AU2019236227B2 - High-purity steviol glycosides - Google Patents
High-purity steviol glycosides Download PDFInfo
- Publication number
- AU2019236227B2 AU2019236227B2 AU2019236227A AU2019236227A AU2019236227B2 AU 2019236227 B2 AU2019236227 B2 AU 2019236227B2 AU 2019236227 A AU2019236227 A AU 2019236227A AU 2019236227 A AU2019236227 A AU 2019236227A AU 2019236227 B2 AU2019236227 B2 AU 2019236227B2
- Authority
- AU
- Australia
- Prior art keywords
- rebaudioside
- udp
- glucosyltransferase
- acid sequence
- sequence identity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- 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
-
- 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
- A23L27/88—Taste or flavour enhancing agents
-
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Seasonings (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
- Cosmetics (AREA)
- Confectionery (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- General Preparation And Processing Of Foods (AREA)
- Manufacture Of Tobacco Products (AREA)
- Non-Alcoholic Beverages (AREA)
Abstract
Methods of preparing highly purified steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside lo, rebaudioside lp, rebaudioside Iq, rebaudioside Ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. The highly purified steviol glycosides are useful as non-caloric sweetener, flavor enhancer, sweetness enhancer, and foaming suppressor in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.
Description
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 rebaudianaBertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%). Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.
Although methods are known for preparing steviol glycosides from Stevia rebaudiana,many of these methods are unsuitable for use commercially.
Accordingly, there remains a need for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.
Reference to any prior art in the specification is not an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art.
la
As used herein, the abbreviation term "reb" refers to "rebaudioside". Both terms have the same meaning and may be used interchangeably.
As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells 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 enzymes, and whole-cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.
As used herein, the term "steviol glycoside(s)" refers to a glycoside of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside ip, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof. As used herein, the term "SvG7" refers to any naturally occurring steviol glycosides or any synthetic steviol glycosides, including enzymatically glucosylated steviol glycosides and combinations thereof, specifically a molecule comprising steviol having seven glucose residues attached covalently including, but not limited to reb a, reb 1b, reb ic, reb id, reb le, reb If reb ig, reb ih, reb Ii, reb ij, reb ik, reb 1, reb im, reb 2a, reb 2b, reb 2c, reb 2d, reb 2e, reb In, reb ,rebip,rebqrebir,rebIs,rebIt,reb 2f reb 2g, reb 2h, reb 2i, reb 2j, reb 2k, reb 21, reb 2m, reb 2n, reb 2o, reb 2p, reb 2q, reb 2r, and/ or reb 2s. SvG7 can refer to a single steviol glycoside having seven glucose residues attached covalently or a mixture of steviol glycosides having seven glucose residues attached covalently.
In a first aspect of the invention, there is provided steviol glycosides with the following formulae:
R2
H R1 HHO H
wherein R Iand R2 sugar chains are defined in the following table;
No. RI R2
HO OH HO -0HO
OH 0 0 HOH HOt HO H
0 HO
OH 0 OH
HO HO 0OH HO OH HO 0
3a
No. Ri R2
IV 0 0 HO"'
0 HOHO H OH 0OH
,r.K OH HO 0 J- QOH 0 0 HO HO1 0" OH HO OH
v Ho" 1 . OHO. H VHO 0
5H 5 H
HOOO HO 0 A "OH HO, HO OH 0 OH
3b
No. RI R2
Ho,,~H,. H, HO HO OH
L HO: :OH VII HO *HO 0q OH 6H 0 0
H HOL O, 0 0HOH HO OH
HO 0, O OHj
HO 0 HO 0 H o<0H HO O
Ho,. HO H OH HO H0 HO OH
HOHO 0 OH H
3c
No. Ri R2
HO HOhOH
xHO, HH H OO OH 0 0 aa - 0, ,,OH O HO HO -OHO OH~i H "OH HO OH H
H HOH HO 0H
xiHO' HOI H- 0 OH H 0 HO 0 ~ H
3d
This page has intentionally been left blank
3e
No. Rl R2
HOiQ HOH
HO q HOHO, (0OH OH 0 cHO 0 _OH
ttj.OH HO- HO 0,. OH HO OH
HO,, )HH HO, H OH 0HO 0.0
XVI HO , HO 0 OH OH 00 0 HO 0, ,OH
0 HO OH H
H O HO HO pH H HOH xvii HO, HO 0H0 OH
HO 0 OH 0HO 0, ,OH
H,/oI.Q H HO1 OH
3f
No. Ri R2 HO HO
HO,,,, OHOH HO HO, OH xvi OH HO 0 OH HOJ'§I HO 0, 0 00 H
H 0HH
H OH HO,,. H 00
HO 0 0 OHZ HO0 ,, HO XIX OH c)0).
H05 OH0 00 0H
H H HO OH00
HO 0 OHH
xx HO 0 0
H 00 H 0OH HO, HO ,
0 HH
3g
No. Rl R2
HO,,, H O* o Hq HO,, ,,OH
H 0-HO 0)%% OH OH 0 CH 2 hO 0, OH
HO 0 11HQO
HO OHO xiHO H . 0 O HOO OH -m
OH 0 HO 0, ~ < -OH OH
HO HO HO 0OH
H ,,OH H~ OHO
3h
No. Rl R2
HO HO HO HOAO HO4 HO v H 0
HO 0 O O OH 0 HO 0 OH
00 HO OH 0 HS.OH
HH O O0 xxv ~H OH OHH HO 0O
0 H( 0
0O OH
HO HO HO 0 OH
3i
No. Ri R2
0N 0
OHil OHH HO 0 0 H
HO OH 7
HO HO 4 ,
H0 H
HO-0 HO 65H HO 0 OH OH xxix HOD,,,,A 0 HO,. II H 0, OH HO : O OH 0 O
Hd/jiOH HO O
No. Rl R2 HO
H-10" HO,,H0 H0 O 0 H
xxx 0 0OH HOim. 0Q 0, OH H 0 OH 0 H HO bi- H
HO0 01-H
HO HO HO0
OH HOH H 0 OH
H OH0
XXXI OH 0 0 HOOHO 0
HO 01H
OH HO HO Hi'
HOXII OHo HO 0 OH
0OH HO 0 HO 0' 0, ' OH
3k
No. Rl R2
HO HO xxxiii HO, HO,0 HO- 0 OH OH
0H HO 0, Q O OH OH9 HO
-. OH OH 0:H
XXXIV HO 0OV HO 0 OH OH
HO 0 O 'OH
OH 0 HKO
No. Rl R2
OH' Axx HO 0, ~ OH
0 0 COH
HO HHH HO 0O XXVIOH t H 0 OH 0 -iOOH
HCOH0 OHH xxxvi O~H OH0 O
HOH 0 HH
HO O HO HO 0HO
HOO H II, HO o t O HO O OH O0 O0H OHvl
4 HO 0 HOK OH HO OH
3m
No. R1 R2 HO HO HO
HO 0 HO' 0 OH XXXVIII H HO H
HO HO H OOH HO,,, 0" HO 0
HOi~~Y, OH 0
HO 0
O OH HO OH 0
O,;~ H HOI 0 OH II OHr XL HO 0, OH 0
0 OQ
In a second aspect of the invention, there is provided a method for producing at least one steviol glycoside of the first aspect, comprising the steps of:
a. providing a starting composition comprising an organic compound with at least one carbon atom, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, other steviol glycosides, polyols, carbohydrates, and combinations thereof;
3n
b. providing an enzyme preparation or microorganism containing at least one enzyme selected from steviol biosynthesis enzymes, NDP-glucosyltransferases and NDP-glucose recycling enzymes; c. contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of the first aspect.
In a third aspect of the invention, there is provided a method for producing at least one steviol glycoside of the first aspect, comprising the steps of:
a. providing a starting composition comprising an organic compound with at least one carbon atom, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, other steviol glycosides, polyols, carbohydrates, and combinations thereof;
b. providing a biocatalyst comprising at least one enzyme selected from steviol biosynthesis enzymes, NDP-glucosyltransferases and NDP-glucose recycling enzymes; c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of the first aspect.
In a fourth aspect of the invention, there is provided a consumable product comprising at least one steviol glycoside of the first aspect, wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition.
In a fifth aspect of the invention, there is provided a method for enhancing the sweetness of a beverage or food product, comprising a sweetener comprising:
3o
a. providing a beverage or food product comprising a sweetener; and b. adding a sweetness enhancer comprising at least one steviol glycoside of the first aspect, wherein at least one steviol glycoside of the first aspect is present in a concentration at or below the sweetness recognition threshold.
In a sixth aspect of the invention, there is provided a method for modifying the flavor of a beverage or food product, comprising
a. providing a beverage or food product, and b. adding a composition comprising at least one steviol glycoside of the first aspect.
In a seventh aspect of the invention, there is provided a method for suppressing foaming of a beverage or food product, comprising
a. providing a beverage or a food product, and b. adding a foam suppressor comprising at least one steviol glycoside of the first aspect.
The present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or enzyme preparation, thereby producing a composition comprising a target steviol glycoside.
The starting composition can be any organic compound comprising at least one carbon atom. In one embodiment, the starting composition is selected from the group consisting of steviol glycosides, polyols or sugar alcohols, various carbohydrates.
The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside 1c, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside 1h, rebaudioside li, rebaudioside 1j,
3p
rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2frebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, SvG7 or a synthetic steviol glycoside.
In one embodiment, the target steviol glycoside is rebaudioside a.
In one embodiment, the target steviol glycoside is rebaudioside ib.
In one embodiment, the target steviol glycoside is rebaudioside ic.
In one embodiment, the target steviol glycoside is rebaudioside id.
In one embodiment, the target steviol glycoside is rebaudioside ie.
In one embodiment, the target steviol glycoside is rebaudioside If
In one embodiment, the target steviol glycoside is rebaudioside ig.
In one embodiment, the target steviol glycoside is rebaudioside h.
In one embodiment, the target steviol glycoside is rebaudioside ii.
In one embodiment, the target steviol glycoside is rebaudioside1j.
In one embodiment, the target steviol glycoside is rebaudioside 1k.
In one embodiment, the target steviol glycoside is rebaudioside ll.
In one embodiment, the target steviol glycoside is rebaudioside im.
In one embodiment, the target steviol glycoside is rebaudioside In.
In one embodiment, the target steviol glycoside is rebaudioside 1o.
In one embodiment, the target steviol glycoside is rebaudioside 1p.
In one embodiment, the target steviol glycoside is rebaudioside 1q.
In one embodiment, the target steviol glycoside is rebaudioside Jr.
In one embodiment, the target steviol glycoside is rebaudioside Is.
In one embodiment, the target steviol glycoside is rebaudioside It.
In one embodiment, the target steviol glycoside is rebaudioside 2a.
In one embodiment, the target steviol glycoside is rebaudioside 2b.
In one embodiment, the target steviol glycoside is rebaudioside 2c.
In one embodiment, the target steviol glycoside is rebaudioside 2d.
In one embodiment, the target steviol glycoside is rebaudioside 2e.
In one embodiment, the target steviol glycoside is rebaudioside 2f
In one embodiment, the target steviol glycoside is rebaudioside 2g.
In one embodiment, the target steviol glycoside is rebaudioside 2h.
In one embodiment, the target steviol glycoside is rebaudioside 2i.
In one embodiment, the target steviol glycoside is rebaudioside 2j.
In one embodiment, the target steviol glycoside is rebaudioside 2k.
In one embodiment, the target steviol glycoside is rebaudioside 21.
In one embodiment, the target steviol glycoside is rebaudioside 2m.
In one embodiment, the target steviol glycoside is rebaudioside 2n.
In one embodiment, the target steviol glycoside is rebaudioside 2o.
In one embodiment, the target steviol glycoside is rebaudioside 2p.
In one embodiment, the target steviol glycoside is rebaudioside 2q.
In one embodiment, the target steviol glycoside is rebaudioside 2r.
In one embodiment, the target steviol glycoside is rebaudioside 2s.
In one embodiment, the target steviol glycoside is rebaudioside M4.
In one embodiment, the target steviol glycoside is SvG7.
In some preferred embodiments enzyme preparation comprising one or more enzymes, or a microbial cell comprising one or more enzymes, capable of converting the starting composition to target steviol glycosides are used. The enzyme can be located on the surface and/or inside the cell. The enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate or as purified enzyme(s). The enzyme preparation can be in free form or immobilized to a solid support made from inorganic or organic materials.
In some embodiments, a microbial cell comprises the necessary enzymes and genes encoding thereof for converting the starting composition to target steviol glycosides. Accordingly, the present invention also provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell comprising at least one enzyme capable of converting the starting composition to target steviol glycosides, thereby producing a medium comprising at least one target steviol glycoside.
The enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes, NDP-glucosyltransferases (NGTs), ADP-glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs), GDP glucosyltransferases (GGTs), TDP-glucosyltransferases (TDPs), UDP glucosyltransferases (UGTs) and/or NDP-recycling enzyme, ADP-recycling enzyme, CDP-recycling enzyme, GDP-recycling enzyme, TDP-recycling enzyme, and/or UDP recycling enzyme.
In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.
In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.
In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4 diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E) butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase etc.
The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.
As used hereinafter, the term "SuSyAT", unless specified otherwise, refers to sucrose synthase having amino-acid sequence "SEQ ID 1" as described in Example 1, 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 1 polypeptide as well as isolated nucleic acid molecules that code for those polypetides.
As used hereinafter, the term "UGTSl2", unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence "SEQ ID 2" as described in Example 1 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 2 polypeptide as well as isolated nucleic acid molecules that code for those polypetides.
As used hereinafter, the term "UGT76G1", unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence "SEQ ID 3" as described in Example 1 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 3 polypeptide as well as isolated nucleic acid molecules that code for those polypetides.
In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell. The microbial cell may be, for example, E coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc. In another embodiment, the UDP-glucosyltransferases are synthesized.
In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS2, EUGTll and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.
In one embodiment, steviol biosynthesis enzymes, UGTs, and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be for example, F coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing an -OH functional group at C13 to give a target steviol glycoside having an -0 glucose beta glucopyranoside glycosidic linkage at C13. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a -COOH functional group at C19 to give a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, 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). In a particular embodiment, the UDP glucosyltransferase is UGTSl2, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to 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->3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to 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). In a particular embodiment, the UDP glucosyltransferase is UGTSl2, or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2. In another particular embodiment, the UDP glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, 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-+6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 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). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1->3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 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). In a particular embodiment, the UDP glucosyltransferase is UGTSl2, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2. In another particular embodiment, the UDP glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1-+6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT74Gl or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form rebaudioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside G. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, the UDP-glucosyltransferase is any UJDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTI, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form rebaudioside G. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76GI.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside B to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the IDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E6. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E4. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E6. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside C to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having
>85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTll. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside I In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside I. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside I to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside AMto form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M4. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT9lD2. In a particular embodiment, the UDP-glucosyltransferase is UGT76GI or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside la. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1c. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside Id In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside le. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside If In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1g. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino- acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside Ii. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1j. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside I. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside im. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside In. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1o. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or aUGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any JDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1p. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1q. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1r. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside Is. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any JDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside It. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2c. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2d In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2e. In a particular embodiment, the IDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2f In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2g. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2i. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2j. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2m. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2n. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2o. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2p. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2q. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2r. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2s. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1q. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTI1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of steviol and/or the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP glucosyltransferase and UDP-glucose.
In one embodiment, the recycling catalyst is sucrose synthase SuSyAt or a sucrose synthase having >85% amino-acid sequence identity with SuSy.At.
In one embodiment, the recycling substrate for UDP-glucose recycling catalyst is sucrose.
Optionally, the method of the present invention further comprises the use of transglycosidases that use oligo- or poly-saccharides as the sugar donor to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosylinvertase, alkaline invertase, acid invertase, fructofuranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose are transferred to the recipient target steviol glycosides. In one embodiment, the recipient steviol glycoside is rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If rebaudioside ig, rebaudioside h, rebaudioside ii, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, and/or rebaudioside 2s. In another embodiment, the recipient steviol glycoside is rebaudioside M4. In another embodiment, the recipient steviol glycoside is SvG7. Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
In one embodiment, the target steviol glycoside can be produced within the microorganism. In another embodiment, the target steviol glycoside can be secreted out in the medium. In one another embodiment, the released steviol glycoside can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the completion of the conversion reaction.
In one embodiment, separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In particular embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside. In other embodiments, the composition comprises greater than about 99% by weight of the target steviol glycoside.
The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
Purified target steviol glycosides can be used in consumable products as a sweetener, flavor modifier, flavor with modifying properties and/or foaming suppressor. Suitable consumer products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.
FIG. la through FIG. It show the chemical structure of some SvG7 steviol glycosides rebaudioside la, rebaudioside 1b, rebaudioside 1c, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside 1h, rebaudioside li, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside ip, rebaudioside 1q, rebaudioside 1r, rebaudioside Is and rebaudioside It respectively.
FIG. 2a through FIG. 2s show the chemical structure of some SvG7 steviol glycosides rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r and rebaudioside 2s respectively.
FIG. 3 shows the chemical structure of rebaudioside M4.
FIG. 4a through FIG. 4s show the pathways of producing rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is and rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, and rebaudioside 2s, rebaudioside M4 and various steviol glycosides from steviol and the various intermediary steviol glycosides.
FIG. 5a through FIG. 5t show the biocatalytic production of rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside 1e, rebaudioside If rebaudioside ig, rebaudioside ih, rebaudioside 1i, rebaudioside j, rebaudioside 1k, rebaudioside il, rebaudioside im and rebaudioside In, rebaudioside io, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is and rebaudioside It respectively, from rebaudioside A using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP glucose via sucrose synthase SuSyAt.
FIG. 6a through FIG. 6t show the biocatalytic production of rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and rebaudioside 1q, respectively, from stevioside using the enzymes UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSyAt.
FIG. 7 shows the biocatalytic production of rebaudioside M4 from stevioside using the enzymes UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSyAt.
FIG. 8a through FIG. 8t show the biocatalytic production of rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2 q, rebaudioside 2r, rebaudioside 2s and rebaudioside lq, respectively, from rebaudioside AM using the enzymes UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP glucose via sucrose synthase SuSyAt.
FIG. 9 shows the biocatalytic production of rebaudioside M4 from rebaudioside AMusing the enzymes UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSyAt.
FIG. 1Oa through FIG. lot show the biocatalytic production of rebaudioside 2a rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and rebaudioside 1q, respectively, from rebaudioside M4 using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSyAt.
FIG. 1la shows the HPLC chromatogram of stevioside. The peak with retention time of 20.958 minutes corresponds to stevioside. The peak with retention time 20.725 minutes corresponds to rebaudioside A. The peak at 32.925 minutes corresponds to rebaudioside B. The peak at 33.930 minutes corresponds to steviolbioside.
FIG. 1lb shows the HPLC chromatogram of the product of the biocatalytic production of SvG7 molecules from stevioside. The peak at 5.089 minutes corresponds to rebaudioside 2m. The peak at 6.459 minutes corresponds to rebaudioside 2a. The peak at 9.825 minutes corresponds to rebaudioside AM. The peak at 13.845 minutes corresponds to rebaudioside
M. The peak at 32.974 minutes corresponds to rebaudioside B. The peak at 33.979 minutes corresponds to steviolbioside.
FIG. 1I shows the HPLC chromatogram of rebaudioside 2a after purification by HPLC. The peak with retention time of 6.261 minutes correspond to rebaudioside 2a.
FIG. lid shows the HPLC chromatogram of rebaudioside 2m after purification by HPLC. The peak with retention time of 5.197 minutes correspond to rebaudioside 2m.
FIG. 12a shows the 1H NMR spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12b shows the HSQC spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12c shows the H,H COSY spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12d shows the HMBC spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12e shows the HSQC-TOCSY spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12f shows the ID-NOESY spectrum of rebaudioside 2a (500 MHz, pyridine-d5).
FIG. 12g shows the LC chromatogram of rebaudioside 2a.
FIG. 12h shows the mass spectrum of rebaudioside 2a.
FIG. 13a shows the 1H NMR spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13b shows the HSQC spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13c shows the H,H COSY spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13d shows the HMBC spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13e shows the HSQC-TOCSY spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13f shows the ID-NOESY spectrum of rebaudioside 2m (500 MHz, pyridine-d5).
FIG. 13g shows the LC chromatogram of rebaudioside 2m.
FIG. 13h shows the mass spectrum of rebaudioside 2m.
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 aspect of the invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside 1c, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside 1h, rebaudioside ii, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside Jo, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2frebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 or a synthetic steviol glycoside from various starting compositions.
Starting Composition
As used herein, "starting composition" refers to any composition (generally an aqueous solution) containing one or more organic compound comprising at least one carbon atom.
In one embodiment, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols and various carbohydrates.
The starting composition steviol glycoside is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, and/or rebaudioside M4 or other glycoside of steviol occurring in Stevia rebaudianaplant, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.
In one embodiment, the starting composition is steviol.
In another embodiment, the starting composition steviol glycoside is steviolmonoside.
In yet another embodiment, the starting composition steviol glycoside is
steviolmonoside A.
In another embodiment, the starting composition steviol glycoside is steviolbioside.
In another embodiment, the starting composition steviol glycoside is steviolbioside D.
In another embodiment, the starting composition steviol glycoside is rubusoside.
In another embodiment, the starting composition steviol glycoside is rubusoside.
In another embodiment, the starting composition steviol glycoside is steviolbioside A.
In another embodiment, the starting composition steviol glycoside is steviolbioside B.
In another embodiment, the starting composition steviol glycoside is rebaudioside B.
In another embodiment, the starting composition steviol glycoside is stevioside.
In another embodiment, the starting composition steviol glycoside is rebaudioside G.
In another embodiment, the starting composition steviol glycoside is stevioside A.
In another embodiment, the starting composition steviol glycoside is stevioside B.
In another embodiment, the starting composition steviol glycoside is stevioside C.
In another embodiment, the starting composition steviol glycoside is rebaudioside A.
In another embodiment, the starting composition steviol glycoside is rebaudioside E.
In another embodiment, the starting composition steviol glycoside is rebaudioside E2.
In another embodiment, the starting composition steviol glycoside is rebaudioside E4.
In another embodiment, the starting composition steviol glycoside is rebaudioside E6.
In another embodiment, the starting composition steviol glycoside is rebaudioside E3.
In another embodiment, the starting composition steviol glycoside is rebaudioside D.
In another embodiment, the starting composition steviol glycoside is rebaudioside I.
In another embodiment, the starting composition steviol glycoside is rebaudioside AM.
In another embodiment, the starting composition steviol glycoside is rebaudioside D7.
In another embodiment, the starting composition steviol glycoside is rebaudioside M
In another embodiment, the starting composition steviol glycoside is rebaudioside M4.
The term "polyol" refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced.
The term "carbohydrate" refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH 2 O)n, wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives", "substituted carbohydrate", and "modified carbohydrates" are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.
Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides
(isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.
The starting composition may be synthetic or purified (partially or entirely), commercially available or prepared.
In one embodiment, the starting composition is glycerol.
In another embodiment, the starting composition is glucose.
In 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 yet another embodiment, the starting composition is cellulose.
In still another embodiment, the starting composition is amylose.
The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein.
Target Steviol Glycoside
The target steviol glycoside of the present method can be any steviol glycoside that can be prepared by the process disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside Ic, rebaudioside id, rebaudioside le, rebaudioside If; rebaudioside ig, rebaudioside ih, rebaudioside 1i, rebaudioside ij, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, SvG7 or other glycoside of steviol occurring in Stevia rebaudianaplant, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof. In one embodiment, the target steviol glycoside is steviolmonoside.
In another embodiment, the target steviol glycoside is steviolmonoside A.
In another embodiment, the target steviol glycoside is steviolbioside.
In another embodiment, the target steviol glycoside is steviolbioside D.
In another embodiment, the target steviol glycoside is rubusoside.
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 rebaudioside B.
In another embodiment, the target steviol glycoside is stevioside.
In another embodiment, the target steviol glycoside is rebaudioside G.
In another embodiment, the target steviol glycoside is stevioside A.
In another embodiment, the target steviol glycoside is stevioside B.
In another embodiment, the target steviol glycoside is stevioside C.
In another embodiment, the target steviol glycoside is rebaudioside A.
In another embodiment, the target steviol glycoside is rebaudioside K
In another embodiment, the target steviol glycoside is rebaudioside E2.
In another embodiment, the target steviol glycoside is rebaudioside E4.
In another embodiment, the target steviol glycoside is rebaudioside E6.
In another embodiment, the target steviol glycoside is rebaudioside E3.
In another embodiment, the target steviol glycoside is rebaudioside D.
In another embodiment, the target steviol glycoside is rebaudioside I.
In another embodiment, the target steviol glycoside is rebaudioside AM
In another embodiment, the target steviol glycoside is rebaudioside D7.
