substrate. The UDP recycling enzyme can be sucrose synthase SuSy_At or a sucrose synthase with greater than 85% amino acid sequence identity to SuSy_At, and the recycling substrate can be sucrose.

Optionally, the methods of the invention further include the use of a transglycosidase using an oligosaccharide or polysaccharide as a sugar donor to modify the recipient target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase , fructosylinevertase, alkaline invertase , acid invertase, and fructofuranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, are transferred to the recipient target steviol glycoside. Ru. In one aspect, the recipient steviol glycoside is rebaudioside AM.

In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to the starting composition steviol glycoside is selected from the list of GenInfo identifier numbers below, preferably as shown in Table 1 and Table 2. It has more than 85% amino acid sequence identity with a UGT selected from the group.

One aspect of the invention is a microbial cell containing an enzyme, an enzyme capable of converting a starting composition to a target steviol glycoside. Accordingly, some embodiments of the present methods include contacting a microorganism with a medium containing a starting composition to obtain a medium containing at least one target steviol glycoside.
The microorganism can be any microorganism that possesses 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 microorganisms include E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. including, but not limited to, Yarrowia sp.
In one aspect, the microorganisms are free when contacted with the starting composition.

In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, microorganisms may be immobilized on solid supports made of inorganic or organic materials. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derivatized cellulose or glass, ceramics, metal oxides or membranes. Microorganisms may be immobilized on solid supports, for example, by covalent bonding, adsorption, cross-linking, entrapment or encapsulation.
In yet another embodiment, an enzyme capable of converting the starting composition to the target steviol glycoside is secreted from the microorganism 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 to obtain a highly purified target steviol glycoside composition. Suitable methods include crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or a combination of such methods.

Use particularly the highly purified target glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used "as is" or in combination with other sweeteners, flavors, food ingredients and combinations thereof. sell.

Non-limiting examples of flavors include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla, and combinations thereof. However, it is not limited to these.
Non-limiting examples of other food ingredients include acidulants, organic acids and amino acids, colorants, fillers, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, enhancers. Including, but not limited to, thickening agents, gelling agents and combinations thereof.

In particular highly purified target glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM include hydrates, solvates, anhydrides, amorphous forms, and combinations thereof, Can be prepared in a variety of polymorphic forms, including but not limited to.

In particular the highly purified target glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used in foods, beverages, pharmaceutical compositions, cosmetics, chewing gums, tabletop products, cereals, dairy products, toothpastes and other oral compositions, etc., as high-intensity natural sweeteners.

Highly purified target glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used as sweetener compounds as the sole sweetener, or Rebaudioside A, Rebaudioside A2, Rebaudioside A3 , rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside O, rebaudioside O2, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Darcoside A, Darcoside C, Stevioside D, Stevioside E, Stevioside E2, Stevioside F, Mogrosides, Blazein, Neohesperidin dihydrochalcone, Glycyrrhizic acid and its salts, Thaumatin , perilartin, pernandurtin, muclodiosides, bayunoside, furomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosifurosides, cyclokaryoside, pterocariosides, polypodoside A, brazilin, hernandurtin, filo Dultin, glycifyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastiribine, trans-cinnamaldehyde, monatin and its salts, serigain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mavinrin, pentadine , miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, cyamenoside, and combinations thereof.

In certain embodiments, steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2. , rebaudioside E3 and/or rebaudioside AM are rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside O, rebaudioside O2, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Darcoside A, Darcoside C, Stevioside D, Stevioside E, Stevioside E2, Stevioside F , NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol, and combinations thereof.

Highly purified target glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may also include sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dultin, suosan, advantame, etc. may be used in combination with synthetic high-intensity sweeteners, such as salts of

In addition, highly purified target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, steviolside A (rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used in combination with natural sweetener inhibitors such as gymnemic acid, fozurusin, didifine, lactisol. Steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/ Or rebaudioside AM may also be combined with various umami taste enhancers. Steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/ Or rebaudioside AM can be mixed with umami and sweet amino acids such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and combinations thereof.

Highly purified target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA) , Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM are carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants , emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

Highly purified target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA) , Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be combined with polyols or sugar alcohols. The term "polyol" refers to a molecule containing two or more hydroxyl groups. Polyols may be diols, triols, or tetraols containing 2, 3, and 4 hydroxyl groups, respectively. Polyols may also contain more than four hydroxyl groups, such as pentaol, hexaol, heptaol, each containing 5, 6, or 7 hydroxyl groups. Additionally, the polyol may also be a sugar alcohol, a polyhydric alcohol, or a polyhydric alcohol that is a reduced form of a carbohydrate, where the carbonyl groups (aldehydes or ketones, reducing sugars) are reduced to primary or secondary hydroxyl groups. has been done. Examples of polyols are erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides. , reduced gentio-oligosaccharides, reduced maltose syrups, reduced glucose syrups, hydrogenated starch hydrolysates, polyglycitols, and sugar alcohols, or any other reducible material that does not adversely affect the taste of the sweetener composition. including, but not limited to, carbohydrates.

