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WO2025048633A2 - Glycosides de stéviol de haute pureté - Google Patents

Glycosides de stéviol de haute pureté Download PDF

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Publication number
WO2025048633A2
WO2025048633A2 PCT/MY2024/050063 MY2024050063W WO2025048633A2 WO 2025048633 A2 WO2025048633 A2 WO 2025048633A2 MY 2024050063 W MY2024050063 W MY 2024050063W WO 2025048633 A2 WO2025048633 A2 WO 2025048633A2
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Prior art keywords
rebaudioside
weight
udp
stevioside
steviolbioside
Prior art date
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WO2025048633A3 (fr
Inventor
Avetik Markosyan
Xiao Juie WONG
Mohamad Afzaal Bin HASIM
Khairul NIZAM BIN NAWI
Saravanan A/l RAMANDACH
Kristina Chkhan
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PureCircle Sdn Bhd
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PureCircle Sdn Bhd
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/36—Terpene glycosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A23L2/60—Sweeteners

Definitions

  • the present invention relates to a process for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.
  • High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted to diabetics and others interested in controlling for their intake of carbohydrates.
  • Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%).
  • steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.
  • methods are known for preparing steviol glycosides from Stevia rebaudiana, many of these methods are unsuitable for use commercially. Accordingly, there remains a need for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.
  • rebaudioside O8 salts thereof, or combinations thereof:
  • a method for producing rebaudioside O8 that includes the steps of: a.
  • a starting composition comprising at least one steviol glycoside
  • weight percentages presented herein are by weight of the total composition.
  • the target steviol glycoside(s) can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
  • Purified target steviol glycosides can be used in consumable products as a sweetener, flavor stabilizer, flavoring with modifying properties (FMP), foaming suppressor and/or solubility enhancing agent.
  • Suitable consumer products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.
  • FIG.1 shows the chemical structure of Steviolbioside A.
  • FIG.2 shows the chemical structure of Steviolbioside B.
  • FIG.3 shows the chemical structure of Steviolbioside C.
  • FIG.4 shows the chemical structure of Steviolbioside D.
  • FIG.5 shows the chemical structure of Dulcoside C.
  • FIG.6 shows the chemical structure of Dulcoside E.
  • FIG.7 shows the chemical structure of Stevioside A.
  • FIG.8 shows the chemical structure of Stevioside B.
  • FIG.9 shows the chemical structure of Stevioside C.
  • FIG.10 shows the chemical structure of Stevioside O.
  • FIG.11 shows the chemical structure of Rebaudioside C5.
  • FIG.12 shows the chemical structure of Rebaudioside C6.
  • FIG.13 shows the chemical structure of Rebaudioside C9.
  • FIG.14 shows the chemical structure of Rebaudioside C10.
  • FIG.15 shows the chemical structure of Rebaudioside C11.
  • FIG.16 shows the chemical structure of Rebaudioside E3.
  • FIG.17 shows the chemical structure of Rebaudioside E4.
  • FIG.18 shows the chemical structure of Rebaudioside E6.
  • FIG.19 shows the chemical structure of Rebaudioside E15.
  • FIG.20 shows the chemical structure of Rebaudioside K.
  • FIG.21 shows the chemical structure of Rebaudioside K3.
  • FIG.22 shows the chemical structure of Rebaudioside K4.
  • FIG.23 shows the chemical structure of Rebaudioside K5.
  • FIG.24 shows the chemical structure of Rebaudioside D7.
  • FIG.25 shows the chemical structure of Rebaudioside D15.
  • FIG.26 shows the chemical structure of Rebaudioside D16.
  • FIG.27 shows the chemical structure of Rebaudioside H4.
  • FIG.28 shows the chemical structure of Rebaudioside H6.
  • FIG.29 shows the chemical structure of Rebaudioside N2.
  • FIG.30 shows the chemical structure of Rebaudioside N9.
  • FIG.31 shows the chemical structure of Rebaudioside N10.
  • FIG.32 shows the chemical structure of Rebaudioside N11.
  • FIG.33 shows the chemical structure of Rebaudioside N12.
  • FIG.34 shows the chemical structure of Rebaudioside N13.
  • FIG.35 shows the chemical structure of Rebaudioside M6.
  • FIG.36-41 show the pathways of producing rebaudioside O5 and various steviol glycosides from steviol.
  • FIG.42-47 show the pathways of producing rebaudioside O8 and various steviol glycosides from steviol.
  • FIG. 48 shows the biocatalytic production of rebaudioside N2 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.49 shows the biocatalytic production of rebaudioside N10 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.50 shows the biocatalytic production of rebaudioside N12 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG. 49 shows the biocatalytic production of rebaudioside N10 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG. 51 shows the biocatalytic production of rebaudioside O5 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG. 52 shows the biocatalytic production of rebaudioside O8 from rebaudioside K using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.53 shows the biocatalytic production of rebaudioside K from rebaudioside C using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG. 54 shows the biocatalytic production of rebaudioside O5 from rebaudioside C using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.53 shows the biocatalytic production of rebaudioside K from rebaudioside C using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG. 55 shows the biocatalytic production of rebaudioside O8 from rebaudioside C using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.56 shows the biocatalytic production of rebaudioside O5 from rebaudioside N2 using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.57 shows the biocatalytic production of rebaudioside O5 from rebaudioside N10 using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.58 shows the biocatalytic production of rebaudioside O8 from rebaudioside N2 using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.59 shows the biocatalytic production of rebaudioside O8 from rebaudioside N12 using the enzyme UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.
  • FIG.60 shows the HPLC chromatogram of initial material of the biocatalytic production of rebaubioside O5 and rebaudioside O8. The peak with retention time of 22.573 minutes corresponds to rebaudioside C.
  • FIG. 61 shows the HPLC chromatogram of the product of the biocatalytic production of rebaudioside O5 and rebaudioside O8 from initial material after 48 hours reaction.
  • FIG. 62 shows the HPLC chromatogram of rebaudioside O5 and rebaudioside O8 after purification by HPLC. The peak with retention time of 8.888 minutes corresponds to both rebaudioside O5 and rebaudioside O8 which co-eluted in HPLC.
  • FIG.63 shows the MSD chromatogram of the mixture containing both rebaudioside O5 and rebaudioside O8.
  • FIG.64 shows the mass spectrum of the mixture containing both rebaudioside O5 and rebaudioside O8.
  • biocatalysis or “biocatalytic” refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells comprising one or more enzyme(s), capable of single or multiple step chemical transformations on organic compounds.
  • Biocatalysis processes include fermentation, biosynthesis, bioconversion and biotransformation processes. The general methods of using isolated enzymes and whole- cell biocatalysts are known in the art.
  • Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.
  • steviol glycoside(s) refers to a glycoside of steviol, including, but not limited to, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16,
  • the present invention provides a process for preparing a composition comprising one or more target steviol glycoside(s) by contacting a starting composition comprising one or more starting steviol glycoside(s) with a microbial cell and/or enzyme preparation, thereby producing a composition comprising the target steviol glycoside(s).
