WO2019090336A1 - Glycosides de stéviol de haute pureté - Google Patents

Glycosides de stéviol de haute pureté Download PDF

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Publication number
WO2019090336A1
WO2019090336A1 PCT/US2018/059461 US2018059461W WO2019090336A1 WO 2019090336 A1 WO2019090336 A1 WO 2019090336A1 US 2018059461 W US2018059461 W US 2018059461W WO 2019090336 A1 WO2019090336 A1 WO 2019090336A1
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Prior art keywords
amino
acid sequence
sequence identity
reb
composition
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PCT/US2018/059461
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English (en)
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Avetik Markosyan
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Purecircle Usa Inc.
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Publication of WO2019090336A1 publication Critical patent/WO2019090336A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/60—Sweeteners
    • 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

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 CI 9.
  • Stevia leaves composing approximately 10% - 20% of the total dry weight.
  • 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 ⁇ (0.3%).
  • Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.
  • the present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or enzyme preparation, thereby producing a composition comprising a target steviol glycoside.
  • the starting composition can be any organic compound comprising at least one carbon atom.
  • the starting composition is selected from the group consisting of steviol glycosides, polyols or sugar alcohols, various carbohydrates.
  • the target steviol glycoside can be any steviol glycoside.
  • the target steviol glycoside is steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside /, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O or a synthetic steviol glycoside.
  • the target steviol glycoside is rebaudioside A.
  • the target steviol glycoside is rebaudioside E.
  • the target steviol glycoside is rebaudioside D.
  • the target steviol glycoside is rebaudioside I.
  • the target steviol glycoside is rebaudioside M.
  • enzyme preparation comprising one or more enzymes, or a microbial cell comprising one or more enzymes, capable of converting the starting composition to target steviol glycosides are used.
  • the enzyme can be located on the surface and/or inside the cell.
  • the enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate or as purified enzyme(s).
  • the enzyme preparation can be in free form or immobilized to a solid support made from inorganic or organic materials.
  • a microbial cell comprises the necessary enzymes and genes encoding thereof for converting the starting composition to target steviol glycosides. Accordingly, the present invention also provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell comprising at least one enzyme capable of converting the starting composition to target steviol glycosides, thereby producing a medium comprising at least one target steviol glycoside.
  • the enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes, UDP-glycosyltransferases (UGTs) 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-l-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
  • the UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to the steviol and or steviol glycoside substrate to provide the target steviol glycoside.
  • steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell.
  • the microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc.
  • the UDP-glucosyltransferases are synthesized.
  • the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G1 , UGT91D2 and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.
  • steviol biosynthesis enzymes, UGTs and UDP-glucose recycling system are present in one microorganism (microbial cell).
  • the microorganism may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
  • the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside.
  • the UDP-glucosyltransferase is UGT91D2 or a UGT having >85% amino- acid sequence identity with UGT91D2.
  • the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A.
  • the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1 (SEQ ID 3).
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D.
  • the UDP-glucosyltransferase is UGT91D2 or a UGT having >85% amino-acid sequence identity with UGT91D2.
  • the UDP-glucosyltransferase is UGTSL2 or a UGT having >85% amino-acid sequence identity with UGTSL2 (SEQ ID 2).
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside
  • the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1 (SEQ ID 3).
  • the method of the present invention further comprises recycling UDP to provide UDP-glucose.
  • the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.
  • the recycling catalyst is sucrose synthase.
  • sucrose synthase is SuSy_At or a sucrose synthase having >85% amino- acid sequence identity with SuSy At (SEQ ID 1).
  • the recycling substrate is sucrose.
  • the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.
  • the target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
  • the target steviol glycoside can be produced within the microorganism. In another embodiment, the target steviol glycoside can be secreted out in the medium. In one another embodiment, the released steviol glycoside can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the completion of the conversion reaction.
  • separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition.
  • separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside.
  • the composition comprises greater than about 95% by weight of the target steviol glycoside.
  • the composition comprises greater than about 99% by weight of the target steviol glycoside.
  • the target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
  • Purified target steviol glycosides can be used in consumable products as a sweetener.
  • 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 is a diagram showing one embodiment of the manufacturing process for steviol glycosides with a high reb M content produced by enzymatic conversion of reb A.