In another embodiment, the target steviol glycoside is rebaudioside M
In another embodiment, the target steviol glycoside is rebaudioside M4.
In another embodiment, the target steviol glycoside is rebaudioside la.
In another embodiment, the target steviol glycoside is rebaudioside lb.
In another embodiment, the target steviol glycoside is rebaudioside ic.
In another embodiment, the target steviol glycoside is rebaudioside Id.
In another embodiment, the target steviol glycoside is rebaudioside le.
In another embodiment, the target steviol glycoside is rebaudioside If
In another embodiment, the target steviol glycoside is rebaudioside 1g.
In another embodiment, the target steviol glycoside is rebaudioside h.
In another embodiment, the target steviol glycoside is rebaudioside li.
In another embodiment, the target steviol glycoside is rebaudioside 1j.
In another embodiment, the target steviol glycoside is rebaudioside 1k.
In another embodiment, the target steviol glycoside is rebaudioside i.
In another embodiment, the target steviol glycoside is rebaudioside im.
In another embodiment, the target steviol glycoside is rebaudioside In.
In another embodiment, the target steviol glycoside is rebaudioside 1o.
In another embodiment, the target steviol glycoside is rebaudioside ip.
In another embodiment, the target steviol glycoside is rebaudioside 1q.
In another embodiment, the target steviol glycoside is rebaudioside 1r.
In another embodiment, the target steviol glycoside is rebaudioside Is.
In another embodiment, the target steviol glycoside is rebaudioside It.
In another embodiment, the target steviol glycoside is rebaudioside 2a.
In another embodiment, the target steviol glycoside is rebaudioside 2b.
In another embodiment, the target steviol glycoside is rebaudioside 2c.
In another embodiment, the target steviol glycoside is rebaudioside 2d
In another embodiment, the target steviol glycoside is rebaudioside 2e.
In another embodiment, the target steviol glycoside is rebaudioside 2f
In another embodiment, the target steviol glycoside is rebaudioside 2g.
In another embodiment, the target steviol glycoside is rebaudioside 2h.
In another embodiment, the target steviol glycoside is rebaudioside 2i.
In another embodiment, the target steviol glycoside is rebaudioside 2j.
In another embodiment, the target steviol glycoside is rebaudioside 2k.
In another embodiment, the target steviol glycoside is rebaudioside 2.
In another embodiment, the target steviol glycoside is rebaudioside 2m.
In another embodiment, the target steviol glycoside is rebaudioside 2n.
In another embodiment, the target steviol glycoside is rebaudioside 2o.
In another embodiment, the target steviol glycoside is rebaudioside 2p.
In another embodiment, the target steviol glycoside is rebaudioside 2q.
In another embodiment, the target steviol glycoside is rebaudioside 2r.
In another embodiment, the target steviol glycoside is rebaudioside 2s.
In another embodiment, the target steviol glycoside is SvG7.
The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside.
In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside A.
In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside.
In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside D.
In one embodiment, the present invention is a biocatalytic process for the production of rubusoside.
In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside A.
In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside B.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside B.
In one embodiment, the present invention is a biocatalytic process for the production of stevioside.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside G.
In one embodiment, the present invention is a biocatalytic process for the production of stevioside A.
In one embodiment, the present invention is a biocatalytic process for the production of stevioside B.
In one embodiment, the present invention is a biocatalytic process for the production of stevioside C.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside A.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E2.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E4.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E6.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside D.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside I.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside AM.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside D7.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside M
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside M4.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside la.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside lb.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside ic.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside id.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside le.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside If
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1g.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside lh.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside li.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1j.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1k.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside ll.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside im.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside In.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1o.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1p.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1q.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1r.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside Is.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside It.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2a.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2b.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2c.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2d.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2e.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2f
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2g.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2h.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2i.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2j.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2k.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 21.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2m.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2n.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2o.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2p.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2q.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2r.
In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2s.
In one embodiment, the present invention is a biocatalytic process for the production of SvG7.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside la from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside ]b from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside ic from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Id from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside le from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside If from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Ig from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside ih from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 11 from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside im from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside In from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Io from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1p from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1r from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Is from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside It from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2a from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2b from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2c from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2d from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2e from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2f from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2g from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2h from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2i from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2j from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2k from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 21 from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2m from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2n from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2o from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2p from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2q from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2r from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2s from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1q from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside M4 from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside AMand UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2g from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2h from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2i from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside AMand UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 21 from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2m from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2n from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2q from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2s from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside M4 from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2g from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2h from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2i from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 21 from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2m from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2n from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2q from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 2s from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside M4 and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside la from a starting composition comprising rebaudioside Mand UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside lb from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Ic from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Id from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside le from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside If from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside ig from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside h from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 11 from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Im from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside In from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1o from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside ip from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside iq from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 1r from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside Is from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside It from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising stevioside and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising stevioside, rebaudioside A and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside AM and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M and UDP-glucose.
In a particular embodiment, the present invention provides for the biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M4 and UDP-glucose.
Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by any suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
In particular embodiments, the process described herein results in a highly purified target steviol glycoside composition. The term "highly purified", as used herein, refers to a composition having greater than about 80% by weight of the target steviol glycoside on an anhydrous (dried) basis. In one embodiment, the highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous (dried) basis, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% target steviol glycoside content on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition having greater than about 90% rebaudioside M4 content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside la, the process described herein provides a composition having greater than about 90% rebaudioside la content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside la, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1b, the process described herein provides a composition having greater than about 90% rebaudioside 1b content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1b, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1c, the process described herein provides a composition having greater than about 90% rebaudioside ic content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1c, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside id, the process described herein provides a composition having greater than about 90% rebaudioside id content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside id, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside le, the process described herein provides a composition having greater than about 90% rebaudioside le content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside le, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside If; the process described herein provides a composition having greater than about 90% rebaudioside If content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside If, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1g, the process described herein provides a composition having greater than about 90% rebaudioside ig content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1g, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1h, the process described herein provides a composition having greater than about 90% rebaudioside h content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1h, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside li, the process described herein provides a composition having greater than about 90% rebaudioside li content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside li, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1j, the process described herein provides a composition having greater than about 90% rebaudioside j content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1j, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1k, the process described herein provides a composition having greater than about 90% rebaudioside 1k content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1k, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside il, the process described herein provides a composition having greater than about 90% rebaudioside 11 content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside ll, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside im, the process described herein provides a composition having greater than about 90% rebaudioside Im content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside im, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside In, the process described herein provides a composition having greater than about 90% rebaudioside In content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside In, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1o, the process described herein provides a composition having greater than about 90% rebaudioside Io content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside lo, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1p, the process described herein provides a composition having greater than about 90% rebaudioside 1p content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1p, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 1q, the process described herein provides a composition having greater than about 90% rebaudioside iq content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 1q, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside ir, the process described herein provides a composition having greater than about 90% rebaudioside 1r content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside ir, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside Is, the process described herein provides a composition having greater than about 90% rebaudioside Is content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside Is, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside It, the process described herein provides a composition having greater than about 90% rebaudioside It content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside It, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition having greater than about 90% rebaudioside 2a content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2b, the process described herein provides a composition having greater than about 90% rebaudioside 2b content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2b, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2c, the process described herein provides a composition having greater than about 90% rebaudioside 2c content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2c, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2d, the process described herein provides a composition having greater than about 90% rebaudioside 2d content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2d, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2e, the process described herein provides a composition having greater than about 90% rebaudioside 2e content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2e, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2f the process described herein provides a composition having greater than about 90% rebaudioside 2f content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2f, the process described herein provides a.composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2g, the process described herein provides a composition having greater than about 90% rebaudioside 2g content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2g, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2h, the process described herein provides a composition having greater than about 90% rebaudioside 2h content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2h, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2i, the process described herein provides a composition having greater than about 90% rebaudioside 2i content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2i, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2j, the process described herein provides a composition having greater than about 90% rebaudioside 2j content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2j, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2k, the process described herein provides a composition having greater than about 90% rebaudioside 2k content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2k, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 21, the process described herein provides a composition having greater than about 90% rebaudioside 21content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 21, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2m, the process described herein provides a composition having greater than about 90% rebaudioside 2m content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2m, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2n, the process described herein provides a composition having greater than about 90% rebaudioside 2n content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2n, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2o, the process described herein provides a composition having greater than about 90% rebaudioside 2o content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2o, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2p, the process described herein provides a composition having greater than about 90% rebaudioside 2 p content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2p, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2q, the process described herein provides a composition having greater than about 90% rebaudioside 2q content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2q, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2r, the process described herein provides a composition having greater than about 90% rebaudioside 2r content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2r, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is rebaudioside 2s, the process described herein provides a composition having greater than about 90% rebaudioside 2s content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2s, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.
In one embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition having greater than about 90% SvG7 content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition comprising greater than about 95% SvG7 content by weight on a dried basis.
Microorganisms and enzyme preparations
In one embodiment of present invention, a microorganism (microbial cell) and/or enzyme preparation is contacted with a medium containing the starting composition to produce target steviol glycosides.
The enzyme can be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme or a combination thereof. In one embodiment, the biocatalyst is a purified enzyme capable of converting the starting composition to the target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising at least one
enzyme capable of converting the starting composition to the target steviol glycoside. In still another embodiment, the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting the starting composition to the target steviol glycoside.
In another embodiment, the biocatalyst is one or more microbial cells comprising enzyme(s) capable of converting the starting composition to the target steviol glycoside. The enzyme can be located on the surface of the cell, inside the cell or located both on the surface of the cell and inside the cell.
Suitable enzymes for converting the starting composition to target steviol glycosides include, but are not limited to, the steviol biosynthesis enzymes, NDP glucosyltransferases (NGTs), ADP-glucosyltransferases (AGTs), CDP glucosyltransferases (CGTs), GDP-glucosyltransferases (GGTs), TDP
glucosyltransferases (TDPs), UDP-glucosyltransferases (UGTs). Optionally it may include NDP-recycling enzyme(s), ADP-recycling enzyme(s), CDP-recycling enzyme(s), GDP recycling enzyme(s), TDP-recycling enzyme(s), and/or UDP-recycling enzyme(s).
In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.
In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.
In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4 diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E) butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase etc.
The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.
In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc. In another embodiment, the UDP-glucosyltransferases are synthesized.
In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS2, EUGTll and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.
In one embodiment, steviol biosynthesis enzymes, UGTs and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing an -OH functional group at C13 to give a target steviol glycoside having an -0 glucose beta glucopyranoside glycosidic linkage at C13. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a -COOH functional group at C19 to give a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, 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). In a particular embodiment, the UDP glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTI1, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to 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 1-3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, 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). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT9lD2, or a UGT having >85% amino- acid sequence identity with UGT91D2. In another particular embodiment, the UDP glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UIDP 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-+6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 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). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP
glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1-3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 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). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2. In another particular embodiment, the UDP glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1-+6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP glucosyltransferase is UGTS2, or a UGT having >85% amino-acid sequence identity with UGTS2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT9D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to form steviolbioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form rebaudioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside G. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form rebaudioside G. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76GI or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbiosideA to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76GI or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside B to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4. In a particular embodiment, the UDP-gucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E6. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E4. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E6. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside C to form rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside I In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside I In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino- acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside I to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside AMto form rebaudioside M4. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the JDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside la. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1c. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside Id In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside le. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside If In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1g. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside ii. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1j. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1l. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTll. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside im. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase isUGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside In. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1o. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1p. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside 1q. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside Ir. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside Mto form rebaudioside Is. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside It. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2c. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2d. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2e. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2f In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2g. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2i. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a
UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2j. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 21. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2m. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2n. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino acid sequence identity with EUGT11. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2o. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2p. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2q. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTll, or a UGT having >85% amino acid sequence identity with EUGTll. In yet another particular embodiment, the UDP glucosyltransferase is UGT9lD2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2r. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino-acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGT1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2s. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1q. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl1, or a UGT having >85% amino acid sequence identity with EUGTl1. In yet another particular embodiment, the UDP glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76Gl.
Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of steviol and/or the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.
In one embodiment, the recycling catalyst is sucrose synthase SuSyAt or a sucrose synthase having >85% amino-acid sequence identity with SuSyAt.
In one embodiment, the recycling substrate for UDP-glucose recycling catalyst is sucrose.