  • One object of the invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K,
  • starting composition refers to any composition (generally an aqueous solution) containing one or more steviol glycoside(s).
  • the starting composition steviol glycoside is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside
  • the starting composition is steviol.
  • the starting composition steviol glycoside is steviolmonoside.
  • the starting composition steviol glycoside is steviolmonoside A.
  • the starting composition steviol glycoside is steviolbioside A.
  • the starting composition steviol glycoside is steviolbioside B.
  • the starting composition steviol glycoside is steviolbioside C.
  • the starting composition steviol glycoside is steviolbioside D.
  • the starting composition steviol glycoside is rubusoside.
  • the starting composition steviol glycoside is stevioside A. In another embodiment, the starting composition steviol glycoside is stevioside B. In another embodiment, the starting composition steviol glycoside is stevioside C. In another embodiment, the starting composition steviol glycoside is stevioside O. In another embodiment, the starting composition steviol glycoside is dulcoside A. In another embodiment, the starting composition steviol glycoside is dulcoside C. In another embodiment, the starting composition steviol glycoside is dulcoside E. In another embodiment, the starting composition steviol glycoside is rebaudioside G. In another embodiment, the starting composition steviol glycoside is rebaudioside C.
  • the starting composition steviol glycoside is rebaudioside C5. In another embodiment, the starting composition steviol glycoside is rebaudioside C6. In another embodiment, the starting composition steviol glycoside is rebaudioside C9. In another embodiment, the starting composition steviol glycoside is rebaudioside C10. In another embodiment, the starting composition steviol glycoside is rebaudioside C11. In another embodiment, the starting composition steviol glycoside is rebaudioside E3. In another embodiment, the starting composition steviol glycoside is rebaudioside E4. In another embodiment, the starting composition steviol glycoside is rebaudioside E6. In another embodiment, the starting composition steviol glycoside is rebaudioside E15.
  • the starting composition steviol glycoside is rebaudioside H. In another embodiment, the starting composition steviol glycoside is rebaudioside H4. In another embodiment, the starting composition steviol glycoside is rebaudioside H6. In another embodiment, the starting composition steviol glycoside is rebaudioside D7. In another embodiment, the starting composition steviol glycoside is rebaudioside D15. In another embodiment, the starting composition steviol glycoside is rebaudioside D16. In another embodiment, the starting composition steviol glycoside is rebaudioside K. In another embodiment, the starting composition steviol glycoside is rebaudioside K3. In another embodiment, the starting composition steviol glycoside is rebaudioside K4.
  • the starting composition steviol glycoside is rebaudioside K5. In another embodiment, the starting composition steviol glycoside is rebaudioside N2. In another embodiment, the starting composition steviol glycoside is rebaudioside N9. In another embodiment, the starting composition steviol glycoside is rebaudioside N10. In another embodiment, the starting composition steviol glycoside is rebaudioside N11. In another embodiment, the starting composition steviol glycoside is rebaudioside N12. In another embodiment, the starting composition steviol glycoside is rebaudioside N13. In another embodiment, the starting composition steviol glycoside is rebaudioside M6.
  • the starting composition may be synthetic or purified (partially or entirely), commercially available or prepared.
  • the starting composition is glycerol. In another embodiment, the starting composition is glucose. In another embodiment, the starting composition is rhamnose. In still another embodiment, the starting composition is sucrose. In yet another embodiment, the starting composition is starch. In another embodiment, the starting composition is maltodextrin. In still another embodiment, the starting composition is cellulose. In yet another embodiment, the starting composition is hemicellulose. In another embodiment, the starting composition is amylose. In still another embodiment, the starting composition is xylose. In yet another embodiment, the starting composition is xylooligosaccharide. In another embodiment, the starting composition is xyloglucan. In still another embodiment, the starting composition is arabinoxylan.
  • the starting composition is glucuronoxylan.
  • the starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein.
  • Target Steviol Glycoside The target steviol glycoside(s) can be any steviol glycoside different from the starting steviol glycoside(s).
  • the target steviol glycoside(s) of the present method can be any steviol glycoside that can be prepared by the process disclosed herein.
  • the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside
  • the target steviol glycoside is steviolmonoside. In another embodiment, the target steviol glycoside is steviolmonoside A. In another embodiment, the target steviol glycoside is steviolbioside A. In another embodiment, the target steviol glycoside is steviolbioside B. In another embodiment, the target steviol glycoside is steviolbioside C. In another embodiment, the target steviol glycoside is steviolbioside D. In another embodiment, the target steviol glycoside is rubusoside. In another embodiment, the target steviol glycoside is stevioside A. In another embodiment, the target steviol glycoside is stevioside B.
  • the target steviol glycoside is stevioside C. In another embodiment, the target steviol glycoside is stevioside O. In another embodiment, the target steviol glycoside is dulcoside A. In another embodiment, the target steviol glycoside is dulcoside C. In another embodiment, the target steviol glycoside is dulcoside E. In another embodiment, the target steviol glycoside is rebaudioside G. In another embodiment, the target steviol glycoside is rebaudioside C. In another embodiment, the target steviol glycoside is rebaudioside C5. In another embodiment, the target steviol glycoside is rebaudioside C6. In another embodiment, the target steviol glycoside is rebaudioside C9.
  • the target steviol glycoside is rebaudioside C10. In another embodiment, the target steviol glycoside is rebaudioside C11. In another embodiment, the target steviol glycoside is rebaudioside E3. In another embodiment, the target steviol glycoside is rebaudioside E4. In another embodiment, the target steviol glycoside is rebaudioside E6. In another embodiment, the target steviol glycoside is rebaudioside E15. In another embodiment, the target steviol glycoside is rebaudioside H. In another embodiment, the target steviol glycoside is rebaudioside H4. In another embodiment, the target steviol glycoside is rebaudioside H6.
  • the target steviol glycoside is rebaudioside D7. In another embodiment, the target steviol glycoside is rebaudioside D15. In another embodiment, the target steviol glycoside is rebaudioside D16. In another embodiment, the target steviol glycoside is rebaudioside K. In another embodiment, the target steviol glycoside is rebaudioside K3. In another embodiment, the target steviol glycoside is rebaudioside K4. In another embodiment, the target steviol glycoside is rebaudioside K5. In another embodiment, the target steviol glycoside is rebaudioside N2. In another embodiment, the target steviol glycoside is rebaudioside N9.
  • the target steviol glycoside is rebaudioside N10. In another embodiment, the target steviol glycoside is rebaudioside N11. In another embodiment, the target steviol glycoside is rebaudioside N12. In another embodiment, the target steviol glycoside is rebaudioside N13. In another embodiment, the target steviol glycoside is rebaudioside M6. In another embodiment, the target steviol glycoside is rebaudioside O5. In another embodiment, the target steviol glycoside is rebaudioside O8. In another embodiment, the target steviol glycoside(s) are a combination of multiple different steviol glycoside(s).
  • the target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
  • the present invention is a biocatalytic process for the production of steviolmonoside.