Highly purified target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA) , Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be combined with low calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L-arabinose and combinations thereof. May be combined.

Highly purified target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA) , Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may also be combined with various carbohydrates. The term "carbohydrate" generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, and oligomers and polymers thereof, of the general formula (CH ₂ O) _n , where n is 3 to 30. Additionally, the carbohydrates of the invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrate includes unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivative,""substitutedcarbohydrate," and "modified carbohydrate" are synonymous. Modified carbohydrate means any carbohydrate in which at least one atom has been added, removed, or substituted, or in which combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate may optionally be deoxygenated and/or hydrogen, at any corresponding C position, provided that the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition. Halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, Can be substituted with one or more moieties such as carbalkoxy, carboxamide, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonate, or any other viable functional group. .

Examples of carbohydrates that may be used according to the invention are psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, Ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose , xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, sugar beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.) ), xylo-oligosaccharides (xylotriose, xylobiose, etc.), xylo-terminal oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructo-oligos Sugars (kestose, nystose, etc.), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulo-oligosaccharides , lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Furthermore, the carbohydrates used herein may be in either the D or L configuration.

In particular the highly purified target steviol glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients generally include carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutritional supplements, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols); polyols; prebiotics, probiotics ; phytoestrogens; soy proteins; sulfides/thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients may also be categorized based on health benefits such as cardiovascular, cholesterol lowering, anti-inflammatory, etc. Exemplary functional ingredients are provided in WO2013/096420, the contents of which are incorporated herein by reference.

In particular the highly purified target steviol glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used to produce zero-calorie, low-calorie or diabetic beverages and food products with improved taste properties. , may be applied as a high intensity sweetener. It may also be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used. Furthermore, highly purified target steviol glycosides, particularly steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used not only as sweeteners in beverages, foods and other products for human consumption, but also in animal and livestock feeds with improved properties. .

In particular the highly purified target steviol glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used to produce zero calorie, low calorie or diabetic beverages and food products with modified flavours. , may be applied as a flavor modifier. When used as a flavor modifier, or flavor with modifying properties (FMP), highly purified target steviol glycosides are used in consumable products at below detectable levels of the flavor modifier or FMP. Flavor modifiers or FMPs do not impart a detectable taste or flavor of their own to a consumable product, but instead modify the consumer's detection of the taste and/or flavor of other ingredients in the consumable product. Helpful. One example of taste and flavor modification is sweetness enhancement, where the flavor modifier or FMP itself does not contribute to the sweetness of the consumable product, but enhances the quality of sweetness that is experienced by the consumer.

Highly purified target steviol glycoside(s), in particular steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, Examples of consumable products in which Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used as flavor modifiers or flavors with modifying properties include vodka, wine, beer, liqueurs, sake, etc. Alcoholic drinks; Natural juices; Soft drinks; Carbonated soft drinks; Diet drinks; Zero calorie drinks; Low calorie drinks and foods; Yogurt drinks; Instant juice; Instant coffee; Powder type instant drinks; Canned goods; Syrup; Bean sauce; Soy sauce; Vinegar; dressing; mayonnaise; ketchup; curry; soup; instant bouillon; soy sauce powder; powdered vinegar; biscuit type; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables ; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sherbet; bottled vegetables and fruit; canned boiled beans; meat and foods cooked in sweet sauces; agricultural vegetable foods; seafood; ham; sausage; fish meat ham; fish meat These include, but are not limited to, sausages; fish paste; fried fish products; dried seafood products; frozen foods; preserved seaweed; preserved meat; tobacco; pharmaceuticals; and many others. Basically, it can have unlimited uses.

In particular the highly purified target steviol glycoside(s) obtained according to the invention, in particular steviol monoside, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, Stevioside A (Rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM can be used as antifoaming agents to produce zero-calorie, low-calorie, or diabetic beverages and foods. May be applied.

Highly purified target steviol glycoside(s), in particular steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, Examples of consumable products in which Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used as sweetener compounds are alcoholic beverages such as vodka, wine, beer, liqueurs, sake; natural juices; refreshing drinks; Beverages; Carbonated soft drinks; Diet drinks; Zero calorie drinks; Low calorie drinks and foods; Yogurt drinks; Instant juice; Instant coffee; Powder type instant drinks; Canned goods; Syrup; Bean sauce; Soy sauce; Vinegar; Dressing; Mayonnaise; Ketchup; curry; soup; instant bouillon; soy sauce powder; powdered vinegar; biscuit type; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; ; flour paste; powdered milk; ice cream; sorbet; bottled vegetables and fruit; canned boiled beans; meat and foods boiled in sweet sauce; agricultural vegetable foods; seafood; ham; sausage; fish ham; fish sausage; fish paste; fried fish products; dried seafood products; frozen foods; preserved seaweed; preserved meat; tobacco; pharmaceuticals; and many others. Basically, it can have unlimited uses.
In the production of products such as food, beverages, pharmaceuticals, cosmetics, tabletop products, and chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, permeating, infiltrating, scattering, spraying, pouring and other methods are used. method may be used.