  • the present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or enzyme preparation, thereby producing a composition comprising a target steviol glycoside.
  • One object of the invention is to provide an efficient biocatalytic method for preparing steviol glycosides, particularly stevioside, reb E, reb A, reb D, and reb from various starting compositions.
  • One particular object of the invention is to provide a manufacturing process for producing a blend of steviol glycosides having greater than about 30% reb M, hereinafter referred to as "steviol glycosides with a high reb M content”.
  • biocatalysis or “biocatalytic” refers to the use of natural or genetically engineered biocatalysis, such as enzymes, or cells comprising one or more enzyme, capable of single or multiple step chemical transformations on organic compounds.
  • Biocatalysis processes include fermentation, biosynthesis, bioconversion and biotransformation processes. Both isolated enzyme, and whole-cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.
  • steviol glycoside(s) refers to a glycoside of steviol, ⁇ including, but not limited to, naturally occurring steviol glycosides, e.g. steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside 7, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.
  • Starting Composition e.g. enzymatically glucosylated steviol glyco
  • starting composition refers to any composition (generally an aqueous solution) containing one or more organic compound comprising at least one carbon atom.
  • the starting composition is selected from the group consisting of steviol glycosides, polyols and various carbohydrates.
  • the starting composition steviol glycoside is selected from the group consisting of steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside /, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other glycoside of steviol occurring in Stevia rebaudiana plant and/or combinations thereof.
  • the starting composition steviol glycoside is stevioside. In another embodiment, the starting composition steviol glycoside is rebaudioside
  • rebaudioside A is extracted from the leaves of Stevia rebaudiana plants, such as Stevia rebaudiana Bertoni plants, and purified to greater than 95% rebaudioside A.
  • the starting composition steviol glycoside is rebaudioside E.
  • the starting composition steviol glycoside is rebaudioside
  • the starting composition steviol glycoside is rebaudioside D.
  • polyol refers to a molecule that contains more than one hydroxyl group.
  • a polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively.
  • a polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively.
  • a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.
  • 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.
  • carbohydrate refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH 2 0) 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
  • 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 starting composition may be synthetic or purified (partially or entirely), commercially available or prepared.
  • the starting composition is glycerol.
  • the starting composition is glucose
  • the starting composition is sucrose.
  • the starting composition is starch. In another embodiment, the starting composition is maltodextrin.
  • the organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein.
  • the target steviol glycoside 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, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside /, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other glycoside of steviol.
  • the target steviol glycoside is stevioside.
  • the target steviol glycoside is rebaudioside A (reb A).
  • the target steviol glycoside is rebaudioside E (reb E).
  • the target steviol glycoside is rebaudioside I (reb I).
  • the target steviol glycoside is rebaudioside D (reb D).
  • the target steviol glycoside is rebaudioside M (reb M).
  • 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 reb D.
  • the present invention is a biocatalytic process for the production of reb E.
  • the present invention is a biocatalytic process for the production of reb I.
  • the present invention is a biocatalytic process for the production of reb M.
  • the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.
  • the target steviol glycoside can be separated by any suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
  • the process described herein results in a highly purified target steviol glycoside composition.
  • the term "highly purified”, as used herein, refers to a composition having greater than about 80% by weight of the target steviol glycoside on an anhydrous (dried) basis.
  • the highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous (dried) basis, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% target steviol glycoside content on a dried basis.
  • the process described herein provides a composition having greater than about 90% reb M content by weight on a dried basis.
  • the process described herein when the target steviol glycoside is reb , the process described herein provides a composition comprising greater than about 95% reb M content by weight on a dried basis. In another embodiment, when the target steviol glycoside is reb 7, the process described herein provides a composition having greater than about 90% reb I content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is reb I, the process described herein provides a composition comprising greater than about 95% reb / content by weight on a dried basis. In yet another embodiment, when the target steviol glycoside is reb D, the process described herein provides a composition greater than about 90% reb D content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is reb
  • the process described herein provides a composition comprising greater than about 95% reb D content by weight on a dried basis.
  • the process described herein provides a composition greater than about 90% reb E content by weight on a dried basis.
  • the target steviol glycoside is reb
  • the process described herein provides a composition comprising greater than about 95% reb E content by weight on a dried basis.
  • the process described herein provides a composition comprising greater than about 90% reb A content by weight on a dried basis.