  • the present invention is a biocatalytic process for the production of steviolmonoside A.
  • the present invention is a biocatalytic process for the production of steviolbioside A.
  • the present invention is a biocatalytic process for the production of steviolbioside B.
  • the present invention is a biocatalytic process for the production of steviolbioside C.
  • the present invention is a biocatalytic process for the production of steviolbioside D. In one embodiment, the present invention is a biocatalytic process for the production of rubusoside. In one embodiment, the present invention is a biocatalytic process for the production of stevioside A. In one embodiment, the present invention is a biocatalytic process for the production of stevioside B. In one embodiment, the present invention is a biocatalytic process for the production of stevioside C. In one embodiment, the present invention is a biocatalytic process for the production of stevioside O. In one embodiment, the present invention is a biocatalytic process for the production of dulcoside A.
  • the present invention is a biocatalytic process for the production of dulcoside C. In one embodiment, the present invention is a biocatalytic process for the production of dulcoside E. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside G. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside C. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside C5. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside C6. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside C9.
  • the present invention is a biocatalytic process for the production of rebaudioside C10. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside C11. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E4. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E6. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E15. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside H.
  • the present invention is a biocatalytic process for the production of rebaudioside K4. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside K5. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside N2. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside N9. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside N10. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside N11. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside N12.
  • the present invention is a biocatalytic process for the production of rebaudioside N13. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside M6. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside O5. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside O8. In one embodiment, the present invention is a biocatalytic process for the production of a combination of multiple different steviol glycosides(s). In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside N2 from a starting composition comprising rebaudioside K and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside N10 from a starting composition comprising rebaudioside K and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside N12 from a starting composition comprising rebaudioside K and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside O5 from a starting composition comprising rebaudioside K and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside O5 from a starting composition comprising rebaudioside N2 and UDP-glucose. In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside O5 from a starting composition comprising rebaudioside N10 and UDP-glucose. In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside O8 from a starting composition comprising rebaudioside K and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside O8 from a starting composition comprising rebaudioside N2 and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside O8 from a starting composition comprising rebaudioside N12 and UDP-glucose.
  • the present invention provides for the biocatalytic process for the production of rebaudioside K from a starting composition comprising rebaudioside C and UDP-glucose.
  • the method of the present invention further comprises separating the target steviol glycoside(s) from the reaction medium to provide a highly purified target steviol glycoside composition.
  • the target steviol glycoside(s) can be separated by any suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
  • the process described herein results in a highly purified target steviol glycoside composition.
  • the term “highly purified”, as used herein, refers to a composition having greater than about 40% by weight of the target steviol glycoside on an anhydrous (dried) basis.
  • the process described herein provides a composition having greater than about 40% rebaudioside N2 content by weight on a dried basis.
  • the process described herein provides a composition comprising greater than about 45% by weight of the target steviol glycoside on an anhydrous (dried) basis, such as, for example, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% target steviol glycoside content on a dried basis.
  • UGTSl2 refers to UDP- glucosyltransferase having amino-acid sequence SEQ ID 4 or a polypetide having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to the SEQ ID 4 polypeptide as well as isolated nucleic acid molecules that code for those polypeptides.
  • Alternative amino- acid sequences can also be obtained through further translocation, inversion, substitution, insertion, deletion and/or duplication of the sequences having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to the SEQ ID 10 polypeptide.
  • enzyme preparation comprising one or more enzymes, or a microbial cell comprising one or more enzymes, capable of converting the starting composition to target steviol glycosides are used.
  • the enzyme can be located on the surface and/or inside the cell.
  • fermentation process can utilize a non-genetically modified or a genetically modified organism that is capable of producing one or more steviol glycosides, such as Reb M and Reb D.
  • a genetically engineered microbial strain contains a set of enzymes that can synthesize one or more of target steviol glycosides.
  • One or more steviol glycosides other than target steviol glycosides can also be produced by the genetically engineered microbial strains or enzyme composition prepared from the genetically engineered microbial strains.
  • enzyme(s) can be produced through fermentation process.
  • fermentation process can utilize a non- genetically modified or a genetically modified organism that is capable of producing one or more enzymes capable of converting the starting composition to the target steviol glycoside(s).
  • a genetically engineered microbial strain can be used for production of steviol glycosides by expressing various enzymes such as geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), kaurene oxidase (KO), kaurene synthase (KS), steviol synthase (KAH), cytochrome P450 reductase (CPR), UGT74G1, UGT85C2, UGT76G1, EUGT11 and UGT91D2.
  • GGPPS geranylgeranyl diphosphate synthase
  • CDPS ent-copalyl diphosphate synthase
  • KO kaurene oxidase
  • KS kaurene synthase
  • KAH cytochrome
  • WO2014/122227 describes a genetically engineered yeast strain that expresses these enzymes.
  • the genetically engineered microbial strain additionally expresses enzymes including UGT74G1, UGT85C2, UGTSl2, UGT76G1, EUGT11, UGT91D2, URhaT12V1, URhaT22V1, URhaT32V1, URhaT42V1, URhaT52V1 and sucrose synthase (SuSy).
  • UDP-glucosyltransferases are genes that encode polypeptides capable of carrying out reactions such as (i) glucosylation of the - OH functional group at the C13 of a steviol or steviol glycoside, (ii) glucosylation of the - COOH functional group at the C19 of a steviol or steviol glycoside, (iii) beta 1,2- glucosylation at the C2’ of the 19-O-glucose of a steviol glycoside, (iv) beta 1,2- glucosylation at the C2’ of the 13-O-glucose of a steviol glycoside, (v) beta 1,3- glucosylation at the C3’ of the 19-O-glucose of a steviol glycoside, (vi) beta 1,3- glucosylation at the C3’ of the 13-O-glucose of a steviol glycoside, (vii) beta 1,4- glucosylation at the C4’ of the
  • UGT85C2 carries out (i); UGT74G1 carries out (ii); UGTSl2 carries out (iii), (iv), (vii), (viii), (ix) and (x); UGT76G1 carries out (v), (vi), (vii) and (viii); EUGT11 carries out (iii), (iv), (vii), (viii), (ix) and (x); UGT91D2 carries out (iii), (iv), (vii), (viii), (ix) and (x).
  • a genetically engineered yeast can be used for production of steviol glycosides includes the following genes encoding a polypeptide capable of (i) synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP), e.g. geranylgeranyl diphosphate synthase (GGPPS); (ii) synthesizing ent-copalyl diphosphate from GGPP, e.g. ent-copalyl diphosphate synthase (CDPS); (iii) synthesizing ent-kaurene from ent-copalyl pyrophosphate, e.g.
  • steviol glycosides can be produced by the genetically engineered microbial strain, such as Reb M, Reb D, Reb A, etc.
  • Fermentation process generally can be carried out under conditions with presence of oxygen (aerobic conditions), a carbon source, a nutritious medium (nitrogen base) and at a neutral or lower pH. Fermentation process can be a continuous or fed batch process.