Furthermore, the highly purified target steviol glycoside(s) obtained in the present invention, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside) KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM may be used in dry or liquid form.

The highly purified targeted steviol glycosides can be added before or after heat processing of the food product. Highly purified targeted steviol glycosides, especially steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E , rebaudioside E2, rebaudioside E3 and/or rebaudioside AM depending on the purpose of use. As mentioned above, it can be added alone or in combination with other compounds.

The present invention also provides steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and/or sweetness enhancement in beverages using rebaudioside AM. Therefore, the present invention provides sweeteners and steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2. , rebaudioside E3, and/or rebaudioside AM as a sweetness enhancer, wherein steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM are present at concentrations below their respective sweet taste perception thresholds.

As used herein, the term "sweetness enhancer" refers to a compound that can enhance or intensify the perception of sweetness in a composition such as a beverage. The term "sweetness enhancer" includes "sweet taste potentiator," "sweetness potentiator," "sweetness amplifier," and "sweetness enhancer." taste sensitization The term sweetness intensifier is synonymous with the term sweetness intensifier.

The term "sweet taste perception threshold concentration" as used generally herein is the lowest known concentration of a sweet tasting compound that can be perceived by the human palate, typically around 1.0% sucrose equivalent (1 .0%SE). In general, a sweetness enhancer may enhance or intensify the sweetness of a sweetener without providing any significant sweetness by itself when present below the sweetness perception threshold concentration of a given sweetness enhancer; however , sweetness enhancers may themselves provide sweetness at concentrations above their sweetness perception threshold concentration. The sweetness perception threshold concentration is specific to the particular enhancer and can vary based on the beverage matrix. Sweetness perception threshold concentrations can be readily determined by taste testing in which the concentration of a given enhancer is increased until more than 1.0% sucrose equivalent is detected in a given beverage matrix. The concentration that provides approximately 1.0% sucrose equivalent is considered the sweet taste perception threshold.

In some embodiments, the sweetener is, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.0%, by weight. 5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0 Weight%, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight %, about 11.0%, about 11.5%, or about 12.0% by weight, from about 0.5% to about 12% by weight.

In certain embodiments, the sweetener is from about 0.5% by weight, such as from about 2% to about 8%, from about 3% to about 7%, or from about 4% to about 6%. It is present in the beverage in an amount of about 10% by weight. In certain embodiments, the sweetener is present in the beverage in an amount from about 0.5% to about 8% by weight. In certain embodiments, the sweetener is present in the beverage in an amount from about 2% to about 8% by weight.

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

In yet another embodiment, the sweetener is a rare sugar. Suitable rare sugars include D-allose, D-psicose, D-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose, D-turanose, D-leuclose, and combinations thereof. Not limited to these.
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 particular aspect, 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% by weight.

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

In a further embodiment, the rare sugar is L-glucose. In more particular embodiments, 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% by weight.
In one aspect, the rare sugar is L-fucose. In a more particular aspect, 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% by weight.
In another embodiment, the rare sugar is L-arabinose. In a more particular aspect, 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% by weight.

In yet another embodiment, the rare sugar is D-turanose. In a more particular embodiment, D-turanose is present in the beverage in an amount from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight.
In yet another embodiment, the rare sugar is D-leuclose. In a more particular aspect, D-leuclose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as about 2% to about 8% by weight.
Addition of a sweetness enhancer at a concentration below the sweetness perception threshold increases the detected sucrose equivalents of a beverage containing the sweetener and sweetness enhancer compared to a corresponding beverage in the absence of any sweetness enhancer. . Additionally, the sweetener can be increased by an amount greater than the detectable sweetness of a solution containing the same concentration of at least one sweetness enhancer in the absence of the sweetener.

The invention therefore also provides a beverage comprising a sweetener, as well as steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B , Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM or combinations thereof. Here, steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and /or the rebaudioside AM is present at a concentration below the sweet taste perception threshold.

Steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside at concentrations below the sweet taste perception threshold. The addition of E3 and/or rebaudioside AM to a beverage containing a sweetener can be, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about The detected sucrose equivalent may be increased by about 1.0% to about 5.0%, such as 3.5%, about 4.0%, about 4.5% or about 5.0%.