  • the process described herein provides a composition comprising greater than about 95% reb A content by weight on a dried basis.
  • the process described herein provides a composition comprising greater than about 90% stevioside content by weight on a dried basis.
  • the process described herein provides a composition comprising greater than about 95% stevioside content by weight on a dried basis.
  • a microorganism (microbial cell) and/or enzyme preparation is contacted with a medium containing the starting composition to produce target steviol glycosides.
  • the enzyme can be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme or a combination thereof.
  • the biocatalyst is a purified enzyme capable of converting the starting composition to the target steviol glycoside.
  • the biocatalyst is a crude lysate comprising at least one enzyme capable of converting the starting composition to the target steviol glycoside.
  • the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting the starting composition to the target steviol glycoside.
  • the biocatalyst is one or more microbial cells comprising enzyme(s) capable of converting the starting composition to the target steviol glycoside.
  • the enzyme can be located on the surface of the cell, inside the cell or located both on the surface of the cell and inside the cell.
  • Suitable enzymes for converting the starting composition to target steviol glycosides include, but are not limited to, the steviol biosynthesis enzymes and UDP- glycosyltransferases (UGTs). Optionally it may include UDP recycling enzyme(s).
  • 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-l -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)- buten
  • steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell.
  • the microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc.
  • the enzymes are produced by microbial fermentation of the E. coli production strain LEI B 109 carrying the expression vector for the corresponding enzyme gene.
  • the UDP-glucosyltransferases are synthesized. In one embodiment, the UDP-glucosyltransferase is selected from group including
  • UGT74G1 , UGT85C2, UGT76G1, UGT91D2 and UGTs having substantial (>85%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.
  • steviol biosynthesis enzymes, UGTs and UDP-glucose recycling system are present in one microorganism (microbial cell).
  • the microorganism may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
  • the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside.
  • the UDP-glucosyltransferase is UGT91D2 or a UGT having >85% amino- acid sequence identity with UGT91D2.
  • the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A.
  • the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1 (SEQ ID 3).
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D.
  • the UDP-glucosyltransferase is UGT91D2 or a UGT having >85% amino-acid sequence identity with UGT91D2.
  • the UDP-glucosyltransferase is UGTSL or a UGT having >85% amino-acid sequence identity with UGTSL.
  • the UDP-glucosyltransferase is EUGT1 1 or a UGT having >85% amino-acid sequence identity with EUGT1 1.
  • the UDP-glucosyltransferase is UGTSL2 or a UGT having >85% amino-acid sequence identity with UGTSL2 (SEQ ID 2).
  • the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside
  • the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1 (SEQ ID 3).
  • the method of the present invention further comprises recycling UDP to provide UDP-glucose.
  • the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.
  • the UDP recycling enzyme can be sucrose synthase and the recycling substrate can be sucrose.
  • the sucrose synthase is SuSy_At or a sucrose synthase having >85% amino-acid sequence identity with SuSy_At (SEQ ID 1).
  • the UDP-glucosyltransferase capable of adding at least one glucose unit to starting composition steviol glycoside has >85% amino-acid sequence identity with UGTs selected from the following listing of Genlnfo identifier numbers, preferably from the group presented in Table 1, and more preferably the group presented in Table 2.
  • One embodiment is a microbial cell comprising an enzyme of the present invention, i.e. an enzyme capable of converting the starting composition to the target steviol glycoside.
  • some embodiments of the present method include contacting a microorganism with a medium containing the starting composition to provide a medium comprising at least one target steviol glycoside.
  • the microorganism can be any microorganism possessing the necessary enzyme(s) for converting the starting composition to target steviol glycoside(s). These enzymes are encoded within the microorganism's genome.
  • Suitable microorganisms include, but are not limited to, E.coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc.
  • the microorganism is free when contacted with the starting composition.
  • the microorganism is immobilized when contacted with the starting composition.
  • the microorganism may be immobilized to a solid support made from inorganic or organic materials.
  • solid supports suitable to immobilize the microorganism include derivatized cellulose or glass, ceramics, metal oxides or membranes.
  • the microorganism may be immobilized to the solid support, for example, by covalent attachment, adsorption, cross-linking, entrapment or encapsulation.
  • the enzyme capable of converting the starting composition to the target steviol glycoside is secreted out of the microorganism and into the reaction medium.