  • fermentation process is generally conducted by growing a genetically engineered microbial strain in a base medium followed by a longer feeding phase using feed medium mainly consists of glucose or sucrose with traces of metals, vitamins and salts.
  • the fermentation medium consists of >5 g/L glucose or sucrose, >5 g/L ammonium sulfate, >3 g/L potassium dihydrogenphosphate, >0.5 g/L magnesium sulphate, trace elements and vitamins. Verduyn, C. et al. (1992) Yeast 8, 501-517, the content of which is hereby incorporated by reference, describes a minimal composition of a fermentation medium.
  • the fermentation medium can be maintained at about pH 5 and temperature at 30 °C.
  • fermentation can be conducted in media containing steviol or steviol glycoside(s).
  • One or more genetically engineered microbial strain(s) to be used with the media expresses genes encoding a functional UGT74G1, a functional UGT85C2, a functional UGTSl2, a functional UGT76G1, a functional EUGT11, a functional UGT91D2, a functional URhaT12V1, a functional URhaT22V1, a functional URhaT32V1, a functional URhaT42V1 and a functional URhaT52V1.
  • Target steviol glycosides, Reb A, Reb M and Reb D may be synthesized in the fermentation media.
  • target steviol glycosides can be produced using an enzyme composition prepared from one or more genetically engineered microbial strain(s).
  • the genetically engineered microbial strain(s) expresses genes encoding geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), kaurene oxidase (KO), kaurene synthase (KS), steviol synthase (KAH), cytochrome P450 reductase (CPR), sucrose synthase (SuSy), UGT74G1, UGT85C2, UGTSl2, UGT76G1, EUGT11, UGT91D2, URhaT12V1, URhaT22V1, URhaT32V1, URhaT42V1 and URhaT52V1 enzymes is used for enzyme composition preparation.
  • GGPPS geranylgeranyl diphosphate synthase
  • CDPS ent-copalyl diphosphate synthase
  • KO kaurene oxidase
  • KS kaurene synthase
  • KAH
  • the organisms can be treated with reagents that disrupt cell membranes to release the enzymes into a composition.
  • the enzymes are secreted into the fermentation media which can be used to prepare the enzyme composition.
  • Various enzymes in the composition react with one or more starting compounds, e.g. steviol or steviol glycoside(s) through one or multiple enzymatic reaction(s) involving a series of intermediates, to provide a composition consisting of one or more of target steviol glycosides.
  • an enzyme composition can be prepared from one cellular extract or combination of multiple cellular extracts from one genetically engineered strain or multiple genetically engineered strains.
  • Each strain expresses a number of enzymes for the bioconversion of one or more of starting steviol glycosides to one or more of target steviol glycosides and/or other steviol glycosides.
  • a composition consisting of steviol glycosides including one or more of target steviol glycosides can be obtained from the culture media using various methods.
  • a permeabilizing agent can be used to enhance the release of the steviol glycosides from the cell into the fermentation media.
  • the genetically engineered microbial strains can be separated from the fermentation media by centrifugation or filtration.
  • membrane dialysis can be carried out to remove low molecular weight components such as glucose, basic nutrients and salts.
  • the obtained composition containing one or more of target steviol glycosides and/or other steviol glycosides can be used.
  • the enzyme composition can be recovered from the growth media using various methods.
  • a permeabilizing agent can be used to enhance the release of enzymes from the cells into the fermentation media.
  • the genetically engineered microbial strains can be separated from the fermentation media by centrifugation or filtration.
  • membrane dialysis can be carried out to remove low molecular weight components such as glucose, basic nutrients and salts.
  • the obtained solution containing enzyme composition can be used for bioconversion of one or more of starting steviol glycosides to one or more of target steviol glycosides and/or other steviol glycosides.
  • the cells can be obtained from the fermentation media by centrifugation or filtration.
  • a lysis buffer can be used to disrupt cell membrane for releasing enzymes from the cells.
  • the lysed cells can be separated from the solution containing enzyme composition by centrifugation or filtration.
  • the solution containing enzyme composition can be used for bioconversion of one or more of starting steviol glycosides to one or more of target steviol glycosides and/or other steviol glycosides.
  • bioconversion process generally can be carried out by the enzyme composition prepared to convert one or more of starting steviol glycosides to one or more steviol glycosides under conditions with presence of substrate, UDP-glucose or UDP-rhamnose, ADP-glucose or ADP-rhamnose, at pH around 6-7 and at a temperature of around 45-55 ⁇ C.
  • the bioconversion substrate can be glucose, sucrose, maltodextrin or liquefied starch.
  • Bioconversion process can be a batch, continuous or fed batch process.
  • the product of bioconversion is a reaction medium consists of one or more of target steviol glycosides and/or other steviol glycosides which can be used as is or further enriched or purified.
  • a composition with steviol glycosides including one or more of target steviol glycosides in enriched or purified form can be prepared by further purification.
  • one or more of target steviol glycosides are separated from other steviol glycosides or separated from one another.
  • Such enrichment or purification of steviol glycosides compounds can be carried out on a composition containing one or more of target steviol glycosides in liquid or dry form. Dry form of a composition containing one or more of target steviol glycosides can be obtained by spray drying, flash drying, oven drying or lyophilization.
  • a dried material containing one or more of target steviol glycosides is used as a starting material for purification.
  • the dried material containing one or more of target steviol glycosides can be dissolved in a solvent or combination of solvents.
  • An exemplary combination of solvents is a mixture of water and an alcohol (water:alcohol ratio from 1:99 to 99:1) for dissolving the dried material.
  • the dissolution of the dried material in the solvent can be facilitated by heating the mixture at a temperature above room temperature, such as 40-80 °C and mechanical disruption by sonication.
  • the undissolved material can be filtered using a micron or sub-micron filter to produce a solution containing one or more of target steviol glycosides which can be further purified by chromatographic system.
  • a dried material containing one or more of target steviol glycosides is used as a starting material for purification.
  • the dried material containing one or more of target steviol glycosides can be suspended in a solvent or combination of solvents.
  • An exemplary combination of solvents is a mixture of water and an alcohol (water:alcohol ratio from 1:99 to 99:1) for suspending the dried material.
  • the suspension of the dried material in the solvent can be facilitated by heating the mixture at a temperature above room temperature, such as 40-80 °C and mechanical disruption by sonication.
  • the undissolved material can be filtered or centrifuged to produce a cake containing one or more of target steviol glycosides which can be further dried to obtain a dry form of a purified composition containing one or more of target steviol glycosides by spray drying, flash drying, oven drying or lyophilization.
  • the cake containing one or more of target steviol glycosides can be dissolved and further purified by a chromatographic system.
  • the solution containing one or more of target steviol glycosides can be purified by chromatographic system, such as reverse phase liquid chromatography, or multi-column chromatographic system with macroporous adsorption resin, etc.
  • a resin is used to adsorb steviol glycoside compounds and remove hydrophilic compounds from the column(s) by using liquid such as water as the mobile phase.
  • Steviol glycosides including one or more of target steviol glycosides can be eluted from the column(s) using a suitable solvent or combination of solvents such as water combined with ethanol, methanol or acetonitrile.