The following examples include highly purified target steviol glycoside(s), specifically steviol monoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA). ), represents a preferred embodiment of the invention for the preparation of Stevioside B, Stevioside C, Rebaudioside E, Rebaudioside E2, Rebaudioside E3 and/or Rebaudioside AM. It will be understood that the materials, ratios, conditions, and procedures described in the Examples are illustrative only and the invention is not limited thereto.

Example (Example 1)
Protein sequence of modified enzyme used in biocatalytic process SEQ ID NO: 1:
>SuSy_At, variant PM1-54-2-E05 (modified sucrose synthase; source of WT gene: Arabidopsis thaliana)

Sequence number 2:
>UGTSl2 variant 0234 (modified glucosyltransferase; source of WT gene: tomato (Solanum lycopersicum))

Sequence number 3:
>UGT76G1 variant 0042 (modified glucosyltransferase; source of WT gene: Stevia rebaudiana)

(Example 2)
Expression and Preparation of the SuSy_At Variant of SEQ ID NO: 1 The gene encoding the SuSy_At variant of SEQ ID NO: 1 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). Using the obtained plasmid, E. E. coli BL21 (DE3) cells were transformed.

Cells were cultured at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l). Gene expression was induced by IPTG (0.2 mM) in log phase and carried out at 30° C. and 200 rpm for 16-18 hours.
Cells were collected by centrifugation (3220 x g, 20 min, 4°C) and lysed for 200 min in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM _MgCl2 , DNA nuclease 20U/mL, lysozyme 0.5mg/mL). (measured at 600 nm (OD ₆₀₀ )). Cells were then disrupted by sonication and the crude extract was separated from cell debris by centrifugation (18000 x g, 40 min, 4°C). The supernatant was filter sterilized through a 0.2 μm filter and diluted 50:50 with distilled water to obtain an enzyme activity preparation.

For the enzymatic activity preparation of SuSy_At, the unit activity 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, 50mM potassium phosphate buffer pH 7.0, 400mM sucrose at _t0 , 3mM _MgCl2 , and 15mM uridine diphosphate (UDP).

(Example 3)
Expression and preparation of the UGTSl2 variant of SEQ ID NO: 2 The gene encoding the UGTSl2 variant of SEQ ID NO: 2 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). Using the obtained plasmid, E. E. coli BL21 (DE3) cells were transformed.
Cells were cultured at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l). Gene expression was induced by IPTG (0.1 mM) in log phase and carried out at 30° C. and 200 rpm for 16-18 hours.

Cells were collected by centrifugation (3220 x g, 20 min, 4°C) and lysed for 200 min in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM _MgCl2 , DNA nuclease 20U/mL, lysozyme 0.5mg/mL). (measured at 600 nm (OD ₆₀₀ )). Cells were then disrupted by sonication and the crude extract was separated from cell debris by centrifugation (18000 x g, 40 min, 4°C). The supernatant was filter sterilized through a 0.2 μm filter and diluted 50:50 with 1M sucrose solution to obtain the enzyme activity preparation.

For the enzymatic activity preparation of UGTSl2, the unit 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. The reaction conditions of the assay were: 30°C, 50mM potassium phosphate buffer pH 7.0, 10mM Reb A at _t0 , 500mM sucrose, 3mM _MgCl2 , 0.25mM uridine diphosphate (UDP) and 3U/mL SuSy_At. be.

(Example 4)
Expression and Preparation of the UGT76G1 Variant of SEQ ID NO: 3 The gene encoding the UGT76G1 variant of SEQ ID NO: 3 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). Using the obtained plasmid, E. E. coli BL21 (DE3) cells were transformed.
Cells were cultured at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l). Gene expression was induced by IPTG (0.1 mM) in log phase and carried out at 30° C. and 200 rpm for 16-18 hours.

Cells were collected by centrifugation (3220 x g, 20 min, 4°C) and lysed for 200 min in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM _MgCl2 , DNA nuclease 20U/mL, lysozyme 0.5mg/mL). (measured at 600 nm (OD ₆₀₀ )). Cells were then disrupted by sonication and the crude extract was separated from cell debris by centrifugation (18000 x g, 40 min, 4°C). The supernatant was filter sterilized through a 0.2 μm filter and diluted 50:50 with 1M sucrose solution to obtain the enzyme activity preparation.
For the enzymatic activity preparation of UGT76G1, the unit activity is defined as follows: 1 mU of UGT76G1 converts 1 nmol of rebaudioside D (RebD) to rebaudioside M (RebM) in 1 minute. The reaction conditions of the assay were: 30°C, 50mM potassium phosphate buffer pH 7.0, 10mM Reb A at _t0 , 500mM sucrose, 3mM _MgCl2 , 0.25mM uridine diphosphate (UDP) and 3U/mL SuSy_At. be.