  • the target steviol glycoside is optionally purified.
  • Purification of the target steviol glycoside from the reaction medium can be achieved by at least one suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.
  • Highly purified target glycoside(s) particularly, reb M, reb D, reb / and/or reb E obtained according to this invention can be used "as-is” or in combination with other sweeteners, flavors, food ingredients and combinations thereof.
  • Non-limiting examples of flavors include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla and combinations thereof.
  • Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents and combinations thereof.
  • Highly purified target glycoside(s) particularly, reb M, reb D, reb / and/or reb E obtained according to this invention can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous, amorphous forms and combinations thereof.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E as a sweetening compound may be employed as the sole sweetener, or it may be used together with at least one naturally occurring high intensity sweeteners such as stevioside, reb A, reb B, reb C, reb F, reb N, reb O, steviolbioside, dulcoside A, rubusoside, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pte
  • reb M, reb D, reb / and/or reb E can be used in a sweetener composition comprising a compound selected from the group consisting of reb A, reb B, reb O, NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E may also be used in combination with synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.
  • synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.
  • highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others, reb M, reb D, reb / and/or reb E may also be combined with various umami taste enhancers, reb , reb D, reb / and/or reb E can be mixed with umatni tasting and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and combinations thereof.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E can be used in combination with one or more additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly- amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.
  • one or more 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,
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E may be combined with polyols or sugar alcohols.
  • polyol refers to a molecule that contains more than one hydroxyl group.
  • a polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively.
  • a polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively.
  • a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.
  • polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto- oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E may be combined with reduced calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L-arabinose and combinations thereof.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E may also be combined with various carbohydrates.
  • the term "carbohydrate” generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH 2 0) 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 As used herein, the phrases “carbohydrate derivatives”, “substituted carbohydrate”, and “modified carbohydrates” are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • the carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E obtained according to this invention can be used in combination with various physiologically active substances or functional ingredients.
  • 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; 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.
  • Highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E obtained according to this invention may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. It may also be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used.
  • highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.
  • Examples of consumable products in which highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods
  • the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.
  • the highly purified target steviol glycoside(s), reb M, reb D, reb / and/or reb E obtained in this invention may be used in dry or liquid forms.
  • the highly purified target steviol glycoside can be added before or after heat treatment of food products.
  • the amount of the highly purified target steviol glycoside(s), particularly, reb M, reb D, reb / and/or reb E depends on the purpose of usage. As discussed above, it can be added alone or in combination with other compounds.
  • the present invention is also directed to sweetness enhancement in beverages using reb , reb D, reb / and/or reb E. Accordingly, the present invention provides a beverage comprising a sweetener and reb M, reb D, reb / and/or reb £ as a sweetness enhancer, wherein reb , reb D, reb / and/or reb E is present in a concentration at or below their respective sweetness recognition thresholds.
  • sweetness enhancer refers to a compound capable of enhancing or intensifying the perception of sweet taste in a composition, such as a beverage.
  • sweetness enhancer is synonymous with the terms “sweet taste potentiator,” “sweetness potentiator,” “sweetness amplifier,” and “sweetness intensifier.”
  • sweetness recognition threshold concentration 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 0.5% to about 12% by weight, such as, for example, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 1 1.0% by weight, about 1 1.5% by weight or about 12.0% by weight.
  • the sweetener is present in the beverage in an amount from about 0.5% of about 10%, such as for example, from about 2% to about 8%, from about 3% to about 7% or from about 4% to about 6% by weight. In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.5% to about 8% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount from about 2% to about 8% by weight. In one embodiment, the sweetener is a traditional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup and high fructose starch syrup.
  • the sweetener is erythritol.
  • the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L- fucose, L-arbinose, D-turanose, D-leucrose and combinations thereof.
  • a sweetener can be used alone, or in combination with other sweeteners.
  • the rare sugar is D-allose.
  • 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. In a more particular embodiment,
  • L-fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
  • 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 addition of the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer.
  • 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 beverage comprising a sweetener comprising providing a beverage comprising a sweetener and adding a sweetness enhancer selected from reb M, reb D, reb / and/or reb E or a combination thereof, wherein reb M, reb D, reb / and/or reb E are present in a concentration at or below their sweetness recognition thresholds.