  • the elution of steviol glycosides including one or more of target steviol glycosides from the column(s) can produce a composition which can be used for variety of uses.
  • the enzymes necessary for converting the starting composition to target steviol glycosides include the NDP-glucosyltransferases (NGTs), such as, ADP- glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs), GDP-glucosyltransferases (GGTs), TDP-glucosyltransferases (TGTs) and/or UDP-glucosyltransferases (UGTs).
  • NDP-glucosyltransferases such as, ADP- glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs), GDP-glucosyltransferases (GGTs), TDP-glucosyltransferases (TGTs) and/or UDP-glucosyltransferases (UGTs).
  • NDP-glucosyltransferases such as, ADP- glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs
  • the enzymes necessary for converting the starting composition to target steviol glycosides include the NDP-rhamnosyltransferases (NRhaTs), such as, ADP-rhamnosyltransferases (ARhaTs), CDP-rhamnosyltransferases (CRhaTs), GDP-rhamnosyltransferases (GRhaTs), TDP-rhamnosyltransferases (TRhaTs), and/or UDP-rhamnosyltransferases (URhaTs).
  • NDP-rhamnosyltransferases such as, ADP-rhamnosyltransferases (ARhaTs), CDP-rhamnosyltransferases (CRhaTs), GDP-rhamnosyltransferases (GRhaTs), TDP-rhamnosyltransferases (TRhaTs), and/or UDP-rhamnosyltransferases (URhaTs).
  • NDP-recycling enzyme such as, ADP-recycling enzyme, CDP-recycling enzyme, GDP-recycling enzyme, TDP- recycling enzyme, and/or UDP-recycling enzyme.
  • the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.
  • the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.
  • the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13–hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl- D-erythritol 2,4- cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4- di
  • the UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.
  • the UDP-rhamnosyltransferase can be any UDP-rhamnosyltransferase capable of adding at least one rhamnose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.
  • steviol biosynthesis enzymes, UDP-glucosyltransferases and UDP-rhamnosyltransferase are produced in a microbial cell.
  • the microbial cell may be, for example, E.
  • UDP-glucosyltransferases and UDP-rhamnosyltransferase are synthesized.
  • the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGTSl2, UGT76G1 and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to SEQ ID 2, SEQ ID 3, SEQ ID 4 and SEQ ID 5, respectively as well as isolated nucleic acid molecules that code for these UGTs.
  • Alternative amino-acid sequences can also be obtained through further translocation, inversion, substitution, insertion, deletion and/or duplication of the sequences having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides.
  • the UDP-rhamnosyltransferase is selected from group including URhaT12V1, URhaT22V1, URhaT32V1, URhaT42V1, URhaT52V1 and URhaTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9 and SEQ ID 10, respectively as well as isolated nucleic acid molecules that code for these URhaTs.
  • Alternative amino-acid sequences can also be obtained through further translocation, inversion, substitution, insertion, deletion and/or duplication of the sequences having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides.
  • steviol biosynthesis enzymes, UGTs, URhaTs, UDP-glucose recycling system, UDP-rhamnose recycling system and UDP-rhamnose synthesis system are present in one microorganism (microbial cell).
  • the microorganism may be for example, E.
  • the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing an -OH functional group at C13 to give a target steviol glycoside having an -O-glucose beta glucopyranoside glycosidic linkage at C13.
  • the UDP- glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with SEQ ID 3.
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a -COOH functional group at C19 to give a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C19.
  • the UDP-glucosyltransferase is UGT74G1, or a UGT having >85% amino- acid sequence identity with SEQ ID 2.
  • the UDP-rhamnosyltransferase is any UDP- rhamnosyltransferase capable of adding at least one rhamnose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional rhamnose bearing at least one alpha 1 ⁇ 2 rhamnopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s).
  • the UDP-rhamnosyltransferase is URhaT12V1, or a URhaT having >85% amino-acid sequence identity with SEQ ID 6.
  • the UDP- rhamnosyltransferase is URhaT22V1, or a URhaT having >85% amino-acid sequence identity with SEQ ID 7.
  • the UDP-rhamnosyltransferase is URhaT32V1, or a URhaT having >85% amino-acid sequence identity with SEQ ID 8.
  • the UDP-rhamnosyltransferase is URhaT42V1, or a URhaT having >85% amino-acid sequence identity with SEQ ID 9.
  • the UDP-rhamnosyltransferase is URhaT52V1, or a URhaT having >85% amino-acid sequence identity with SEQ ID 10.
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1 ⁇ 2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s).
  • the UDP- glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with SEQ ID 5.
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1 ⁇ 4 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s).
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside C to form rebaudioside H4.
  • the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with SEQ ID 5.
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside C to form rebaudioside K.
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside N2 to form rebaudioside O8.
  • the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with SEQ ID 5.
  • the method of the present invention further comprises recycling UDP to provide UDP-glucose.
  • the sweetener is present in the beverage in an amount from about 0.00001% by weight to about 8% by weight, such as for example, from about 0.00001% by weight to about 0.00003% by weight, from about 0.00003% by weight to about 0.00005% by weight, from about 0.00005% by weight to about 0.00007% by weight, from about 0.00007% by weight to about 0.00009% by weight, from about 0.0001% by weight to about 0.0005% by weight, from about 0.0005% by weight to about 0.001% by weight, from about 0.001% by weight to about 0.005% by weight, from about 0.005% by weight to about 0.01% by weight, from about 0.01% by weight to about 0.05% by weight, from about 0.05% by weight to about 0.1% by weight, from about 0.1% by weight to about 0.5% by weight, from about 0.1% by weight to about 0.5% by weight, from about 0.5% by weight to about 1% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 3% by weight,
  • steviolmonoside steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4, rebaudioside K5,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K3, rebaudio
  • steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly, steviolmonoside, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, reb
  • polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo- oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • carbohydrate generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH2O)n, wherein n is 3- 30, as well as their oligomers and polymers.
  • the carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions.
  • Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates.
  • carbohydrate derivatives “substituted carbohydrate”, and “modified carbohydrates” are synonymous.
  • Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof.
  • carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • the carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxi
  • the carbohydrates as used herein may be in either the D- or L-configuration.
  • Functional ingredients generally are classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols, polyols, prebiotics, probiotics, postbiotics, phytoestrogens, soy protein, sulfides/thiols, amino acids, proteins, vitamins, and minerals.
  • Functional ingredients also may be classified based on their health benefits, such as cardiovascular, cholesterol-reducing, and anti-inflammatory. Exemplary functional ingredients are provided in WO2013/096420, the contents of which is hereby incorporated by reference.
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, reb
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, reb
  • the highly purified target steviol glycoside can be added before or after heat treatment of food products.