(Example 5)
Synthesis of rebaudioside AM from stevioside in a one-pot reaction with simultaneous addition of UGTSl2, SuSy_At and UGT76G1 Three enzymes: UGTSl2 (variant of SEQ ID NO: 2), SuSy_At- (variant of SEQ ID NO: 1) and UGT76G1 (variant of SEQ ID NO: 3) (see Examples 1, 2, 3, and 4), rebaudioside AM (reb Am) was synthesized directly from stevioside in a one-pot reaction (Figure 3). The final reaction solution contained 105 U/L UGTSl2, 405 U/L SuSy_At, 3 U/L UGT76G1, 5 mM stevioside, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl ₂ , and phosphorus. It contained a potassium acid buffer (pH 6.6). First, 207 mL of distilled water was mixed with 0.24 g of MgCl ₂ .6H ₂ O, 103 g of sucrose, 9.9 mL of 1.5 M potassium phosphate buffer (pH 6.6), and 15 g of stevioside. After the components were dissolved, the temperature was adjusted to 45° C. and UGTSl2, SuSy_At, UGT76G1, and 39 mg of UDP were added. The reaction mixture was incubated on a shaker at 45°C for 24 hours. An additional 39 mg of UDP was added at 8 and 18 hours. At several time points, the contents of reb AM, reb E, stevioside, reb M, reb B, steviolbioside, and reb I were analyzed by HPLC.

For analysis, the biotransformation samples were inactivated by adjusting the reaction mixture to pH 5.5 using 17% H ₃ PO ₄ and then boiling for 10 min. The resulting 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, autosampler, UV detector with background correction, and data acquisition system. Analytes were separated using an Agilent Poroshell 120 SB-C18, 4.6 mm x 150 mm, 2.7 μm, 40°C. The mobile phase consists of two premixes:
- Premix 1 containing 75% 10mM phosphate buffer (pH 2.6) and 25% acetonitrile, and - Premix 2 containing 68% 10mM phosphate buffer (pH 2.6) and 32% acetonitrile. .

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

Table 3 shows the conversion of stevioside to the identified rebaudioside species (area percentage) for each time point. The chromatograms of stevioside and reaction mixture after 24 hours are shown in Figures 5 and 6, respectively. Those skilled in the art will appreciate that retention times can sometimes vary due to changes in solvent and/or equipment.

(Example 6)
Synthesis of Rebaudioside AM from Rebaudioside E in a Simultaneous One-Pot Reaction of SuSy_At and UGT76G1 Two enzymes: SuSy_At- (variant of SEQ ID NO: 1) and UGT76G1 (variant of SEQ ID NO: 3) (see Examples 1, 2, and 4) Using this method, rebaudioside AM (reb AM) was directly synthesized from rebaudioside E (reb E) in a one-pot reaction (Figure 4). The final reaction solution contained 405 U/L SuSy_At, 3 U/L UGT76G1, 5 mM reb E, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl ₂ 6H ₂ O, and phosphoric acid. Contained potassium buffer (pH 6.6). First, 37 mL of distilled water was mixed with 40.3 mg MgCl ₂ , 17.12 g sucrose, 1.65 mL 1.5 M potassium phosphate buffer (pH 6.6), and 5.04 g reb E. After dissolving the components, the temperature was adjusted to 45° C. and SuSy_At, UGT76G1, and 6.5 mg of UDP were added. The reaction mixture was incubated on a shaker at 45°C for 24 hours. An additional 6.5 mg of UDP was added at 8 and 18 hours. At several time points, the contents of reb AM, reb E, stevioside, reb A, reb M, reb B, and steviolbioside were analyzed by HPLC.

For analysis, the biotransformation samples were inactivated by adjusting the reaction mixture to pH 5.5 using 17% H ₃ PO ₄ and then boiling for 10 min. The resulting 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, autosampler, UV detector with background correction, and data acquisition system. Analytes were separated using an Agilent Poroshell 120 SB-C18, 4.6 mm x 150 mm, 2.7 μm, 40°C. The mobile phase consists of two premixes:
- Premix 1 containing 75% 10mM phosphate buffer (pH 2.6) and 25% acetonitrile, and - Premix 2 containing 68% 10mM phosphate buffer (pH 2.6) and 32% acetonitrile. .

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

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

(Example 7)
Purification of Rebaudioside AM After 24 hours, the reaction mixture of Example 5 was inactivated by adjusting the pH to pH 5.5 with H ₃ PO ₄ and then boiled for 10 minutes. After boiling, the reaction mixture was filtered and diluted to 5% solids with RO water. The diluted solution was passed through a 1 L column packed with YWD03 macroporous adsorption resin (Cangzhou Yuanwei, China). The adsorbed steviol glycosides were eluted with 5 L of 70% ethanol. The resulting eluate was evaporated to dryness to obtain 16 g of dry powder, which was dissolved in 80 mL of 70% methanol. The solution was crystallized at 20° C. for 3 days. The crystals were separated by filtration and dried in a vacuum oven at 80° C. for 18 hours to yield 10.4 g of pure reb AM crystals with a purity of 95.92% as determined by HPLC assay. A chromatogram of reb AM is shown in FIG. Those skilled in the art will appreciate that retention times can sometimes vary due to changes in solvent and/or equipment.