  • Addition of reb M, reb D, reb / and/or reb E in a concentration at or below the sweetness recognition threshold to a beverage containing a sweetener may increase the detected sucrose equivalence from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.
  • the gene coding for the SuSy_At variant of SEQ ID 1 was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.
  • 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 60 o)) 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 ⁇ 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 MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL.
  • activity in Units is defined as follows: 1 mU of SuSy turns over 1 nmol of sucrose into fructose in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose at to, 3 mM MgCl 2 , and 15 mM uridin diphosphate (UDP).
  • Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose at to, 3 mM MgCl 2 , and 15 mM uridin diphosphate (UDP).
  • EXAMPLE 1 The gene coding for the UGTS1 variant of SEQ ID 2 (EXAMPLE 1) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.
  • 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 6 oo)) 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 ⁇ 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 UGTS1 turns over 1 nmol of rebaudioside A (RebA) into rebaudioside D (RebD) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridin diphosphate (UDP) and 3 U/mL of SuSy_At.
  • RebA rebaudioside A
  • RebD rebaudioside D
  • Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridin diphosphate (UDP) and 3 U/mL of SuSy_At.
  • EXAMPLE 1 The gene coding for the UGTSr variant of SEQ ID 3 (EXAMPLE 1) was cloned into the expression vector pLEl A17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21 (DE3) cells.
  • 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 ⁇ 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 UGTSr turns over 1 nmol of rebaudioside A (RebA) into rebaudioside I (Rebl) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridin diphosphate (UDP) and 3 U/mL of SuSy_At.
  • RebA rebaudioside A
  • Rebl rebaudioside I
  • Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridin diphosphate (UDP) and 3 U/mL of SuSy_At.
  • Rebaudioside M was synthesized directly from rebaudioside A (RebA) in a one-pot reaction, utilizing the three enzymes (see EXAMPLES 1 , 2, 3 and 4): UGTS1 (variant of SEQ ID 2), SuSy_At-(variant of SEQ ID 1) and UGTSr (variant of SEQ ID 3).
  • the final reaction solution contained 20 mU/mL UGTS1, 160 mU/mL SuSy_At, 10 mU/mL UGTSr, 25 mM rebaudioside A, 0.5 mM uridin diphosphate (UDP), 1 M sucrose, 4 mM MgCl 2 and 50 mM potassium phosphate buffer (buffer stock prepared at pH 7.5), prepared in distilled water to a total volume of 1.6 mL.
  • biotransformation samples were inactivated by mixing 100 ⁇ , of reaction solution with 10 ⁇ L ⁇ 1M H 2 S0 4 , and adding 90 ⁇ L ⁇ of 60% MeOH (in H 2 0). Resulting samples were diluted a further 10-fold in 30% MeOH (in H 2 0), centrifuged at 18 x g for 10 min at 4°C, and supernatants were used as samples for HPLC injection.
  • HPLC was carried out on a Shimadzu 20A series unit equipped with two pump units, an auto sampler, and a thermostat column compartment. Mobile phases A (10 mM NaH 2 P0 4 , pH 2.6) and B (Acetonitrile, HPLC grade) were mixed on-line in different ratios at different times.
  • Table 3 shows for each time point the conversion of rebA into identified rebaudioside species (percentages calculated from molarities).
  • Rebaudioside M was synthesized directly from rebaudioside A (RebA) in a one-pot reaction, utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4); UGTSl
  • the final reaction solution contained 20 mU/mL UGTSl, 160 mU/mL SuSy At, 10 mU/mL UGTSr, 25 mM rebaudioside A, 0.5 mM uridin diphosphate (UDP), 1 M sucrose, 4 mM MgCl 2 and 50 mM potassium phosphate buffer (buffer stock prepared at pH 7.5), prepared in distilled water to a total volume of 1.6 mL.
  • biotransformation samples were inactivated by mixing 100 of reaction solution with 10 ⁇ , 1M H 2 S0 4 , and adding 90 ⁇ . of 60% MeOH (in H 2 0). Resulting samples were diluted a further 10-fold in 30% MeOH (in H 2 0), centrifuged at 18 x g for 10 min at 4°C, and supernatants were used as samples for HPLC injection.
  • HPLC was carried out on a Shimadzu 20A series unit equipped with two pump units, an auto sampler, and a thermostat column compartment. Mobile phases A (10 mM NaH 2 P0 4 , pH 2.6) and B (Acetonitrile, HPLC grade) were mixed on-line in different ratios at different times.