Optionally, the method of the present invention further comprises the use of transglycosidases that use oligo- or poly-saccharides as the sugar donor to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosylinvertase, alkaline invertase, acid invertase, fructofranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose are transferred to the recipient target steviol glycosides. In one embodiment, the recipient steviol glycoside is rebaudioside la, rebaudioside ib, rebaudioside ic, rebaudioside Id, rebaudioside 1e, rebaudioside If rebaudioside ig, rebaudioside h, rebaudioside ii, rebaudioside 1j, rebaudioside 1k, rebaudioside Il, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2 q, rebaudioside 2r, and/ or rebaudioside 2s. In another embodiment, the recipient steviol glycoside is rebaudioside M4. In another embodiment, the recipient steviol glycoside is SvG7. In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to starting composition steviol glycoside has >85% amino-acid sequence identity with UGTs selected from the following listing of Gennfo identifier numbers, preferably from the group presented in Table 1, and Table 2.
397567 30680413 115480946 147798902 218193594 225443294 454245 32816174 116310259 147811764 218193942 225444853 1359905 32816178 116310985 147827151 219885307 225449296 1685003 34393978 116788066 147836230 222615927 225449700 1685005 37993665 116788606 147839909 222619587 225454338 2191136 37993671 116789315 147846163 222623142 225454340 2501497 37993675 119394507 147855977 222625633 225454342 2911049 39104603 119640480 148905778 222625635 225454473 4218003 41469414 122209731 148905999 222636620 225454475 4314356 41469452 125526997 148906835 222636621 225458362 13492674 42566366 125534279 148907340 222636628 225461551 13492676 42570280 125534461 148908935 222636629 225461556 15217773 42572855 125540090 148909182 224053242 225461558 15217796 44890129 125541516 148909920 224053386 225469538 15223396 46806235 125545408 148910082 224055535 225469540 15223589 50284482 125547340 148910154 224056138 226316457 15227766 51090402 125547520 148910612 224056160 226492603 15230017 51090594 125554547 148910769 224067918 226494221 15231757 52839682 125557592 156138791 224072747 226495389 15234056 56550539 125557593 156138797 224080189 226495945 15234195 62734263 125557608 156138799 224091845 226502400 15234196 62857204 125559566 156138803 224094703 226507980 15238503 62857206 125563266 165972256 224100653 226531147 15239523 62857210 125571055 168016721 224100657 226532094 15239525 62857212 125579728 171674071 224101569 238477377 15239543 75265643 125588307 171906258 224103105 240254512 15239937 75285934 125589492 183013901 224103633 242032615 15240305 75288884 125599469 183013903 224103637 242032621 15240534 77550661 125601477 186478321 224109218 242038423 15982889 77556148 126635837 187373030 224114583 242043290 18086351 82791223 126635845 187373042 224116284 242044836 18418378 83778990 126635847 190692175 224120552 242051252 18418380 89953335 126635863 194701936 224121288 242056217 18418382 110741436 126635867 195620060 224121296 242056219 19743740 110743955 126635883 209954691 224121300 242056663 19911201 115438196 126635887 209954719 224130358 242059339 20149064 115438785 133874210 209954725 224140703 242059341 20260654 115441237 133874212 209954733 224143404 242060922 21435782 115454819 145358033 210063105 224143406 242067411 21553613 115456047 147772508 210063107 224144306 242067413 21593514 115457492 147776893 212275846 224285244 242076258 22759895 115459312 147776894 216296854 225431707 242076396 23955910 115464719 147776895 217074506 225435532 242084750 26452040 115471069 147786916 218185693 225436321 242091005 28393204 115471071 147798900 218187075 225440041 242095206 30679796 115474009 147798901 218189427 225441116 242345159
Table 1
GI number Accession Origin 190692175 ACE87855.1 Stevia rebaudiana 41469452 AAS07253.1 Oryza sativa 62857204 BAD95881.1 Ipomoea nil 62857206 BAD95882.1 Ipomoeapurperea 56550539 BAD77944.1 Bellis perennis 115454819 NP 001051010.1 Oryza sativa JaponicaGroup 115459312 NP 001053256.1 Oryza sativa JaponicaGroup 115471069 NP 001059133.1 Oryza sativa JaponicaGroup 115471071 NP 001059134.1 Oryza sativa JaponicaGroup 116310985 CAH67920.1 Oryza sativa Indica Group
116788066 ABK24743.1 Picea sitchensis 122209731 Q2V6J9.1 Fragariax ananassa 125534461 EAY81009.1 Oryza sativaIndica Group 125559566 EAZ05102.1 Oryza sativaIndica Group 125588307 EAZ28971.1 Oryza sativaJaponicaGroup 148907340 ABR16806.1 Picea sitchensis 148910082 ABR18123.1 Picea sitchensis 148910612 ABR18376.1 Picea sitchensis 15234195 NP_194486.1 Arabidopsisthaliana 15239523 NP_200210.1 Arabidopsis thaliana 15239937 NP 196793.1 Arabidopsis thaliana 1685005 AAB36653.1 Nicotiana tabacum 183013903 ACC38471.1 Medicago truncatula 186478321 NP_172511.3 Arabidopsis thaliana 187373030 ACD03249.1 Avena strigosa 194701936 ACF85052.1 Zea mays 19743740 AAL92461.1 Solanum lycopersicum 212275846 NP_001131009.1 Zea mays 222619587 EEE55719.1 Oryza sativaJaponicaGroup 224055535 XP 002298527.1 Populus trichocarpa 224101569 XP 002334266.1 Populus trichocarpa 224120552 XP 002318358.1 Populus trichocarpa 224121288 XP_002330790.1 Populus trichocarpa 225444853 XP_002281094 Vitis vinifera 225454342 XP_002275850.1 Vitis vinifera 225454475 XP_002280923.1 Vitis vinifera 225461556 XP_002285222 Vitis vinifera 225469540 XP_002270294.1 Vitis vinifera 226495389 NP_001148083.1 Zea mays 226502400 NP_001147674.1 Zea mays 238477377 ACR43489.1 Triticum aestivum 240254512 NP 565540.4 Arabidopsis thaliana 2501497 Q43716.1 Petuniax hybrida 255555369 XP_002518721.1 Ricinus communis 26452040 BAC43110.1 Arabidopsisthaliana 296088529 CB137520.3 Vitis vinifera 297611791 NP_001067852.2 Oryza sativa JaponicaGroup 297795735 XP_002865752.1 Arabidopsislyrata subsp. lyrata 297798502 XP 002867135.1 Arabidopsislyratasubsp. lyrata 297820040 XP 002877903.1 Arabidopsislyrata subsp. lyrata 297832276 XP_002884020.1 Arabidopsislyrata subsp. lyrata 302821107 XP_002992218.1 Selaginella moellendorffii 30680413 NP_179446.2 Arabidopsisthaliana 319759266 ADV71369.1 Puerariamontana var. lobata 326507826 BAJ86656.1 Hordeum vulgare subsp. Vulgare 343457675 AEM37036.1 Brassicarapasubsp. oleifera 350534960 NP_001234680.1 Solanum lycopersicum 356501328 XP 003519477.1 Glycine max 356522586 XP 003529927.1 Glycine max 356535480 XP 003536273.1 Glycine max 357445733 XP_003593144.1 Medicago truncatula 357452783 XP_003596668.1 Medicago truncatula 357474493 XP_003607531.1 Medicago truncatula 357500579 XP_003620578.1 Medicago truncatula 357504691 XP_003622634.1 Medicago truncatula 359477998 | XP_003632051.1 Vitis vinifera 359487055 XP 002271587 Vitis vinifera 359495869 XP 003635104.1 Vitis vinifera
387135134 AFJ52948.1 Linum usitatissimum 387135176 AFJ52969.1 Linum usitatissimum 387135192 AFJ52977.1 Linum usitatissimum 387135282 AFJ53022.1 Linum usitatissimum 387135302 AFJ53032.1 Linum usitatissimum 387135312 AFJ53037.1 Linum usitatissimum 388519407 AFK47765.1 Medicago truncatula 393887646 AFN26668.1 Barbareavulgaris subsp. arcuata 414888074 DAA64088.1 Zea mays 42572855 NP_974524.1 Arabidopsis thaliana 449440433 XP 004137989.1 Cucumis sativus 449446454 XP_004140986.1 Cucumis sativus 449449004 XP_004142255.1 Cucumis sativus 449451593 XP 004143546.1 Cucumis sativus 449515857 XP_004164964.1 Cucumis sativus 460382095 XP_004236775.1 Solanum lycopersicum 460409128 XP 004249992.1 Solanum lycopersicum 460409461 XP_004250157.1 Solanum lycopersicum 460409465 XP_004250159.1 Solanum lycopersicum 462396388 EMJ02187.1 Prunuspersica 462402118 EMJ07675.1 Prunuspersica 462409359 EMJ14693.1 Prunuspersica 462416923 EMJ21660.1 Prunuspersica 46806235 BAD17459.1 Oryza sativa JaponicaGroup 470104266 XP_004288529.1 Fragariavesca subsp. vesca 470142008 XP 004306714.1 Fragariavesca subsp. vesca 475432777 EMT01232.1 Aegilops tauschii 51090402 BAD35324.1 Oryza sativa JaponicaGroup
Table 2
GI number Accession Origin Internal reference 460409128 XP.004249992.1 Solanum lycopersicum UGTSI 460386018 XP.004238697.1 Solanum lycopersicum 460409134 XP.004249995.1 Solanum lycopersicum 460410132 XP.004250485.1 Solanum lycopersicum UGTS12 460410130 XP.004250484.1 Solanum lycopersicum 460410128 XP.004250483.1 Solanum lycopersicum 460378310 XP.004234916.1 Solanum lycopersicum 209954733 BAG80557.1 Lycium barbarum UGTLB 209954725 BAG80553.1 Lycium barbarum
One embodiment of the present invention is a microbial cell comprising an enzyme, i.e. an enzyme capable of converting the starting composition to the target steviol glycoside. Accordingly, some embodiments of the present method include contacting a microorganism with a medium containing the starting composition to provide a medium comprising at least one target steviol glycoside.
The microorganism can be any microorganism possessing the necessary enzyme(s) for converting the starting composition to target steviol glycoside(s). These enzymes are encoded within the microorganism's genome.
Suitable microorganisms include, but are not limited to, E.coli, Saccharomyces sp., Aspergillus sp., Pichiasp., Bacillus sp., Yarrowia sp. etc.
In one embodiment, the microorganism is free when contacted with the starting composition.
In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, the microorganism may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the microorganism include derivatized cellulose or glass, ceramics, metal oxides or membranes. The microorganism may be immobilized to the solid support, for example, by covalent attachment, adsorption, cross-linking, entrapment or encapsulation.
In still another embodiment, the enzyme capable of converting the starting composition to the target steviol glycoside is secreted out of the microorganism and into the reaction medium.
The target steviol glycoside is optionally purified. Purification of the target steviol glycoside from the reaction medium can be achieved by at least one suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.
Uses
Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside 1c, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 obtained according to this invention can be used "as-is" or in combination with other sweeteners, flavors, food ingredients and combinations thereof. Non-limiting examples of flavors include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla and combinations thereof.
Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents and combinations thereof.
Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside ii, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 obtained according to this invention can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous, amorphous forms and combinations thereof.
Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside 1c, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside lh, rebaudioside li, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside Ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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.
In some embodiments, the highly purified target glycoside(s) of present invention are present in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc in an amount from about 0.0001% to about 12% by weight, such as, for example, 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, about 11.5% by weight or about 12.0% by weight.
In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.0001% by weight to about 8% by weight, such as for example, 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 0.5% 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 about 3% by weight to about 4% by weight, from about 4% by weight to about 5% by weight, from about 5% by weight to about 6% by weight, from about 6% by weight to about 7% by weight, and from about 7% by weight to about 8% by weight.
Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside j, rebaudioside 1k, rebaudioside il, rebaudioside im,
rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s SvG7 and/or combinations thereof, obtained according to this invention, may be employed as a sweetening compound, or it may be used together with at least one naturally occurring high intensity sweeteners such as dulcoside A, dulcoside B, dulcoside C, dulcoside D, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B2, rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D8, rebaudioside E5, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside H, rebaudioside H2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside 0, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zi, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin, monatin salts, other indole derivative sweeteners, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, siamenoside, siratose and combinations thereof.
In a particular embodiment, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside f, rebaudioside ig, rebaudioside ih, rebaudioside li, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside 1m, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, 2 q, rebaudioside 2r, rebaudioside 2s and/or SvG7 can be rebaudioside 2p, rebaudioside used in a sweetener composition comprising a compound selected from the group consisting of dulcoside A, dulcoside B, dulcoside C, dulcoside D, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B2, rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D8, rebaudioside E5, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside H, rebaudioside H2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside 0, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Ti, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, NSF-02, Mogroside V, siratose, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.
Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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 advantage, salts thereof, and combinations thereof.
Moreover, highly purified target steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If; rebaudioside ig, rebaudioside ih, rebaudioside li, rebaudioside lj, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside lj, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 may also be combined with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside f, rebaudioside ig, rebaudioside ih, rebaudioside li, rebaudioside ij, rebaudioside ik, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside it, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 can be mixed with umami tasting and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and combinations thereof.
Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside ie, rebaudioside f rebaudioside ig, rebaudioside h, rebaudioside Ii, rebaudioside ij, rebaudioside ik, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside io, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2, rebaudioside 2, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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.
Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside i, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 may be combined with polyols or sugar alcohols. The term "polyol" refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.
Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside b, rebaudioside ic, rebaudioside Id, rebaudioside 1e, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside ij, rebaudioside ik, rebaudioside i1, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 may be combined with reduced calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L-arabinose and combinationsthereof.
Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside ia, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside li, rebaudioside ij, rebaudioside ik, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside io, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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 2O)n, wherein n is 3 30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives", "substituted carbohydrate", and "modified carbohydrates" are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.
Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.
Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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 W02013/096420, the contents of which is hereby incorporated by reference.
Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside h, rebaudioside ii, rebaudioside lj, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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. In addition, highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside ij, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside ip, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 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.
Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside ik, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside ip, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 obtained according to this invention may be applied as a foaming suppressor to produce zero calorie, reduced calorie or diabetic beverages and food products.
Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside f, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside ij, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside ip, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.
Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside f, rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, 2 q, rebaudioside 2r, rebaudioside 2s and/or SvG7 may be rebaudioside 2p, rebaudioside used as a flavor modifier or flavor with modifying properties include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.
During the manufacturing of products such as foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, and chewing gum, the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.
Moreover, the highly purified target steviol glycoside(s) steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside ij, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 obtained in this invention may be used in dry or liquid forms.
The highly purified target steviol glycoside can be added before or after heat treatment of food products. The amount of the highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside ie, rebaudioside If; rebaudioside ig, rebaudioside ih, rebaudioside ii, rebaudioside Ij, rebaudioside ik, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 depends on the purpose of usage. As discussed above, it can be added alone or in combination with other compounds.
The present invention is also directed to sweetness enhancement in beverages using steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside ie, rebaudioside If; rebaudioside ig, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside i1, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 as a sweetness enhancer, wherein steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside ie, rebaudioside If; rebaudioside ig, rebaudioside ih, rebaudioside ii, rebaudioside lj, rebaudioside 1k, rebaudioside il, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 is present in a concentration at or below their respective sweetness recognition thresholds.
As used herein, the term "sweetness enhancer" refers to a compound capable of enhancing or intensifying the perception of sweet taste in a composition, such as a beverage. The term "sweetness enhancer" is synonymous with the terms "sweet taste potentiator," "sweetness potentiator," "sweetness amplifier," and "sweetness intensifier."
The term "sweetness recognition threshold concentration," as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE). Generally, the sweetness enhancers may enhance or potentiate the sweet taste of sweeteners without providing any noticeable sweet taste by themselves when present at or below the sweetness recognition threshold concentration of a given sweetness enhancer; however, the sweetness enhancers may themselves provide sweet taste at concentrations above their sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific for a particular enhancer and can vary based on the beverage matrix. The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.
In some embodiments, sweetener is present in the beverage in an amount from about 0.0001% to about 12% by weight, such as, for example, 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, about 11.5% by weight or about 12.0% by weight.
In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.0001% by weight to about 10% by weight, such as for example, 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 0.5% 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 about 3% by weight to about 4% by weight, from about 4% by weight to about 5% by weight, from about 5% by weight to about 6% by weight, from about 6% by weight to about 7% by weight, from about 7% by weight to about 8% by weight, from about 8% by weight to about 9% by weight, or from about 9% by weight to about 10% by weight. In a particular embodiment, 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 and combinations thereof.
It is contemplated that a sweetener can be used alone, or in combination with other sweeteners.
In one embodiment, the rare sugar is D-allose. In a more particular embodiment, D-allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In another embodiment, the rare sugar is D-psicose. In a more particular embodiment, D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In still another embodiment, the rare sugar is D-ribose. In a more particular embodiment, D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-tagatose. In a more particular embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In a further embodiment, the rare sugar is L-glucose. In a more particular embodiment, L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In one embodiment, the rare sugar is L-fucose. In a more particular embodiment, L-fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In another embodiment, the rare sugar is L-arabinose. In a more particular embodiment, L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-turanose. In a more particular embodiment, D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-leucrose. In a more particular embodiment, D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.
The addition of the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer. Moreover, sweetness can be increased by an amount more than the detectable sweetness of a solution containing the same concentration of the at least one sweetness enhancer in the absence of any sweetener.
Accordingly, the present invention also provides a method for enhancing the sweetness of a beverage comprising a sweetener comprising providing a beverage comprising a sweetener and adding a sweetness enhancer selected from steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside ia, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If rebaudioside ig, rebaudioside ih, rebaudioside ii, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 or a combination thereof, wherein steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside ]a, rebaudioside 1b, rebaudioside ic, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside 1g, rebaudioside ih, rebaudioside Ii, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside im, rebaudioside In, rebaudioside lo, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2, rebaudioside 2k, 2p, rebaudioside 2, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 are present in a concentration at or below their sweetness recognition thresholds.
Addition of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside a, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside 1e, rebaudioside If, rebaudioside 1g, rebaudioside ih, rebaudioside li, rebaudioside Ij, rebaudioside ik, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f; rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 in a concentration at or below the sweetness recognition threshold to a beverage containing a sweetener may increase the detected sucrose equivalence from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.
The following examples illustrate preferred embodiments of the invention for the preparation of highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside 1b, rebaudioside ic, rebaudioside id, rebaudioside le, rebaudioside If, rebaudioside ig, rebaudioside ih, rebaudioside li, rebaudioside 1j, rebaudioside 1k, rebaudioside ll, rebaudioside im, rebaudioside In, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside ir, rebaudioside Is, rebaudioside It, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are only illustrative.
EXAMPLE1 Protein sequences of engineered enzymes used in the biocatalytic process SEQID1: >SuSyAt, variant PM-54-2-E05 (engineered sucrose synthase; source of WT gene: Arabidopsisthaliana) MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKI QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL DMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCG ERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVEL SKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDI YWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHT AFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEI EELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL
SEQ ID 2: >UGTSL2 variant 0234 (engineered glucosyltransferase; source of WT gene: Solanum lycopersicum) MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN FSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSF RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENS TVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP EGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVP IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGE TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK
SEQID3: >UGT76G1 variant 0042 (engineered glucosyltransferase; source of WT gene: Stevia rebaudiana) MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSC LITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQAS GFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAP SFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLV DSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWN STLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEG EYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL
EXAMPLE2 Expression and formulation of SuSyAt variant of SEQ ID 1
The gene coding for the SuSyAt variant of SEQ ID 1 (EXAMPLE 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of Ecoli 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 37C. 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 (OD6oo)) 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, 4C). 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. For enzymatic active preparations of SuSyAt, activity in Units is defined as follows: 1 mU of SuSyAt 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, 3mM MgCl2, and 15 mM uridine diphosphate (UDP).
EXAMPLE3 Expression and formulation of UGTS12 variant of SEQ ID 2
The gene coding for the UGTS12 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 cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/l) at 37C. 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 (OD6oo)) 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 tm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation. For enzymatic active preparations of UGTS12, activity in Units is defined as follows: 1 mU of 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 to, 500 mM sucrose, 3mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSyAt.
EXAMPLE4 Expression and formulation of UGT76G1 variant of SEQ ID 3
The gene coding for the UGT76G1 variant of SEQ ID 3 (EXAMPLE 1) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, 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 (OD6 0 0 )) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgC2, 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 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation. For enzymatic active preparations of UGT76G1, activity in Units is defined as follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D) into rebaudioside M (Reb M)in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb D at to, 500 mM sucrose, 3mM MgC 2 , 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSyAt.
EXAMPLE5 Synthesis of SvG7 in a one-pot reaction, adding UGTS12, SuSyAt and UGT76G1 at the same time.
Various SvG7 molecules were synthesized directly from stevioside (see Fig. 1la) in a one-pot reaction, utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4): UGTS12 (variant of SEQ ID 2), SuSyAt (variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 348 U/L UGTS2, 1341 U/L SuSyAt, 10 U/L UGT76G1, 47 mM stevioside, 0.32 mM uridine diphosphate (UDP), 0.99 M sucrose, 3.9 mM MgCl 2 and potassium phosphate buffer (pH 6.6). First, 206 mL of distilled water were mixed with 0.24 g MgCl 2 .6H2 0, 102 g sucrose, 9.8 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 15 g stevioside. The final volume of the reaction mixture was adjusted to 300 mL. After dissolving the components, the temperature was adjusted to 45 °C and UGTS2, SuSyAt, UGT76G1 and 39 mg UDP were added. The reaction mixture was incubated at 45°C shaker for 24 hrs. Additional 39 mg UDP was added at 12 hours, 24 hours, and 36 hours. The content of reb 2a, reb 2m and various SvG7 at the end of the reaction (48 hours) was analyzed by HPLC.
EXAMPLE6 HPLC Analysis
For analysis, biotransformation samples were inactivated by adjusting the reaction mixture to pH5.5 using 17% H 3PO4 and then boiled for 10 minutes. Resulting samples were filtered, the filtrates were diluted 10 times and used as samples for HPLC analysis. HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a pump, a column thermostat, an auto sampler, a UV detector capable of background correction and a data acquisition system. Analytes were separated using Agilent Poroshell 120 SB- C18, 4.6 mm 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 °C. The injection volume was 5 pL. Rebaudioside species were detected by UV at 210 nm. Table 3 shows for each time point the conversion of stevioside into identified rebaudioside species (area percentage). The chromatograms of the starting material stevioside and the reaction mixture at 48 hours are shown in Fig. la and Fig. lb respectively. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.
Table 3 Biotransformation of stevioside to reb 2a (rt 6.459), reb 2m (rt 5.089) and various SvG7
% conversion from stevioside Peak reaction time 0 reaction time 48 hr hr rt 2.651 0 0.83 rt2.874 0 6.79 rt 3.275 0 0.15 rt 3.570 0 0.05 rt 3.798 0 0.18 rt 4.279 0 0.08 rt 4.477 0 0.05 rt 4.798 0 0.04 rt 5.089 0 0.68 rt 5.350 0 0.86 rt 5.758 0 0.28 rt 5.896 0 1.06 rt 6.261 0 0.22 rt 6.459 0 1.37 rt 6.842 0 0.57 rt 7.952 0 0.5 rt 8.775 0 1.07 reb AM 0 65.7 rt 10.346 0 0.83 rt 11.217 0 0.54 rt 12.425 0 0.2 reb M 0 14.97 rt 15.174 0 0.4 reb A 12.97 0 stevioside 82.83 0 reb B 0.38 1.47 steviolbioside 3.82 1.11
EXAMPLE7 Purification of rebaudiosides 2a, 2m and various SvG7 300 mL of the reaction mixture of EXAMPLE 5, (after 48 hrs), was inactivated by adjusting the pH to pH 5.5 with H3 PO4 and then boiled for 10 minutes and filtered. The filtrate was loaded into a column containing 500 mL YWD03 (Cangzhou Yuanwei, China) resin pre-equilibrated with water. The resin was washed with 2.5 L water and the water effluent from this step was discarded. The steviol glycosides were eluted from the YWD03 resin column by elution with 2.5 L 70 % v/v ethanol/water. The effluent from this step was collected and dried under vacuum at 60 0C to yield 20g of dried solid product. This sample was dissolved in water and subjected to further fractionation and separation by HPLC, using the conditions listed in Table 4 below. HPLC fractions that corresponded to individual compounds from multiple runs were combined according to retention time. The fractions were freeze-dried.
Table 4 Conditions for HPLC Column Agilent Prodigy 3u ODS(3) 1OA, 4.6mm x 250mm, 3 micron Temperature 40 °C
Mobile Phase Isocratic - Water 77% Acetonitrile 23% Flow rate 0.5 mL/min Injection 10 pL Stop time 45 mins Autosampler Ambient temperature
Detection UV at 210 nm
The purity of obtained fractions was evaluated by analytical HPLC method described in EXAMPLE 6. The chromatogram of purified rebaudioside 2a is shown in Fig. 1Ic. The chromatogram of purified rebaudioside 2m is shown in Fig. lId.
EXAMPLE8 Structure elucidation of rebaudioside 2a NMR experiments were performed on a Bruker 500 MHz spectrometer, with the sample dissolved in pyridine-d5. Along with signals from the sample, signals from pyridine-d5 at 8c 123.5, 135.5, 149.9 ppm and 8H 7.19, 7.55, 8.71 ppm were observed. 1H-NMR spectrum of rebaudioside 2a recorded in pyridine-ds confirmed the excellent quality of the sample (see Fig. 12a). HSQC (see Fig. 12b) shows the presence of an exo methylene group in the sugar region with a long-range coupling to C-15, observable in the H,H-COSY (Fig. 12c). Other deep-fielded signals of the quaternary carbons (C-13, C-16 and C-19) are detected by the HMBC (Fig. 12d). Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure:
11 12 13 OR2 20 -:16 1 1 5 9 14: 1 2 Z%~15 1 8
R190 H 6 RO 0 Correlation of HSQC and HMBC shows the presence seven anomeric signals, marked with li, lii, liii, liv, lv, vi andlvii. The coupling constant of the anomeric protons of about 8 Hz, the broad signals of their sugar linkage and the NOE-correlations of
the anomeric protons allow the identification of these seven sugars as p-D glucopyranosides. Combined data from HSQC and HMBC reveal the sugar-sugar linkages and sugar
aglycone linkages. The assignment of the sugar sequence was confirmed by using the combination of HSQC-TOCSY (Fig. 12e) and NOESY (Fig. 12f). Altogether, results from NMR experiments above were used to assign the chemical shifts of the protons and carbons of the structure of rebaudioside 2a (see Table 5).
Table 5 Chemical shifts of rebaudioside 2a Position 8 c [ppm] | S [ppm] J [Hz]/ (INT) HMBC (H-> C) Aglycone moiety 40.1 t 0.67 m 1 1.62 m 1.36 m 2 19.5 t 2.10 m 37.1 t 1.05 m 3 2.77 m 4 44.5 s 5 57.1 d 0.94 m 21.8 t 1.92 m 6 2.13 m 7 40.9 t 1.36 m 8 42.1 s 9 53.4 d 0.82 m 10 39.1 s 11 20.4 t 1.57 m
12 37.4 t 1.90 m _______2.13 m 13 86.5 s 43.4 t 1.77 d 11.1 14 2.47 d 11.1 15 47.2 t 1.99 d 16.0 7,8,9,14 16 153.7 s 17 104.4 t 4.99 brs 13,15,16 5.67 br s 18 28.5 q 1.40 s (3H) 3,4,5,19 19 175.7 s 20 16.0 q 1.04 s (3H) 1,5,9, 10
Table 5 (continued) Chemical shifts of rebaudioside 2a HMBC NOE Position Sc[ppm] SH [ppm] J[Hz]!(Int) (H-> C) (H-> H) Sugar moiety Sugar I: f-D-Glucopyranoside 1' 97.0 d 5.09 d 7.7 13 21 81.9 d 4.16 m 3i 75.8 d 4.37 m 4i 81.9 d 3.85 m 51 75.6 d 3.97 m 4.20 m 6' 61.8 t 4.80 m ______ _ _ _ _ _ _
Sugar II: P-D-Glucopyranoside 1 105.0 d 5.28 d 8.0 2i 2i 2i 76.3 d 4.09 m 3" 78.0 d 4.29 m 4" 70.8 d 4.24 m 5" 78.1 d 3.91 m 61.4 t 4.39 m 6ii 4.50 m Sugar III: 3-D-Glucopyranoside liii 102.4 d 4.98 d 8.1 4i 4i 2ii 84.8 d 4.05 m 3111 77.9 d 4.22 m 41i 70.9 d 4.12 m 5M 77.6 d 3.92 m 62.5 t 4.31 m 6iii 4.53 m Sugar IV:/-D-Glucopyranoside liv 106.4 d 5.22 d 7.8 21i 2ii 2iv 76.1 d 4.12 m 3Wv 78.1 d 4.06 m 4iv 70.4 d 4.25 m 51V 78.3 d 3.81 m 61.6 t 4.24 m 6iv 4.50 m Sugar V: -D-Glucopyranoside lV 93.0 d 6.25 d 8.1 19 2" 76.8 d 4.47 m 3" 88.2 d 4.28 m 4v 69.1 d 4.12 m 5v 78.2 d 3.92 m 61.5 t 4.16 m 6v 4.33 m
Table 5 (continued) Chemical shifts of rebaudioside 2a HMBC NOE Position 8c [ppm] 8H [ppm] J [Hz]/ (Int) H -C H -H
Sugar moiety Sugar VI: -D-Glucopyranoside 1vi 103.5 d 5.77 d 7.8 2v 2V 2vi 75.4 d 4.00 m 3vi 77.6 d 4.27 m 4vi 71.1 d 4.27 m 5Vi 78.0 d 3.96 m 62.7 t 4.42 m 6Vi 4.58 m Sugar VII:/-D-Glucopyranoside PH 104.4 d 5.28 d 8.0 3 3 2vil 75.0 d 4.01 m 3Vii 77.2 d 4.17 m 4vii 71.5 d 4.10 m 5vii 77.9 d 4.00 m 61.8 t 4.42 m 6vil 4.52 m
Correlation of all NMR results indicates rebaudioside 2a with seven p-D-glucoses attached to steviol aglycone, as depicted with the following chemical structure: HO OH HO HO,, '0 H IV HO O O O OH OH
HO H 0O 0 ' ,O 111 OH
12 O O "OH 11 HO HO 20 16 H OH 2 1 8'0 HO,, HO' H 15 VIf V HO 0 . O 18 OH 0 0 0 VI *-,OH HO HO OH
LCMS (Fig. 12g and Fig. 12h) analysis of rebaudioside 2a showed a [M-H]- ion at m/z 1451.6, in good agreement with the expected molecular formulaof C 62 H1 00 0 3 8
(calculated for [C 62 H99 0 38 1- monoisotopic ion: 1451.6). The MS data confirms that rebaudioside 2a has a molecular formulaof C62HooO38. LCMS analysis was performed in the following conditions listed in Table 6. Table 6 Conditions for LCMS analysis Column Agilent Poroshell 120 SB-C18, 4.6mm x 150mm, 2.7 m Temperature 40 0 C Mobile Phase A: Mobile Phase Premix Solution - 25 % Acetonitrile: 75 %Formic Acid (0.1% in Water) B: Mobile Phase Premix Solution - 32 % Acetonitrile: 68 % Formic Acid (0.1% in Water) Gradient Time (min) A (%) B (%) 0 100 0 12.0 100 0 12.5 50 50 13.0 0 100 60.0 0 100 Flow rate 0.5 mL/min Injection 2 pL Run time 45 mins Post time 5 mins Autosampler temperature Ambient Detection MSD at Negative Scan mode MSD Setting Mode: ES-API, Negative Polarity Drying gas flow: 13.0 L/min Nebulizer Pressure : 30 psig Drying gas temperature : 270°C Fragmentor :50V Scan ranges :500 to 1500 of mass Sample Preparation 1 mg/ml (30% ACN in water)
EXAMPLE9 Structure elucidation of rebaudioside 2m NMR experiments were performed on a Bruker 500 MHz spectrometer, with the sample dissolved in pyridine-d5. Along with signals from the sample, signals from pyridine-d5 at 6 c 123.5, 135.5, 149.9 ppm and 6 H 7.19, 7.55, 8.71 ppm were observed. 1H-NMR spectrum of rebaudioside 2m recorded in pyridine-ds confirmed the excellent quality of the sample (see Fig. 13a). HSQC (see Fig. 13b) shows the presence of an exo methylene group in the sugar region with a long-range coupling to C-15, observable in the H,H-COSY (Fig. 13c). Other deep-fielded signals of the quaternary carbons (C-13, C-16 and C-19) are detected by the HMBC (Fig. 13d). Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure:
11 12 13 OR1
R201 37
0 Correlation of HSQC and HMBC shows the presence seven anomeric signals, marked withili, lii, liii, liv,lv, lviand lvii.The coupling constant ofthe anomeric protons of about 8Hz, the broad signals of their sugar linkage and the NOE-correlations of the anomeric protons allow the identification of these seven sugars as p3-D glucopyranosides. Combined data from HSQC and HMBC reveal the sugar-sugar linkages and sugar aglycone linkages. The assignment of the sugar sequence was confirmed by using the combination of HSQC-TOCSY(Fig.13e)andNOESY(Fig.13f). Altogether, results from NMR experiments above were used to assign the chemical shifts of the protons and carbons of the structure of rebaudioside 2m (see Table 7).
Table 7 Chemical shifts of rebaudioside 2m Position | c [ppm] | Hppm] J [Hz]/ (INT) HMBC (H ->C) Aglycone moiety 0.68 m 1 40.0 t 1.63 m 1.38 m 2 19.3 t 2.06 m
37.1 t 1.04 m 3 2.81 m 4 44.0 s 5 56.9 d 0.96 m 1.93 m 6 21.8 t 2.13 m 41.1 t 1.25 m 7 1.36 m 8 41.6 s 9 53.6 d 0.82 m 10 40.4 s 11 19.8 t 1.61 m
37.5 t 1.96 m 12 1.99 m 13 86.2 s 43.1 t 1.73 d 10.9 14 2.34 d 10.9
15 47.5 't 2.02 d 15.9 7,8,9, 14 16 153.4 s 17 104.3 t 4 brs 13, 15,16 18 28.6 q 1.42 s (3H) 3, 4, 5, 19 19 175.3 s 20 16.3 q 1.01 s (3H) 1, 5, 9, 10
Table 7 (continued) Chemical shifts of rebaudioside 2m
c [ppm] S[ppm) J [HZl (Int) HMN )BC NOE Position Sugar moiety Sugar I: §-D-Glucopyranoside 11 97.3 d 4.88 d 7.8 13 31, 51 2i 81.9 d 3.75 m 3' 75.3 d 4.18 m 4i 79.6 d 4.40 m 5' 74.0 d 3.50 m 61 67.9 t 4.55 m (2H) 5i, liv Sugar II: §-D-Glucoyranoside li 105.7 d 5.10 d 8.0 2i 2i, 3", 5 2i 74.6 d 4.02 m 13" 77.9 d 4.18 m 41 71.4 d 4.20 m 5" 78.1 d 3.87 m 4.19 m 6" 61.8 t 4.46 m Sugar III:/-D-Glucopyranoside luii 104.3 d 5.57 d 8.1 4i 4i, 3iii, 5iii 21" 75.2 d 4.03 i 3ii 77.1 d 4.35 n 4ii 71.4 d 4.23 n 5"' 76.8 d 4.19 m 63.3 t 4.34 m 6ii 4.56 m Sugar IV: -D-Glucopyranoside liv 104.9 d 5.37 d 8.0 61 6', 31v, 51v 21v 75.0 d 4.06 |m 3iv 78.3 d 4.29 |m 4 1v 71.1 d 4.27 m 51v 78.0 d 3.99 m 62.1 t 4.22 m 6iV 4.36 m Sugar V: -D-Glucopyranoside lV 92.8 d 6.27 d 8.1 19 3v, 5v 2" 77.0 d 4.47 m 3v 88.2 d 4.28 m 4v 69.0 d 4.10 n 5v 78.1 d 3.88 m 64 61.7 ti 54.36
Table 7 (continued) Chemical shifts of rebaudioside 2m HMBC NOE Position Sc[ppm] SH[ppm] J [Hz]/(Int) (H C) (H - H) Sugar moiety Sugar VI:/-D-Glucopyranoside lvi 103.2 d 5.78 d 7.8 2v 2v, 3v, 5 2vi 75.6 d 3.99 m 3Vi 78.2 d 4.25 m 4vi 72.7 d 4.10 m 5vi 78.0 d 3.93 m 62.6 t 4.29 m 6i t 4.40 m Sugar VII:f-D-Glucopyranoside lPil 104.4 d 5.30 d 8.0 3' 3v, 3Vii, 5vi1 2vii 75.3 d 4.00 m 3vii 78.1 d 4.12 m 4vii 71.2 d 4.12 m 5`if 78.1 d 3.97 m 62.0 t 4.26 m 6'11. 4.46 m
Correlation of all NMR results indicates rebaudioside 2m with seven p-D-glucoses attached to steviol aglycone, as depicted with the following chemical structure:
HO HcO
HO 11 0 OH O"H HO OH
110
HO HO 120 16 OH
HO, H0O,, 0 15 O VII V HO H
H H OH 0 VI .aO H HO ,
LCMS (Fig. 13g and Fig. 13h) analysis of rebaudioside 2m showed a[M-H]- ion at m/z 1451.6, in good agreement with the expected molecular formula ofC 6 2 H10038 (calculated for [C 62 H99 0 38 -monoisotopic ion: 1451.6). The MS data confirms that rebaudioside 2m has amolecular formula of C62 H 100 038. LCMS analysis was performed in the conditions listed in Table 6. Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the application is not intended tobe limited to the particular embodiments of the invention described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the invention, the compositions, processes, methods, and steps, presently existing or later tobe developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the invention.
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt SEQUENCE LISTING SEQUENCE LISTING
<110> PureCircle USA Inc. <110> PureCircle USA Inc. <120> HIGH‐PURITY STEVIOL GLYCOSIDES <120> HIGH-PURITY STEVIOL GLYCOSIDES
<130> 39227‐77WO <130> 39227-77WO
<160> 3 <160> 3
<170> PatentIn version 3.5 <170> PatentIn version 3.5
<210> 1 <210> 1 <211> 808 <211> 808 <212> PRT <212> PRT <213> Arabidopsis thaliana Arabidopsis thaliana 223 <400> 1 <400> 1
Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu 1 5 10 15 1 5 10 15
Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu 20 25 30 20 25 30
Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln 35 40 45 35 40 45
Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu 50 55 60 50 55 60
Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile 65 70 75 80 70 75 80
Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 85 90 95
Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu 100 105 110 100 105 110
Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val Page 1 Page 1
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt 115 120 125 115 120 125
Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 130 135 140
Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155 160 145 150 155 160
Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser 165 170 175 165 170 175
Leu Leu Pro Leu Leu Asp Phe Leu Arg Leu His Ser His Gln Gly Lys Leu Leu Pro Leu Leu Asp Phe Leu Arg Leu His Ser His Gln Gly Lys 180 185 190 180 185 190
Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His 195 200 205 195 200 205
Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr 210 215 220 210 215 220
Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg 225 230 235 240 225 230 235 240
Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu 245 250 255 245 250 255
Leu Asp Leu Leu Glu Ala Pro Asp Pro Ser Thr Leu Glu Thr Phe Leu Leu Asp Leu Leu Glu Ala Pro Asp Pro Ser Thr Leu Glu Thr Phe Leu 260 265 270 260 265 270
Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280 285 275 280 285
Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln 290 295 300 290 295 300
Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu Page 2 Page 2
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt 305 310 315 320 305 310 315 320
Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile 325 330 335 325 330 335
Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg 340 345 350 340 345 350
Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro 355 360 365 355 360 365
Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu 370 375 380 370 375 380
Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu 385 390 395 400 385 390 395 400
Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser 405 410 415 405 410 415
Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr 420 425 430 420 425 430
Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser 435 440 445 435 440 445
Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln 450 455 460 450 455 460
Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr 465 470 475 480 465 470 475 480
Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr 485 490 495 485 490 495
Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His Page 3 Page 3
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt 500 505 510 500 505 510
Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala 515 520 525 515 520 525
Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr 530 535 540 530 535 540
Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn 545 550 555 560 545 550 555 560
Asp Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe Asp Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe 565 570 575 565 570 575
Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu 580 585 590 580 585 590
Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Val Asn Leu Val Val Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Val Asn Leu Val Val 595 600 605 595 600 605
Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala 610 615 620 610 615 620
Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly 625 630 635 640 625 630 635 640
Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu 645 650 655 645 650 655
Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala 660 665 670 660 665 670
Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly 675 680 685 675 680 685
Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val Page 4 Page 4
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt 690 695 700 690 695 700
His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala 705 710 715 720 705 710 715 720
Ala Asp Leu Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser Ala Asp Leu Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser 725 730 735 725 730 735
His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys 740 745 750 740 745 750
Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val 755 760 765 755 760 765
Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu His Arg Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu His Arg 770 775 780 770 775 780
Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln 785 790 795 800 785 790 795 800
Ala Val Pro Leu Ala Gln Asp Asp Ala Val Pro Leu Ala Gln Asp Asp 805 805
<210> 2 <210> 2 <211> 442 <211> 442 <212> PRT <212> PRT <213> Solanum lycopersicum <213> Solanum lycopersicum
<400> 2 <400> 2
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly 1 5 10 15 1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly 20 25 30 20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile 35 40 45 35 40 45
Page 5 Page 5
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu 50 55 60 50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn 65 70 75 80 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met 85 90 95 85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu 100 105 110 100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu 115 120 125 115 120 125
Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe 130 135 140 130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe 145 150 155 160 145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile 165 170 175 165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys 180 185 190 180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile 195 200 205 195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro 210 215 220 210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile 225 230 235 240 225 230 235 240
Page 6 Page 6
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt
Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe 245 250 255 245 250 255
Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe 260 265 270 260 265 270
Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro 275 280 285 275 280 285
Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Glu Gly Phe Leu Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Glu Gly Phe Leu 290 295 300 290 295 300
Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln 305 310 315 320 305 310 315 320
Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys 325 330 335 325 330 335
Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile 340 345 350 340 345 350
Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val 355 360 365 355 360 365
Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile 370 375 380 370 375 380
His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu 385 390 395 400 385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu 405 410 415 405 410 415
Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile 420 425 430 420 425 430 Page 7 Page 7
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt
Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys 435 440 435 440
<210> 3 <210> 3 <211> 458 <211> 458 <212> PRT <212> PRT <213> Stevia rebaudiana <213> Stevia rebaudiana
<400> 3 <400> 3
Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 1 5 10 15
Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 20 25 30
Ala Asn Val Leu Tyr Ser Lys Gly Phe Ala Ile Thr Ile Leu His Thr Ala Asn Val Leu Tyr Ser Lys Gly Phe Ala Ile Thr Ile Leu His Thr 35 40 45 35 40 45
Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 50 55 60
Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 70 75 80
Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 85 90 95
Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 100 105 110
Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 115 120 125
Phe Ala Gln Asp Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu Phe Ala Gln Asp Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 130 135 140
Page 8 Page 8
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.tx
Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 145 150 155 160
Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 165 170 175
Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 180 185 190
Ala Tyr Ser Asn Trp Gln Ile Gly Lys Glu Ile Leu Gly Lys Met Ile Ala Tyr Ser Asn Trp Gln Ile Gly Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 195 200 205
Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 210 215 220
Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 225 230 235 240
Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 245 250 255
Leu Leu Asp His Asp Arg Thr Val Phe Glu Trp Leu Asp Gln Gln Ala Leu Leu Asp His Asp Arg Thr Val Phe Glu Trp Leu Asp Gln Gln Ala 260 265 270 260 265 270
Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 275 280 285
Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Gly Gln Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Gly Gln 290 295 300 290 295 300
Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 305 310 315 320
Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Lys Ile Val Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Lys Ile Val 325 330 335 325 330 335
Page 9 Page 9
PC_77WO_Final_ST25.txt PC_77WO_Final_ST25.txt
Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Pro Ala Ile Gly Ala Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Pro Ala Ile Gly Ala 340 345 350 340 345 350
Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 355 360 365
Gly Val Pro Met Ile Phe Ser Ser Phe Gly Gly Asp Gln Pro Leu Asn Gly Val Pro Met Ile Phe Ser Ser Phe Gly Gly Asp Gln Pro Leu Asn 370 375 380 370 375 380
Ala Arg Tyr Met Ser Asp Val Leu Arg Val Gly Val Tyr Leu Glu Asn Ala Arg Tyr Met Ser Asp Val Leu Arg Val Gly Val Tyr Leu Glu Asn 385 390 395 400 385 390 395 400
Gly Trp Glu Arg Gly Glu Val Val Asn Ala Ile Arg Arg Val Met Val Gly Trp Glu Arg Gly Glu Val Val Asn Ala Ile Arg Arg Val Met Val 405 410 415 405 410 415
Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 420 425 430
Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 435 440 445
Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 450 455
Page 10 Page 10
Claims (16)
1. Steviol glycosides with the following formulae:
O R2
H R1 OH
wherein RI and R2 sugar chains are defined in the following table;
No. R1 R2
HO HO .HO HO OH 0"0HO,, HO 0 HO% HHH
HO - 0 OH aH OH HO H O0
HOO
HO OH H,OH H IH - OH
HO HO HO Hg
IIHO~r 0 0 H 0 HOH H H 0H
HO -OH HO 0 H O 9 OH
HO OH HO 0 *OH
0 H
No. Ri R2
OH OH HO HO HO
IV HO, HO H" HO OH HO 0 * HO OHO0 HO 0y HOHO 0 OH
HO OH
H
HO H HO HO
- HO S ll O 0HOHO 0
OH 0 HO L
HO 0 OHH
H
No. RI R2
HO HO
Ho,, H, HO HO OH L HO: :OH VII HO *HO 0q OH 6H 0 0
QH
HO HO
H O OH 0H.-O 0 0HO
HO 0, O OHj
HO 0 HO 0 04* H o<0H HO O
HO HO0 HO OH
HO,,. HO,, H% HO I H
HO'O
Ix HO 0H 0 HO 0 , .,OH
HO 'OH
No. Rl R2
HO HO HO O HOH H HO HO, X HO 0 OH HO0 0 0, 0 0 HO 0 ,H HO
HO" "OH d OH HO OHH
HOHO HO O
H HO 00 , 01O, O HO xiHO-., 0 0) 0 OH
OH 0 H"0 OH HO O0H
Hd 0 HH
This page has intentionally been left blank
No. Rl R2
HO HOHO HOk
H- O, . H0,, HO 0 xv ' 0' 0HO O HO . H0 HO, ~0 H H
H H
5HOH HO 1. HO HO 0H
HO OHH
HO,,~ HOO
O. O XVIHO 0OX HO 0 OH HO 0 H xv HO OHH
O HO HO HO0 HqH
HO 0 0' HH
HO H HO
HOt Hl H H$H HO OH
No. Rl R2 HO HO
HO O O HO, HOHO
xviiiHO OH 0 0 HO 0) 0 0 OH
HO 0, OH
HO' O OH
O HH
HOH
HO HO OH
H - HO HHO0 HO,, O OH
HO OHo HO 0 OH" XIX O H 0
0 ,O OH H O H
HJO HO O Hgk HO HOX
HO HHO 0 H
HOI HO0
HO2 OH 0
OH HO
No. Rl R2
HO HO
H . 0 O 0 OH 0 OHH OH
H 01 "OH
HOH OH
HO HO
HO, OH xxii H D.0OH HO- 0 OH -OH
- Hd OH 0OH
HO HO
HO 0 O
xxii HOz HO 0, OH
HO OOH HO OH H OH
No. R1 R2
HO HO
HO HO XXIV HO4 O t HOA HO OH O OH H 0 HOH OH 0 OH
H HO HO 0H0 H H HH
HO OH OH
HO HO
HO
HO O -%O HO
xxv OH 0 HO 0 OH OH
0~ OH
-OH H
HO H HOH
0 HO HO 0 OH HO H5 0O O 0H OHH HO 0O
HOHO OHO O HO OH
OH
No. Rl R2
HO HO HHO
HO 0 HO 0 O 0H
HOZ H 0't 0 HO4 HO/- O-OO OH
HO H
H 0HO
xxviii HO0 H 0 O OH HOY'` "OHOHH OH~~~ HOH-.. 0,H
HO OH - o
HO
HO,,L HO
H HO - 0 HO
OH HO 0 OH HO,9 H0 OH 0 XXIX HO0tH 0 0 OH
OH 0* 0 <OH
HO O'H
No. Rl R2
HO
HO HO HO
HO," HC, HO HO HO 0 OH
xxx a OH
H H 0O'1~O 5OH HO OH"I
H HOH
H 0 OHH
OH H XXXIOHHO OH HO 0,
O H
HQxHO 0
H XXXII~ H 0HO0
0 OH
OH"O HO 0,HO
HO OH
No. Rl R2
HO HO HO HO
xxxiii HO,,, HO, H 0 OH OH 4. 0 . 0H , O O
5H H
HQ < -OH OH H 0H0OH
OH OH HO H
HOVH
XXXIV HO oHO 0 OH OH 0 HO,. O HO 0, OH
HO o 0 H ",H OH 0
HO OH
No. RI R2
HO HO HO 1,0 HO
XXX 0," xxv0 0HO HO 0 0, 02OH 'H
OH 0
4 HO/K OH H O
OMHOH HO OH
, HO
HO HOH
HO 0 OH
OOH
0CH
OH r-I OH HO
HO
HO I HO 0 OH
OOH
0HH HOA -
OHH
HO OH
No. R1 R2 HO H HO
HO 0 HO 0 OH
HO HO XXXVOIOH HO HO H H 5H H
HO H HOH HOO
HO HO HO_ 0
H~ ~O HO.HC, HO0
OHi H OH H OH
HOH OH 6 HO 0 0 H
HOH HO OH HO
.IOH H
XL OH HO 0,OH
OH 0 a-H 0
HO
HO OH
ofH Htvoseilo Osie seiloodestvlbsdseOboieD
2. A method for producing at least one steviol glycoside of claim 1, comprising the steps of:
a. providing astarting composition comprising an organic compound with at least one carbon atom, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside
E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, other steviol glycosides, polyols, carbohydrates, and combinations thereof; b. providing an enzyme preparation or microorganism containing at least one enzyme selected from steviol biosynthesis enzymes, NDP-glucosyltransferases and NDP glucose recycling enzymes; c. contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of claim 1.
3. A method for producing at least one steviol glycoside of claim 1, comprising the steps of:
a. providing a starting composition comprising an organic compound with at least one carbon atom, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, other steviol glycosides, polyols, carbohydrates, and combinations thereof; b. providing a biocatalyst comprising at least one enzyme selected from steviol biosynthesis enzymes, NDP-glucosyltransferases and NDP-glucose recycling enzymes; c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of claim 1.
4. The method of claim 2 or 3 further comprising the step of: d. separating at least one steviol glycoside of claim 1 from the medium to provide a highly purified composition of at least one steviol glycoside of claim 1.
5. The method of claim 2, wherein the microorganism is selected from the group consisting of colii, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., and Yarrowia sp.
6. The method of claim 3, wherein the biocatalyst is an enzyme, or a cell comprising one or more enzyme, capable of converting the starting composition to at least one steviol glycoside of claim 1.
7. The method of claim 2, wherein 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-erythritol 2,4 cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, UGT74G1, UGT85C2, UGT91D2, EUGT11, UGTS12, UGT76G1, UGlyT91C1 or mutant variant thereof having >85% amino-acid sequence identity, >86% amino-acid sequence identity, >87% amino-acid sequence identity, >88% amino-acid sequence identity, >89% amino-acid sequence identity, >90% amino acid sequence identity, >91% amino-acid sequence identity, >92% amino-acid sequence identity, >93% amino-acid sequence identity, >94% amino-acid sequence identity, >95% amino-acid sequence identity, >96% amino-acid sequence identity, >97% amino-acid sequence identity, >98% amino-acid sequence identity, >99% amino-acid sequence identity.
8. The method of claim 4, wherein the content of at least one steviol glycoside in the highly purified composition of at least one steviol glycoside of claim 1 is greater than about 95% by weight on a dry basis.
9. A consumable product comprising at least one steviol glycoside of claim 1, wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition.
10. The consumable product of claim 9, wherein the product is selected from the group consisting of beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco and medicinal products.
11. The consumable product of claim 9, further comprising 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, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.
12. The consumable product of claim 9, further comprising 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, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.
13. The consumable product of claim 9, further comprising a compound selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, steviolbioside E, rubusoside, dulcoside A, dulcoside B, dulcoside C, dulcoside D, stevioside, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B, rebaudioside B2, rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D7, rebaudioside D8, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside E4, rebaudioside E5, rebaudioside E6, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside H2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside I, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside 0, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Ti, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zi, rebaudioside Z2, rebaudioside AM, SvG7, Mogroside V, siratose, Luo Han Guo, allulose, D-allose, D-tagatose, erythritol, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside, sucralose, potassium acesulfame, aspartame, alitame, saccharin, cyclamate, neotame, dulcin, suosan advantame, gymnemic acid, hodulcin, ziziphin, lactisole, glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, 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, sugar alcohols, L-sugars, L-sorbose, L arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, talose, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannosamine, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, soybean oligosaccharides, D-psicose, D-ribose, L-glucose, L-fucose, D turanose,D-leucrose.
14. A method for enhancing the sweetness of a beverage or food product, comprising a sweetener comprising: a. providing a beverage or food product comprising a sweetener; and b. adding a sweetness enhancer comprising at least one steviol glycoside of claim 1, wherein at least one steviol glycoside of claim 1 is present in a concentration at or below the sweetness recognition threshold.
15. A method for modifying the flavor of a beverage or food product, comprising a. providing a beverage or food product, and b. adding a composition comprising at least one steviol glycoside of claim 1.
16. A method for suppressing foaming of a beverage or food product, comprising a. providing a beverage or a food product, and b. adding a foam suppressor comprising at least one steviol glycoside of claim 1.
OH
OH
OH "OH OH
Rebaudioside 1a
HO
HO
HOM HO H H OH -
HO HO111 O OH
OH HO
HO
HO HO
OH HO FIG. 1a
HO HO111
HO
OH
OH
OH OH OH
O Rebaudioside 1b
HO
HO O
HOW HO HO
H H O OH
OH HO HO111. OH HO
HO HO ,
HO
OH - HO
FIG. 1b HO HOIII,
HO
FIG. 1c
HO HOW
HO O
HO OH HOW HO OH HO O OH HO O "xy OH
HO HO OH HO111. HO H
H HO OH Rebaudioside 1c OH HO HO OH
OH
OH
OH OH OH OH Rebaudioside 1d
OH O
HO) HO HO O HOW HO
HO H H
OH
O OH
HO
HO HO
O OH HO
HO HO111
HO FIG. 1d
H
H I H
I I I
H
I
OH OH OH OH
OH
OH OH OH Rebaudioside
HO
HO
HO
HO H H
"OH
OH
HO HO HO FIG. 1f OH HO HO HOM HO
OH
OH
Rebaudioside 1g
OH OH OH
HO
HO O HO!! HO OH OH HO H OH H H O O O O OH
HO
HO HO
OH HO
HO HOII. FIG. 1g
HO
OH
OH
OH
OH
OH "OH OH
Rebaudioside 1i
HO
HO O
HOW HO
HO H H
O OH
OH
HO
HO HO
OH -
OH HO HO111
FIG. li HO O HOI.. HO
HO
OH
OH OH OH OH
HO,
Rebaudioside 1j
HO
HO HO
HO H H
OH OH
HO HO , O HO
HO HO HOIII 'OH HO
OH FIG. 1j
HO HO
OH OH
OH OH OH
HO
Rebaudioside 1k
HO O
HOW HO
HO H H
O OH
OH
HO
HO O HO
OH HO
HO HO111
O OH
FIG. 1k O HO
HOI HO
Applications Claiming Priority (13)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201862644065P | 2018-03-16 | 2018-03-16 | |
| US62/644,065 | 2018-03-16 | ||
| US201862644407P | 2018-03-17 | 2018-03-17 | |
| US62/644,407 | 2018-03-17 | ||
| AUPCT/US2018/026920 | 2018-04-10 | ||
| PCT/US2018/026920 WO2019177634A1 (en) | 2018-03-16 | 2018-04-10 | High-purity steviol glycosides |
| US201862682461P | 2018-06-08 | 2018-06-08 | |
| US62/682,461 | 2018-06-08 | ||
| US201862771937P | 2018-11-27 | 2018-11-27 | |
| US62/771,937 | 2018-11-27 | ||
| US201962806646P | 2019-02-15 | 2019-02-15 | |
| US62/806,646 | 2019-02-15 | ||
| PCT/US2019/022456 WO2019178471A1 (en) | 2018-03-16 | 2019-03-15 | High-purity steviol glycosides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2019236227A1 AU2019236227A1 (en) | 2020-10-08 |
| AU2019236227B2 true AU2019236227B2 (en) | 2024-06-06 |
Family
ID=72663825
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2019236227A Active AU2019236227B2 (en) | 2018-03-16 | 2019-03-15 | High-purity steviol glycosides |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP3764815A4 (en) |
| JP (2) | JP2021518369A (en) |
| KR (1) | KR20200133251A (en) |
| CN (1) | CN112512337A (en) |
| AU (1) | AU2019236227B2 (en) |
| CA (1) | CA3094203A1 (en) |
| MX (1) | MX2020009626A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20230157339A1 (en) * | 2021-11-12 | 2023-05-25 | Epc Natural Products Co., Ltd. | Sweetener and flavor composition comprising glycosylated high purity steviol glycosides |
| MX2020009626A (en) * | 2018-03-16 | 2021-05-27 | Purecircle Usa Inc | High-purity steviol glycosides. |
| CA3130257A1 (en) | 2019-02-15 | 2020-08-20 | Purecircle Usa Inc. | High-purity steviol glycosides |
| CN115478060B (en) * | 2021-06-16 | 2023-11-28 | 弈柯莱生物科技(集团)股份有限公司 | A kind of glycosyltransferase and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017218036A1 (en) * | 2016-06-17 | 2017-12-21 | Cargill, Incorporated | Steviol glycoside compositions for oral ingestion or use |
| WO2017218888A1 (en) * | 2016-06-17 | 2017-12-21 | Cargill, Incorporated | Steviol glycoside compositions for oral ingestion or use |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6855376B2 (en) * | 2014-12-17 | 2021-04-07 | カーギル インコーポレイテッド | Steviol glycoside compounds, compositions for oral ingestion or use, and methods for enhancing steviol glycoside solubility. |
| CA2973674A1 (en) * | 2015-01-30 | 2016-08-04 | Evolva Sa | Production of steviol glycosides in recombinant hosts |
| US11344051B2 (en) * | 2015-04-03 | 2022-05-31 | Dsm Ip Assets B.V. | Steviol glycosides |
| EP4159050A1 (en) * | 2015-11-30 | 2023-04-05 | Cargill, Incorporated | Steviol glycoside compositions for oral ingestion or use |
| CN105838759A (en) * | 2016-03-28 | 2016-08-10 | 南京诺云生物科技有限公司 | Preparation method of stevia sugar derivative |
| WO2018213290A1 (en) * | 2017-05-15 | 2018-11-22 | Purecircle Usa Inc. | High-purity steviol glycosides |
| MX2020009626A (en) * | 2018-03-16 | 2021-05-27 | Purecircle Usa Inc | High-purity steviol glycosides. |
| CA3120867A1 (en) * | 2018-11-27 | 2020-06-04 | Purecircle Usa Inc. | High-purity steviol glycosides |
-
2019
- 2019-03-15 MX MX2020009626A patent/MX2020009626A/en unknown
- 2019-03-15 EP EP19767008.6A patent/EP3764815A4/en active Pending
- 2019-03-15 CN CN201980032858.7A patent/CN112512337A/en active Pending
- 2019-03-15 AU AU2019236227A patent/AU2019236227B2/en active Active
- 2019-03-15 KR KR1020207029495A patent/KR20200133251A/en active Pending
- 2019-03-15 CA CA3094203A patent/CA3094203A1/en active Pending
- 2019-03-15 JP JP2020549747A patent/JP2021518369A/en active Pending
-
2024
- 2024-05-09 JP JP2024076669A patent/JP2024109659A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017218036A1 (en) * | 2016-06-17 | 2017-12-21 | Cargill, Incorporated | Steviol glycoside compositions for oral ingestion or use |
| WO2017218888A1 (en) * | 2016-06-17 | 2017-12-21 | Cargill, Incorporated | Steviol glycoside compositions for oral ingestion or use |
Also Published As
| Publication number | Publication date |
|---|---|
| BR112020018931A2 (en) | 2021-01-05 |
| CA3094203A1 (en) | 2019-09-19 |
| JP2024109659A (en) | 2024-08-14 |
| KR20200133251A (en) | 2020-11-26 |
| MX2020009626A (en) | 2021-05-27 |
| EP3764815A1 (en) | 2021-01-20 |
| AU2019236227A1 (en) | 2020-10-08 |
| EP3764815A4 (en) | 2022-01-26 |
| JP2021518369A (en) | 2021-08-02 |
| CN112512337A (en) | 2021-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11542537B2 (en) | High-purity steviol glycosides | |
| US20250263768A1 (en) | High-purity steviol glycosides | |
| AU2019282411B2 (en) | High-purity steviol glycosides | |
| JP7634477B2 (en) | High Purity Steviol Glycosides | |
| US12378590B2 (en) | High-purity steviol glycosides | |
| CA3094205A1 (en) | High-purity steviol glycosides | |
| AU2019236227B2 (en) | High-purity steviol glycosides | |
| WO2019178471A1 (en) | High-purity steviol glycosides | |
| US20210002318A1 (en) | High-purity steviol glycosides | |
| RU2848318C2 (en) | High purity steviol glycosides | |
| US20240360489A1 (en) | High-purity steviol glycosides | |
| WO2025048633A2 (en) | High-purity steviol glycosides | |
| BR112020018931B1 (en) | STEVIOL GLYCOSIDES, METHODS FOR PRODUCING AT LEAST ONE STEVIOL GLYCOSIDE AND CONSUMPABLE PRODUCT |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) |