(Example 8)
Structural elucidation of rebaudioside AM NMR experiments were performed on a Bruker 500 MHz spectrometer using samples dissolved in pyridine-d5. In addition to the signals from the sample, signals from pyridine-d5 were observed at δ _C 123.5, 135.5, 149.9 ppm and δ _H 7.19, 7.55, 8.71 ppm.

The ¹ H-NMR spectrum of rebaudioside AM in pyridine- _d5 reveals the excellent quality of the sample (see Figure 10). HSQC (see Figure 11) shows the presence of an exomethylene group in the sugar region, with long range coupling to C-15 observable in H,H-COSY (Figure 12). Other deep field signals of quaternary carbons (C-13, C-16, and C-19) are detected by HMBC (Figure 13). Correlation of signals in HSQC, HMBC, and H,H-COSY reveals the presence of steviol glycosides with the following aglycone structure:

Correlation of HSQC and HMBC signals reveals five anomeric signals. The binding constant of the anomeric protons of about 8 Hz and the broad signal of their sugar binding allow identification of these five sugars as β-D-glucopyranosides.
Observation of anomeric protons in combination with HSQC and HMBC reveals a correlation with sugar bonds and aglycones. Sugar sequence assignments were confirmed using a combination of HSQC-TOCSY (Figure 14) and HSQC.

The above NMR experiments were applied to assign proton and carbon chemical shifts, main coupling constants, and main HMBC correlations (see Table 5).

The correlation of all NMR data points to rebaudioside AM with five β-D-glucopyranoses attached to the steviol aglycone, as shown in the chemical structure below:

The chemical formula of rebaudioside AM is C ₅₀ H ₈₀ O ₂₈ , which corresponds to a calculated monoisotopic molecular weight of 1128.5. For LCMS analysis, rebaudioside AM was dissolved in methanol and analyzed using a Shimadzu Nexera 2020 UFLC LCMS instrument on a Cortecs UPLC C18 1.6 μm, 50×2.1 mm column. The observed LCMS (negative ESI mode) results for 1127.3 (see Figures 15a and 15b, respectively) are consistent with rebaudioside AM and correspond to the ion (MH) ^- .

Solubility, sweetness and flavor modification properties of Reb AM (Example 9)
Reb AM was evaluated for solubility and solution stability. Tables 6a and 6b below show the composition of the test samples, with the percentage of total steviol glycosides (TSG) shown in the last column of Table 6b.

Solution stability:
The dissolution properties were measured as follows. The following aqueous solutions are prepared and each is stirred at 700 rpm. If necessary, heat at 2 minutes and 30 seconds of stirring. Use a stopwatch to determine the time it takes for all the powder to completely dissolve and record the temperature at which it dissolves. The table below summarizes the solubility properties of rebaudioside D, M, and AM. Surprisingly, Reb AM exhibits significantly higher solubility than other minor and major steviol glycosides.

(Example 10)
Reb AM was evaluated for its sensory properties.

Sensory Properties Steviol glycoside molecules are known to vary in their sweetness profile as a function of the sugar moieties present in their structure. Because steviol glycosides contain hydrophobic (steviol) and hydrophilic (sugar moieties), they can exhibit flavor modification at certain dosage levels without significantly contributing to any detectable sweetness perception. can.

Isosweetness determination of Reb AM and other steviol glycosides:
A panel of 40 participants was employed to conduct a two-alternative forced-choice (2-AFC) test at each concentration level. were identified to correspond to sucrose equivalents of 2.5%, 5%, 7.5%, and 10% in
- Once 50% of the panelists selected the sucrose sample as sweeter and 50% selected the stevia sample as sweeter, the samples were evaluated and the isosweetness point was determined.
- Concentration-response relationships were fitted using the Beidler model using four equal sweetness concentrations and their corresponding target sweetness values as data.
- Sweetness potency is calculated as the ratio of sugar concentration to sweetness equivalent. As an example, Reb AM was evaluated.

Effect of Reb AM on Taste & Flavor Profiles for Food and Beverage Applications A series of experiments were conducted to evaluate the effect of Reb AM on taste and flavor profiles. Sweetness and taste/flavor modification can influence each other in food and beverage applications. In order to determine the impact of taste and flavor modification in different applications, FEMA (Flavor and Extract Manufacturing Association) has defined a sensory method for determining sweetness perception threshold measurements as shown in Experiment 1, described below. ing.

Experiment 1 provides an estimate of Reb AM concentration in water that contributes little to sweetness perception. The sweetness perception threshold concentration provides significantly less sweetness than 1.5% sugar in water. Sweetness perception thresholds for selected steviol glycosides are summarized in Table 11 below.