  • the present invention is also directed to sweetness enhancement in beverages using steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K,
  • the present invention is also directed to flavor stabilization of food and beverages using steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside
  • the present invention is also directed to modification (including enhancing or suppressing) of flavor and/or taste profile of food and beverages using steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K
  • the present invention is also directed to foaming suppression of food and beverages using steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside
  • the present invention is also directed to solubility enhancement of insoluble material in food and beverages using steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3,
  • sweetness enhancer refers to a compound or a mixture of compounds capable of enhancing or intensifying the perception of sweet taste in food and beverage products.
  • sweetness enhancer is synonymous with the terms “sweet taste potentiator,” “sweetness potentiator,” “sweetness amplifier,” and “sweetness intensifier.”
  • flavor stabilizer refers to a compound or a mixture of compounds capable of stabilizing the flavor in food and beverage products. It is contemplated that a flavor stabilizer can be used alone, or in combination with other flavor stabilizers.
  • flavoring with modifying properties refers to a compound or a mixture of compounds that enhance, subdue or otherwise affect the taste and/or flavor profile without themselves being sweeteners or flavorings.
  • the Flavor and Extracts Manufacturing Association (FEMA) has developed a protocol published in the November 2013 Edition of Food Technology. It is contemplated that a flavoring with modifying properties (FMP) can be used alone, or in combination with other flavorings.
  • sweetness recognition threshold concentration as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE).
  • the sweetness enhancers may enhance or potentiate the sweet taste of sweeteners without providing any noticeable sweet taste by themselves when present at or below the sweetness recognition threshold concentration of a given sweetness enhancer; however, the sweetness enhancers may themselves provide sweet taste at concentrations above their sweetness recognition threshold concentration.
  • the sweetness recognition threshold concentration is specific for a particular enhancer and can vary based on the beverage matrix. The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.
  • sweetener is present in the beverage in an amount from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight,
  • the sweetener is present in the beverage in an amount from about 0.00001% by weight to about 10% by weight, such as for example, from about 0.00001% by weight to about 0.00003% by weight, from about 0.00003% by weight to about 0.00005% by weight, from about 0.00005% by weight to about 0.00007% by weight, from about 0.00007% by weight to about 0.00009% by weight, from about 0.0001% by weight to about 0.0005% by weight, from about 0.0005% by weight to about 0.001% by weight, from about 0.001% by weight to about 0.005% by weight, from about 0.005% by weight to about 0.01% by weight, from about 0.01% by weight to about 0.05% by weight, from about 0.05% by weight to about 0.1% by weight, from about 0.1% by weight to about 0.5% by weight, from about 0.1% by weight to about 0.5% by weight, from about 0.5% by weight to about 1% by weight, from about 1% by weight to about 2% by weight, from about 2% by weight to about 3% by weight, from
  • the sweetener is present in the beverage in an amount from about 0.5% by weight to about 10% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount from about 2% by weight to about 8% by weight. In one embodiment, the sweetener is a traditional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup and high fructose starch syrup. In another embodiment, the sweetener is erythritol. In still another embodiment, the sweetener is a rare sugar.
  • Suitable rare sugars include, but are not limited to, D-allose, D-psicose, D-ribose, D-tagatose, L-glucose, L- fucose, L-arabinose, D-turanose, D-leucrose, 5-ketofructose and combinations thereof. It is contemplated that a sweetener can be used alone, or in combination with other sweeteners.
  • the rare sugar is D-allose.
  • D- allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is D-psicose.
  • D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is D-ribose.
  • D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is D-tagatose.
  • D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is L-glucose.
  • L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is L-fucose.
  • L- fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is L-arabinose.
  • L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is D-turanose.
  • D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is D-leucrose.
  • D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • the rare sugar is 5-ketofructose.
  • 5-ketofructose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer.
  • sweetness can be increased by an amount more than the detectable sweetness of a solution containing the same concentration of the at least one sweetness enhancer in the absence of any sweetener.
  • the present invention also provides a method for enhancing the sweetness of a food or beverage comprising a sweetener comprising providing a food or beverage comprising a sweetener and adding a sweetness enhancer selected from steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D
  • the present invention also provides a method for stabilizing the flavor of a food or beverage comprising providing a food or beverage and adding a flavor stabilizer selected from steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudior
  • the present invention also provides a method for modification (including enhancing or suppressing) of flavor and/or taste profile of a food or beverage comprising providing a food or beverage and adding a flavoring with modifying properties (FMP) selected from steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6,
  • the present invention also provides a method for adding steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • the present invention also provides a method for adding steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6, rebaudioside D7, rebaudioside D15, rebaudioside D16, rebaudioside K, rebaudioside K3, rebaudioside K4,
  • This invention provides rebaudioside O8, including salts thereof, or combinations thereof.
  • This disclosure provides a method for producing rebaudioside O5, including salts thereof, or combinations thereof, comprising the steps of providing a starting composition comprising at least one steviol glycoside; providing an enzyme preparation or microorganism containing at least one enzyme selected from the group consisting of steviol biosynthesis enzymes, NDP-glucosyltransferases, NDP-rhamnosyltransferases and NDP recycling enzymes; contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising rebaudioside O5.
  • this disclosure also provides a method for producing rebaudioside O8 comprising the steps of providing a starting composition comprising at least one steviol glycoside; providing an enzyme preparation or microorganism containing at least one enzyme selected from the group consisting of steviol biosynthesis enzymes, NDP- glucosyltransferases, NDP-rhamnosyltransferases and NDP recycling enzymes; contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising rebaudioside O8.
  • the method above further comprises the step of separating rebaudioside O5 from the reaction medium to provide a highly purified composition of rebaudioside O5.
  • the method above also further comprises the step of separating rebaudioside O8 from the reaction medium to provide a highly purified composition of rebaudioside O8.
  • the starting composition is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, rubusoside, stevioside A, stevioside B, stevioside C, stevioside O, dulcoside A, dulcoside C, dulcoside E, rebaudioside G, rebaudioside C, rebaudioside C5, rebaudioside C6, rebaudioside C9, rebaudioside C10, rebaudioside C11, rebaudioside E3, rebaudioside E4, rebaudioside E6, rebaudioside E15, rebaudioside H, rebaudioside H4, rebaudioside H6,
  • the microorganism is selected from the group consisting of E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., and Yarrowia sp.
  • the enzyme is selected from the group consisting of a mevalonate (MVA) pathway enzyme, a 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzyme, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13–hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4- diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C- methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythri
  • the content of rebaudioside O5 in the reaction medium is from about 0.05% to about 0.1% by weight, about 0.1% to about 2% by weight, from about 2% to about 4% by weight, from about 4% to about 6% by weight, from about 6% to about 8% by weight, from about 8% to about 10% by weight, from about 10% to about 12% by weight, from about 12% to about 14% by weight, from about 14% to about 16% by weight, from about 16% to about 18% by weight or from about 18% to about 20% by weight on a dried basis.
  • the content of rebaudioside O8 in the reaction medium is from about 0.05% to about 0.1% by weight, about 0.1% to about 2% by weight, from about 2% to about 4% by weight, from about 4% to about 6% by weight, from about 6% to about 8% by weight, from about 8% to about 10% by weight, from about 10% to about 12% by weight, from about 12% to about 14% by weight, from about 14% to about 16% by weight, from about 16% to about 18% by weight or from about 18% to about 20% by weight on a dried basis.