  • Table 4 shows for each time point the conversion of RebA into identified rebaudioside species (percentages calculated from molarities).
  • the production strain LE1B 109 is a genetically modified derivative strain of the laboratory strain E. coli K-12 W31 10.
  • the parental strain E. coli K-12 W31 10 has been modified by site-directed recombination at different chromosomal loci to suit production purposes in terms of genetic stability, especially plasmid stability, and efficiency of expression and biotransformation.
  • the expression of a number of proteases has been eliminated by deletion of the corresponding genes.
  • Antibiotic-free selection of target clones has been enabled through deletion of one gene.
  • One further gene has been deleted to prevent unwanted recombination effects.
  • coli K-12 W31 10 have been inserted into the genome of W31 10 to achieve a strong and regulated enzyme expression. Furthermore, the strain might carry certain deletions of endogenous enzyme genes connected to the degradation of biotransformation reactants in order to avoid side reactions. Insertions and deletions of chromosomal DNA are in general performed by integration of plasmid-based fragments carrying antibiotic resistance genes. After selection of the correct chromosomal mutants, resistance genes are excised and all plasmids are removed. No residual vector sequences or antibiotic resistance genes are left in the final cell. The final production strain used for manufacturing each enzyme is created from the LE1B 109 recipient strain by introducing an expression vector carrying the specific gene for one of the enzymes listed in Table 5. The plasmids used to transform the E.
  • coli recipient strain are based on the well-known vector pRSF-lb (Merck KGaA, Darmstadt, Germany). The plasmids have been fully sequenced and do not carry antibiotic resistance genes or any other sequences of concern. The production strain LE1B109 has been sequenced to confirm absence of antibiotic resistance genes or any other sequences of concern. Table 5
  • Sucrose synthase Catalyzes the formation of UDP-glucose Arabidopsis thaliana
  • UDP-glucosyltransferase UGT-Sr Catalyzes the addition of glucose to steviol Stevia rebaudiana
  • UDP-glucosyltransferase UGT-SI Catalyzes the addition of glucose to steviol Solarium lycopersicum
  • Fig. 1 One embodiment of the manufacturing process for steviol glycosides with a high reb M content produced by enzymatic conversion of reb A is shown in Fig. 1.
  • the steviol glycoside purification processes utilized prior to and following the enzymatic conversion are consistent with the methodologies for the manufacture of steviol glycosides as described in the Chemical and Technical Assessment published by FAO/JECFA (FAO, 2016).
  • stage 1 S. rebaudiana leaves are placed in hot water at 50 to 60°C for 1 to 2 hours in continuous countercurrent extractors.
  • the filtrate is separated using mesh screens, collected in a holding tank, and treated with flocculant (calcium hydroxide) to remove the mechanical particles, proteins, polysaccharides, and coloring agents.
  • a plate-and-frame filter press is used to separate the resulting precipitate from the filtrate, and the filtrate is deionized by ion-exchange resins in (H+) and (OH-) form.
  • the deionized filtrate is fed to a column system packed with macroporous adsorption resin that retains the glycosides.
  • the column is washed with deionized water to remove impurities that did not adsorb to the resin and then the glycosides are desorbed using aqueous ethanol.
  • the obtained glycoside solution is treated with activated carbon and the carbon is separated from the solution by plate-and-frame filter press.
  • a standard evaporator is used to remove the ethanol, and the resulting aqueous solution is deionized again by ion-exchange resins in (H+) and (OH-) forms.
  • the refined solution is concentrated using a nanofiltration membrane and the concentrated solution is spray dried to yield stevia extract powder containing >50% reb A (RA50).
  • the RA50 powder is further purified by dissolving in aqueous ethanol and incubating at low temperature for several hours to allow for reb A to crystallize.
  • the reb A crystals containing >95% reb A are separated by conventional centrifugation and dried in a rotary drum vacuum dryer at 1 10°C and 10 mbar.
  • the obtained powder is sifted through US 80 mesh stainless steel screens and passed through metal detectors to be packed in aluminum foil bags.
  • E. coli production strain LEI B 109 carrying the expression vector for the corresponding enzyme is inoculated in sterilized culture medium composed of the ingredients listed in Table 6, and fermented.