Experiment 2, described further below, investigates the effect of Reb AM on the flavor profile of non-alcoholic beverages. To determine the influence of Reb AM on different taste attributes of the beverage, commercially available raspberry watermelon coconut water samples were used without Reb AM (control) and with Reb AM (test). The results showed that the test samples containing Reb AM had significantly higher mango peach flavor, coconut water flavor, and overall palatability compared to the control sample (95% confidence level).

Experiment 3, described further below, investigates the effect of Reb AM on the taste & flavor profile of sweetened dairy products. In the sensory panel, samples of unsweetened chocolate flavored milk protein shake sweetened with Stevia (Reb A) were tested without (control) and with Reb AM. The panel found that test samples containing 50 ppm Reb AM had significantly lower bitterness, metallic aroma, whey protein, and lower bitter aftertaste than the control (95% confidence), and also had higher cocoa flavor, Found to have dairy aroma, vanilla flavor, and overall palatability (95% confidence).

A panel of trained and experienced taste panel members evaluated zero-calorie lemon-lime carbonated soft drinks (CSD) sweetened with 500 ppm Reb AM, Reb D, or Reb M samples. Panel members found that the CSD using Reb AM was less sweet, but had significantly less residual bitterness and sweetness compared to CSD sweetened with other samples, particularly Reb M.

Experiment 1 of Example 10:
Sweet taste perception threshold of Reb AM Application: Neutral water 1.5% sugar solution and sweetness perception of various solutions of Reb AM were tested on a sensory panel, and 50 ppm Reb AM aqueous solution was significantly higher than 1.5% sugar solution. was found to provide lower sweetness perception. Therefore, we selected 50 ppm Reb AM as the recognition threshold concentration.

Method

The following table (Table 13) is based on the ``FEMAGRAS™'' program published by FEMA (Flavor and Extract Manufacturers Association https://www.femaflavor.org/) for sensory testing of flavorants with modified properties. The evaluation of the recognition threshold concentration according to the method outlined in Section 1.4.2 of the ``Guidance for Human Research and Human Research'' is presented.

Experiment 2 of Example 10:
Raspberry Watermelon Coconut Water Containing Reb AM Application: Non-Alcoholic Beverage Overview Thirty panel members evaluated two samples of raspberry watermelon flavored coconut water in two sessions for overall acceptability and attribute strength ( Sweet taste, raspberry flavor, watermelon flavor, coconut water flavor, salty taste, bitter taste, sweet aftertaste, bitter aftertaste) were evaluated. For Session 1, the two samples included: 1) a store-bought raspberry-watermelon-coconut water control sample, and 2) a store-bought raspberry-watermelon-coconut water test sample containing Reb AM. The purpose of the study was to determine whether the addition of Reb AM affects the flavor profile of non-alcoholic beverages. Results showed that the Reb AM test sample had significantly higher mango peach flavor, coconut water flavor, and overall palatability compared to the control sample (95% confidence level).

Purpose The purpose of the project is to evaluate whether the addition of stevia extract solids affects key flavor attributes in various beverage applications.

Purpose of the Test The purpose of the test was to determine whether the flavor profile and overall acceptability of a control sample of flavored coconut water differed from a test sample of the same beverage containing Reb AM.

Method

sample

result

The results show that the Reb AM test sample has significantly higher watermelon flavor and overall palatability compared to the control sample (95% confidence level). The test sample Reb AM had significantly lower sweet aftertaste intensity (90% confidence) compared to the control sample.

Conclusion Thirty panelists evaluated two samples of raspberry-watermelon flavored coconut water in two sessions for overall acceptability and attribute intensity (sweetness, raspberry flavor, watermelon flavor, coconut water flavor, astringency, artificial flavor). chemical aroma, bitterness, and sweet and bitter aftertaste). For Session 1, the two samples included: 1) a store-bought raspberry-watermelon-coconut water control sample, and 2) a store-bought raspberry-watermelon-coconut water test sample containing Reb AM. The purpose of the study was to determine whether the addition of Reb AM affects the flavor profile of non-alcoholic beverages. Results showed that the Reb AM test sample had significantly higher watermelon flavor and overall palatability compared to the control sample (95% confidence level). A graph of the results is shown in FIG.
The test sample Reb AM had significantly lower sweet aftertaste intensity (90% confidence) compared to the control sample.