  • the content of rebaudioside O5 in the highly purified composition of rebaudioside O5 that this invention provides is greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99% by weight on a dried basis.
  • the content of rebaudioside O8 in the highly purified composition of rebaudioside O8 that this invention provides is greater than 40%, greater than 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95% or greater than about 99% by weight on a dried basis.
  • This invention also provides a consumable product comprising rebaudioside O5, wherein the product is selected from the group consisting of food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.
  • This invention also provides a consumable product comprising rebaudioside O8, wherein the product is selected from the group consisting of food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.
  • the consumable product that this invention provides is selected from the group consisting of foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes, other oral cavity compositions, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc., natural juices, refreshing drinks, carbonated soft drinks, diet drinks, zero calorie drinks, reduced calorie drinks and foods, yogurt drinks, instant juices, instant coffee, powdered types of instant beverages, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchups, curry, soup, instant bouillon, powdered soy sauce, powdered vinegar, types of biscuits, rice biscuit, crackers, bread, chocolates, caramel, candy, chewing gum,
  • the consumable product that this invention provides further comprises at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavorants and flavoring ingredients, flavorings with modifying properties (FMP), astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.
  • at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavorants and flavoring ingredients, flavorings
  • the consumable product that this invention provides further comprises at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, postbiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.
  • at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, postbiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.
  • the consumable product that this invention provides further comprises a compound selected from the group consisting of dulcoside A, dulcoside B, dulcoside C, dulcoside D, dulcoside E, rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudio
  • This invention also provides a method for enhancing the sweetness of a beverage or food product, comprising a sweetener, comprising providing a beverage or food product comprising a sweetener; and adding a sweetness enhancer comprising rebaudioside O5, wherein the concentration of rebaudioside O5 in beverage or food product is at or below the sweetness recognition threshold or in an amount from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00005% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0%
  • This invention also provides a method for enhancing the sweetness of a beverage or food product, comprising a sweetener, comprising providing a beverage or food product comprising a sweetener; and adding a sweetness enhancer comprising rebaudioside O8, wherein the concentration of rebaudioside O8 in beverage or food product is at or below the sweetness recognition threshold or in an amount from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00005% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0%
  • This invention also provides a method for stabilizing the flavor of a beverage or food product, comprising providing a beverage or food product; and adding a flavor stabilizer comprising rebaudioside O5, wherein the concentration of rebaudioside O5 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight
  • This invention also provides a method for stabilizing the flavor of a beverage or food product, comprising providing a beverage or food product; and adding a flavor stabilizer comprising rebaudioside O8, wherein the concentration of rebaudioside O8 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight
  • This invention also provides a method for modification (including enhancing or suppressing) of flavor and/or taste profile of a beverage or food product, comprising providing a beverage or food product; and adding a flavoring with modifying properties (FMP) comprising rebaudioside O5, wherein the concentration of rebaudioside O5 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by
  • This invention also provides a method for modification (including enhancing or suppressing) of flavor and/or taste profile of a beverage or food product, comprising providing a beverage or food product; and adding a flavoring with modifying properties (FMP) comprising rebaudioside O8, wherein the concentration of rebaudioside O8 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by
  • This invention also provides a method for suppressing foaming of a beverage or food product, comprising providing a beverage or food product; and adding a foam suppressor comprising rebaudioside O8, wherein the concentration of rebaudioside O8 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight
  • This invention also provides a method for enhancing the solubility of insoluble material in a beverage or food product, comprising providing a beverage or food product containing insoluble material; and adding a solubility enhancing agent comprising rebaudioside O5, wherein the concentration of rebaudioside O5 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0%
  • This invention also provides a method for enhancing the solubility of insoluble material in a beverage or food product, comprising providing a beverage or food product containing insoluble material; and adding a solubility enhancing agent comprising rebaudioside O8, wherein the concentration of rebaudioside O8 in beverage or food product is from about 0.00001% to about 12% by weight, such as, for example, about 0.00001% by weight, about 0.00003% by weight, about 0.00005% by weight, about 0.00007% by weight, about 0.00009% by weight, about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0%
  • the resulting plasmid was used for transformation of E.coli BL21(DE3) cells.
  • Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l) at 37°C.
  • kanamycin 50 mg/l
  • Expression of the genes was induced at logarithmic phase by IPTG (0.2 mM) and carried out at 30°C and 200 rpm for 16-18 hours.
  • Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD 600 )) with cell lysis buffer (100 mM Tris- HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 ⁇ m filter and diluted 50:50 with distilled water, resulting in an enzymatic active preparation.
  • cell lysis buffer 100 mM Tris- HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL.
  • the resulting plasmid was used for transformation of E.coli BL21(DE3) cells.
  • Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l) at 37°C.
  • kanamycin 50 mg/l
  • Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.
  • Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD 600 )) with cell lysis buffer (100 mM Tris- HCl pH 7.0; 2 mM MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 ⁇ m filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.
  • cell lysis buffer 100 mM Tris- HCl pH 7.0; 2 mM MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL.
  • activity in Units is defined as follows: 1 mU of UGTSl2 turns over 1 nmol of rebaudioside A (Reb A) into rebaudioside D (Reb D) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb A at t0, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.
  • Reb A rebaudioside A
  • Reb D rebaudioside D
  • Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb A at t0, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.
  • EXAMPLE 4 Expression and formulation of UGT76G1 variant of SEQ ID 5
  • the gene coding for the UGT76G1 variant of SEQ ID 5 (EXAMPLE 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells. Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.
  • Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris- HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 ⁇ m filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.
  • cell lysis buffer 100 mM Tris- HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL.
  • activity in Units is defined as follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D) into rebaudioside M (Reb M) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb D at t0, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.
  • EXAMPLE 5 Synthesis of rebaudioside O5 and rebaudioside O8 in a one-pot reaction, adding SuSy_At, UGTSl2 and UGT76G1 at the same time.
  • Rebaudioside O5, rebaudioside O8 and various steviol glycoside molecules were synthesized from steviol glycoside composition (ML2) produced by PureCircle Malaysia (see Fig.60) in a one-pot reaction, utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4): SuSy_At (SEQ ID 1), UGTSl2 (SEQ ID 4) and UGT76G1 (SEQ ID 5).
  • the final reaction solution contained approximately 521.4 U/L UGTSl2, 2010.9 U/L SuSy_At, 14.85 U/L UGT76G1, 100 g/L of ML2, 1.27 mM of 87% uridine diphosphate (UDP), 1000 mM sucrose, 4.0 mM MgCl 2 and 50 mM potassium phosphate buffer (pH 6.5).
  • UDP uridine diphosphate
  • 1000 mM sucrose 1000 mM sucrose
  • 4.0 mM MgCl 2 and 50 mM potassium phosphate buffer (pH 6.5).
  • 700 mL of distilled water were mixed with 0.81 g MgCl2•6H2O, 342.3 g sucrose, 33.3 mL of 1.5 M potassium phosphate buffer (pH 6.5) and 100 g ML2.
  • the final volume of the reaction mixture was adjusted to 1000 mL.
  • HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a pump, a column thermostat, an auto sampler, a UV detector capable of background correction and a data acquisition system. Analytes were separated using Agilent Poroshell 120 SB- C18, 4.6 mm x 150 mm, 2.7 ⁇ m at 40°C.
  • the mobile phase consisted of two premixes: - premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile, and - premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32% acetonitrile.
  • Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5 minute, changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes.
  • the column temperature was maintained at 40 oC.
  • the injection volume was 5 ⁇ L.
  • Steviol glycoside species were detected by UV at 210 nm. Table 1 shows for each time point the conversion of ML2 into identified steviol glycoside species (area percentage).
  • chromatograms of the starting material ML2 and the reaction medium at 48 hours are shown in Fig.60 and Fig.61 respectively.
  • retention times can occasionally vary with changes in solvent and/or equipment.
  • the total content of rebaudioside O5 and rebaudioside O8 in the reaction medium is estimated to be 0.2% by weight on a dried basis based on the HPLC analysis result.
  • the filtrate was loaded into a column containing 1.5 L YWD03 (Cangzhou Yuanwei, China) resin pre-equilibrated with water.
  • the resin was washed with 7.5 L water and the water effluent from this step was discarded.
  • the steviol glycosides were eluted from the YWD03 resin column with 7.5 L 70 % v/v ethanol/water.
  • the effluent from this step was collected and subjected to evaporation on a rotary evaporator to remove ethanol, and the sample was further concentrated.
  • the concentrated sample was subjected to further fractionation and separation by preparative HPLC, using the conditions listed in Table 2 below.
  • EXAMPLE 8 Structure elucidation of rebaudioside O5 NMR experiments were performed on a Bruker 400 MHz spectrometer, with the sample dissolved in pyridine-d5. Along with signals from the sample, signals from pyridine- d5 at ⁇ C 123.5, 135.5, 149.9 ppm and ⁇ H 7.19, 7.55, 8.71 ppm were observed. The sample, which appeared as a single peak from HPLC analysis, was revealed to be a 4:5-mixture of two steviol glycosides based on the 1H-NMR spectra.
  • rebaudioside O5 The mixture was then identified as rebaudioside O5 and rebaudioside O8 through structure elucidation as described in Example 8 and 9 respectively.
  • the HSQC shows the presence of an exo-methylene group in the sugar region, with a long-range coupling to C-15, observable in the H,H-COSY.
  • Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure:
  • Correlation of HSQC and HMBC shows the presence seven anomeric signals, marked as 1a, 1b, 1c, 1d, 1e, 1f and 1g.
  • Sugar B was assigned as ⁇ -L-rhamnopyranoside based on coupling constants of H5b, H6b and NOESY correlations.
  • the coupling constant of the anomeric protons of about 8 Hz and/or the NOE- correlations of the anomeric protons allow the identification of these six sugars as ⁇ -D- glucopyranosides.
  • Combined data from HSQC and HMBC reveal the sugar-sugar linkages and sugar- aglycone linkages. The assignment of the sugar sequence was further supported by HSQC- TOCSY, HMBC.
  • Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure: Correlation of HSQC and HMBC shows the presence seven anomeric signals, marked as 1a, 1b, 1c, 1d, 1e, 1f and 1g.
  • Sugar B was assigned as ⁇ -L-rhamnopyranoside based on coupling constants of H5b, H6b and NOESY correlations.
  • the coupling constant of the anomeric protons of about 8 Hz and/or the NOE- correlations of the anomeric protons allow the identification of these six sugars as ⁇ -D- glucopyranosides.

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Abstract

L'invention concerne des procédés de préparation de glycosides de stéviol. Les procédés comprennent l'utilisation de préparations enzymatiques et de micro-organismes recombinés pour convertir diverses compositions de départ en glycosides de stéviol cibles. Les glycosides de stéviol sont utiles en tant qu'édulcorant non calorique, exhausteur de goût, exhausteur de sucrosité, stabilisateur d'arôme, aromatisant avec des propriétés de modification (FMP), suppresseur de moussage et agent améliorant la solubilité dans des produits consommables tels que tout aliment, des boissons, des compositions pharmaceutiques, des produits de tabac, des compositions nutraceutiques et des compositions d'hygiène buccale.
PCT/MY2024/050063 2023-08-29 2024-08-28 Glycosides de stéviol de haute pureté Pending WO2025048633A2 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060123505A1 (en) 2002-05-30 2006-06-08 National Institute Of Agrobiological Sciences Full-length plant cDNA and uses thereof
WO2013096420A1 (fr) 2011-12-19 2013-06-27 The Coca-Cola Company Procédés de purification de stéviol glycosides et utilisations de ceux-ci
WO2014122227A2 (fr) 2013-02-06 2014-08-14 Evolva Sa Procédés pour la production améliorée de rébaudioside d et de rébaudioside m
US20170332673A1 (en) 2014-11-05 2017-11-23 Manus Biosynthesis, Inc. Microbial production of steviol glycosides
WO2018190378A1 (fr) 2017-04-12 2018-10-18 サントリーホールディングス株式会社 Rhamnose synthase dérivée de stévia et gène
WO2020205685A1 (fr) 2019-03-29 2020-10-08 Conagen Inc. Production biosynthétique d'udp-rhamnose

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CN110022691A (zh) * 2016-10-20 2019-07-16 可口可乐公司 从甜叶菊中分离的二萜糖苷、组合物和方法
CN112367854A (zh) * 2018-06-08 2021-02-12 谱赛科美国股份有限公司 高纯度甜菊醇糖苷
WO2024228615A1 (fr) * 2023-05-03 2024-11-07 Purecircle Sdn. Bhd. Glycosides de stéviol de haute pureté

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060123505A1 (en) 2002-05-30 2006-06-08 National Institute Of Agrobiological Sciences Full-length plant cDNA and uses thereof
WO2013096420A1 (fr) 2011-12-19 2013-06-27 The Coca-Cola Company Procédés de purification de stéviol glycosides et utilisations de ceux-ci
WO2014122227A2 (fr) 2013-02-06 2014-08-14 Evolva Sa Procédés pour la production améliorée de rébaudioside d et de rébaudioside m
US20170332673A1 (en) 2014-11-05 2017-11-23 Manus Biosynthesis, Inc. Microbial production of steviol glycosides
WO2018190378A1 (fr) 2017-04-12 2018-10-18 サントリーホールディングス株式会社 Rhamnose synthase dérivée de stévia et gène
WO2020205685A1 (fr) 2019-03-29 2020-10-08 Conagen Inc. Production biosynthétique d'udp-rhamnose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
F. WILLIAM STUDIER, PROTEIN EXPRESSION AND PURIFICATION, vol. 41, 2005, pages 207 - 234
VERDUYN, C ET AL., YEAST, vol. 8, 1992, pages 501 - 517

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