  • Fermentation Nutrient Permitted for use in food as food additive, food substance, ingredient, flavor enhancer, flavoring agent, processing aid or nutrient supplement, with no limitations apart from cGMP, each being selected from 21 CFR Parts ⁇ 184, ⁇ 172, ⁇ 573, ⁇ 182, ⁇ 582.
  • Nuclease i.e., NuCLEANase, food- Processing aid
  • the fermentation conditions are a pH of between 6 to 8 and a temperature of between 25 to 37°C.
  • the fermentation process is continued until laboratory test data shows the desired enzyme production yield. Usually, after at least 15 hours, the fermentation is stopped.
  • the enzyme is isolated from the biomass.
  • the biomass is separated from the culture broth by standard techniques (e.g., is centrifuged and/or filtered).
  • the biomass is homogenized to disrupt the bacterial cells and treated with a nuclease (e.g., NuCLEANase, c-LEcta, für, Germany) to degrade the DNA/RNA nucleic acids released upon cell disruption. This is followed by solid/liquid separation steps to further remove cell debris and other insoluble matter.
  • the cell-free supernatant is filtered to obtain the purified enzyme preparation. All raw materials used for fermentation and recovery are of food-grade quality or have been assessed to be fit for their intended use.
  • CFU colony- forming unit
  • NS not specified
  • TOS total organic solids
  • U units [1 unit correspond conversion of 1 ⁇ reb A/minute at 30°C and pH 7.0]
  • CFU colony-forming unit
  • NS not specified
  • TOS total organic solids
  • U units [1 unit corresponds to the conversion of 1 pmol reb A/minute at 30°C and pH 7.0]
  • CFU colony- forming unit
  • NS not specified
  • TOS total organic solids
  • U units [1 unit corresponds to the conversion of 1 ⁇ reb A/minute at 30°C and pH 7.0]
  • stage 3 the products of stage 1 (reb A, >95%) and stage 2 (UGTSr, UGTS1, and SuSy At enzymes) are mixed to initiate the enzymatic conversion process.
  • the reb A >95%) powder and sucrose are dissolved in reverse-osmosis water.
  • 5'-UDP-Na2 and UGTSr, UGTS1, and SuSy_At enzymes are added to formulate the reaction mixture.
  • the reaction mixture is incubated at 40 to 50°C for 10 to 48 hours.
  • the use of different reaction times yields steviol glycoside mixtures with different ratios of starting glycoside reb A, intermediate glycosides such as reb D, and the primary final glycoside product reb M.
  • the resulting reaction mixture containing a mixture of steviol glycosides, including those listed in Table 2.2-1 is heated to 80 to 100°C and for 10 minutes to inactivate the enzymes.
  • the reaction mixture is treated with a flocculant (calcium hydroxide) to remove the mechanical particles, proteins, polysaccharides, and other impurities.
  • a flocculant calcium hydroxide
  • a plate-and-frame filter press is used to separate the resulting precipitate from the filtrate, and the filtrate is deionized by ion-exchange resins in (H+) and (OH-) form.
  • the deionized filtrate is fed to a column system packed with macroporous adsorption resin that retains the reb M and other steviol glycosides.
  • the column is washed with deionized water to remove impurities that did not adsorb to the resin and then the glycosides are desorbed using aqueous ethanol.
  • the filtrate is maintained at low temperatures for several hours to allow reb M to crystallize.
  • the reb M crystals containing >30% reb M are separated by conventional centrifugation and dried in a rotary drum vacuum at 1 10°C and 10 mbar.
  • the obtained powder is sifted through US 80 mesh stainless steel screens and passed through metal detectors to be packed in aluminum foil bags.
  • the bags are placed in high-density polyethylene drums sealed with tamper evident seals.
  • the physical and chemical specifications for certain embodiments of steviol glycosides with a high reb M content produced by enzymatic conversion of reb A are based on those established by JECFA for steviol glycosides following their 82nd meeting (JECFA, 2016a).
  • the physical and chemical specifications for steviol glycosides with a high reb M content produced by enzymatic conversion are presented in Table 10. All analytical methods used to measure each specification parameter are internationally- recognized methods (e.g., United States Pharmacopeia [USP], Association of Official Analytical Chemists [AOAC], or JECFA).
  • Total steviol glycoside content is measured using the high-performance liquid chromatography (HPLC) method described in the most recent JECFA specification monograph for steviol glycosides from S. rebaudiana Bertoni (JECFA, 2016a).
  • FCC Food Chemicals Codex
  • HPLC high performance liquid chromatography
  • NA not applicable
  • NS not specified
  • PCR polymerase chain reaction
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • USP United States Pharmacopeia
  • steviol glycosides consists of a mixture of compounds containing a steviol backbone conjugated to any number or combination of the principal sugar moieties in any of the orientations occurring in the leaves of Stevia rebaudiana Bertoni including, glucose, rhamnose, xylose, fructose, deoxyglucose, galactose, and arabinose. (JECFA, 2016a, 2017).
  • microbiological specification parameters listed in Table 1 1 have been established for steviol glycosides with a high reb M content produced by enzymatic conversion of reb A to ensure safe use in food and standard microbial tests appropriate for food ingredients are employed .
  • CFU colony forming units
  • MPN most probable number
  • Cadmium (as Cd) ⁇ 1.0 ppm ⁇ 0.005 ppm ⁇ 0.005 ppm ⁇ 0.005 ppm
  • Example data from 2 production lots (SK BU2D1 , SK-BU3D1) presented in Table 13 demonstrates that as the enzyme reaction time proceeds from 10 to 40 hours the steviol glycoside distribution changes, with increasing amounts of reb M being produced as the reaction proceeds.
  • Example intermediate glycosides include rebaudiosides D and I, as reported in Table 14.
  • the final product contains >95% steviol glycosides, comprised of >30% reb M and other steviol glycosides such as those listed in Table 15.
  • the steviol glycoside distribution, measured by HPLC, is provided for 3 non-consecutive lots of final product manufactured with a 40-hour enzyme reaction time is shown in Table 16 and demonstrates that the manufacturing process produces a product with a consistent steviol glycoside distribution and that the total steviol glycosides measured is consistently >95%.
  • PCR polymerase chain reaction
  • Genomic DNA is extracted using a DNA extraction kit according to manufacturer's protocol.
  • the genomic DNA is quantified using a spectrophotometer and the extracted genomic DNA is evaluated for the presence of the gene of interest.

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Abstract

L'invention concerne des procédés de préparation de glucosides de stéviol hautement purifiés, en particulier les rébaudiosides M, D, E etI. Les procédés incluent l'utilisation de préparations enzymatiques et de micro-organismes de recombinaison pour convertir diverses compositions de départ en glycosides de stéviol cibles. Les rébaudiosides hautement purifiés sont utiles en tant qu'édulcorants non caloriques dans des compositions comestibles et à mâcher comme des boissons, des confiseries, des produits de boulangerie, des biscuits et des gommes à mâcher.
PCT/US2018/059461 2017-11-06 2018-11-06 Glycosides de stéviol de haute pureté WO2019090336A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975972A (zh) * 2022-12-20 2023-04-18 杭州力文所生物科技有限公司 一种糖基转移酶突变体及其编码基因
EP4117452A4 (fr) * 2020-03-13 2024-03-27 Amyris, Inc. Compositions d'édulcorant à base de rébaudioside m

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134292A1 (en) * 2004-12-21 2006-06-22 Stevian Biotechnology Corporation Sdn. Bhd. Malaysia Extraction, separation and modification of sweet glycosides from the stevia rebaudiana plant
US20140357588A1 (en) * 2013-05-28 2014-12-04 Purecircle Sdn Bhd High-purity steviol glycosides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134292A1 (en) * 2004-12-21 2006-06-22 Stevian Biotechnology Corporation Sdn. Bhd. Malaysia Extraction, separation and modification of sweet glycosides from the stevia rebaudiana plant
US20140357588A1 (en) * 2013-05-28 2014-12-04 Purecircle Sdn Bhd High-purity steviol glycosides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4117452A4 (fr) * 2020-03-13 2024-03-27 Amyris, Inc. Compositions d'édulcorant à base de rébaudioside m
CN115975972A (zh) * 2022-12-20 2023-04-18 杭州力文所生物科技有限公司 一种糖基转移酶突变体及其编码基因
CN115975972B (zh) * 2022-12-20 2023-07-25 杭州力文所生物科技有限公司 一种糖基转移酶突变体及其编码基因

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