Experiment 3 of Example 10:
Chocolate Protein Shake Containing Reb AM Application: Milk/Dairy Product Overview Thirty trained panelists evaluated the overall acceptability and attribute intensity (cocoa flavor, milk The product was evaluated for aroma, whey protein, vanilla, metallic, sweetness, bitterness and aftertaste). The two samples included: 1) a "control" sample with no added sugar containing 300 ppm PureCircle Reb A, and 2) added sugar containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. "Test" samples that have not been tested. The purpose of the study was to determine whether the addition of Reb AM affects the flavor profile of dairy products. The panel found that test samples containing 50 ppm Reb AM had significantly lower bitterness, metallic aroma, whey protein, and lower bitter aftertaste than the control (95% confidence), and also had higher cocoa flavor, Found to have dairy aroma, vanilla flavor, and overall palatability (95% confidence). Furthermore, no significant effect on sweetness intensity was observed.

Purpose The purpose of the project is to evaluate whether the addition of stevia extract solids affects key flavor attributes in various beverage applications.

Purpose of the Test The purpose of the test is to determine whether the flavor profile and overall acceptability of a control sample for a dairy beverage application differs from a test sample of the same beverage containing Reb AM.

Method

sample

The panel found that test samples containing 50 ppm Reb AM had significantly lower bitterness, metallic aroma, whey protein, and lower bitter aftertaste than the control (95% confidence level).

The panel found that test samples containing 50 ppm Reb AM had significantly higher cocoa flavor, dairy aroma, vanilla flavor, and overall palatability (95% confidence level).

Conclusion Thirty panelists evaluated the overall acceptability and intensity of attributes (cocoa flavor, dairy aroma, whey protein, vanilla, metallic, sweetness, bitterness and aftertaste) for two samples of chocolate flavored dairy protein shakes. was evaluated. The two samples included: 1) a "control" sample with no added sugar containing 300 ppm PureCircle Reb A, and 2) added sugar containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. "Test" samples that have not been tested. The purpose of the study was to determine whether the addition of Reb AM affects the flavor profile of dairy products. The panel found that test samples containing 50 ppm Reb AM had significantly lower bitterness, metallic aroma, whey protein, and lower bitter aftertaste than the control, and also had higher cocoa flavor, dairy aroma, vanilla flavor, and overall palatability (95% confidence). Furthermore, no significant effect on sweetness intensity was observed. A graph of the results is shown in FIG.

Having described the invention and its advantages in detail, it is understood that various modifications, substitutions, and changes may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. should be understood. Furthermore, the scope of this application is not intended to be limited to the particular embodiments of the invention described herein. As one of ordinary skill in the art will readily understand from the present disclosure, there are currently existing or future embodiments that perform substantially the same function or achieve substantially the same results as corresponding embodiments described herein. The compositions, processes, methods, and steps developed may be utilized in accordance with the present invention.

Claims (5)

A method for enhancing flavor in a consumable product, the method comprising adding highly purified rebaudioside AM to said product at a concentration below the sweetness perception threshold concentration of rebaudioside AM, wherein rebaudioside AM has the formula:
2. The method of claim 1, wherein the product is selected from the group consisting of foods, beverages, pharmaceutical compositions, tobacco products, nutritional compositions, oral hygiene compositions, and cosmetic compositions. The products may contain carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic acid salts and organic base salts. selected from the group consisting of organic salts, inorganic salts, bitter compounds, caffeine, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof. 3. The method of claim 2 , further comprising at least one additive. If the product contains saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, 3. The method of claim 2 , further comprising at least one functional component selected from the group consisting of chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof. The products include rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside O, rebaudioside O2, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, Rebaudioside R, Rebaudioside S, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Darcoside A, Darcoside C, Rubusoside, Steviolbioside , steviolbioside A, steviolbioside B, steviol monoside, steviol monoside A, stevioside, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E, stevioside E2, stevioside F, NSF-02, mogroside V, Luo Han Guo, allulose, D-allose, D-tagatose, erythritol, brazein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartin, pernanzursin, muclodiosides, bayunoside, furomisoside-I, dimethyl-hexahydro Fluorene dicarboxylic acid, abrusosides, periandrin, carnosyfurosides, cyclokaryoside, pterocariosides, polypodoside A, brazilin, hernanzultin, phyllodultin, glycifyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neo Astiribine, trans-cinnamaldehyde, monatin and its salts, Serigain A, hematoxylin, monellin, osladin, pterocarioside A, pterocarioside B, mavinlin, pentadine, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside, sucralose, potassium acesulfame, Aspartame, alitame, saccharin, cyclamate, neotame, dultin, suosan, advantame, gymnemic acid, fozurucin, didifine, lactisole, glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, maltitol, mannitol, Sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalt-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced Glucose syrup, hydrogenated starch hydrolysates, polyglycitols, sugar alcohols, L-saccharides, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various Types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose , gulose, talose, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannosamine, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, sugar beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.) ); kestose, nystose, etc.), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulo-oligosaccharides, lactulose , melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, soybean oligosaccharides, D-psicose, D-ribose, L-glucose, L-fucose, D-turanose, D-leuclose 3. The method of claim 2 , further comprising a compound selected from the group consisting of: