AU2019236279B2

AU2019236279B2 - High-purity steviol glycosides

Info

Publication number: AU2019236279B2
Application number: AU2019236279A
Authority: AU
Inventors: Mohamad AFZAAL BIN HASIM; Siew Yin CHOW; Avetik Markosyan; Khairul NIZAM BIN NAWI; Marcia Petit; Siddhartha Purkayastha; Saravanan A/l RAMANDACH
Original assignee: PureCircle USA Inc
Current assignee: PureCircle USA Inc
Priority date: 2018-03-16
Filing date: 2019-03-15
Publication date: 2023-11-23
Anticipated expiration: 2039-03-15
Also published as: BR112020018972A2; EP3765472A4; CN112513059A; CA3094205A1; JP7432533B2; KR20200132940A; JP2021518163A; AU2024201079A1; MX2020009635A; AU2024201079B2; JP2024033006A; WO2019178541A1; AU2019236279A1; EP3765472A1

Abstract

Methods of using highly purified rebaudioside

Description

HIGH-PURITY STEVIOL GLYCOSIDES TECHNICAL FIELD

The present invention relates to compositions comprising steviol glycosides, including highly purified steviol glycoside compositions, and processes for making the same.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted to diabetics and others interested in controlling for their intake of carbohydrates.

Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A(0.3%). 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.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

SUMMARY OF THE INVENTION

According to the present invention there is provided a method for enhancing flavor in a consumable product, comprising adding highly purified Rebaudioside AMhaving greater

than about 90% Rebaudioside AM content by weight on a dried basis to the product at a concentration below a sweetness recognition threshold concentration of Rebaudioside AM, wherein Rebaudioside AM has the formula:

OH OH HO HO OH OC "HOH HO HO HO HO, HO,. H HO O O

H C HO 0 0

OH O O

HO aO HO OH

In another aspect, the present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic

la 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AMor a synthetic steviol glycoside.

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

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, UDP-glucosyltransferases (UGTs) 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.

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.

As used hereinafter, the term "UGT76Gl", unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence "SEQ ID 3" as described in Example 1.

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, UGTS12, 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 UGT74G1.

In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C19 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 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 UGT9lD2.

In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C19 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 the existing glucose at C13 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 EUGT 1, 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 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 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 EUGT1, 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 JDP 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.

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 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 another 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.

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 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 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 5 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.

In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to form 10 stevioside C. 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 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 rubusoside to form stevioside A (rebaudioside KA). 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 rubusoside to form stevioside.

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 UGT74Gl.

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 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 embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2.

In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) 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 A (rebaudioside KA) to form rebaudioside E.

In another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to stevioside 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.

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 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 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 E3 to form rebaudioside AM

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 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 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.

Optionally, the method of the present invention further comprises using more than one UGT on a starting composition, to give a target steviol glycoside(s) having more than one glucose unit than the starting composition. In a particular embodiment, the UDP glucosyltransferases are UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT 1and/or UGT91D2 or any UGT having >85% amino-acid sequence identity with UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT I1and/or UGT91D2 or any combination thereof, capable of adding more than one glucose unit to a starting composition to give a steviol glycoside(s) having more than one glucose unit than the starting composition.

In one embodiment, the UDP-glucosyltransferases are any UDP glucosyltransferases capable of adding overall two glucose unit to stevioside to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferases are selected from UGTSl2, EUGT1, UGT91D2, UGT76G1 or any UGT having >85% amino-acid sequence identity with UGTSl2, EUGTll, UGT91D2, UGT76G1 or any combination thereof. In another particular embodiment, the UDP-glucosyltransferases are UGTSl2 and 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 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 AM.

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 consumable products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of rebaudioside AM.

FIG. 2 shows the pathways of producing rebaudioside AM and various steviol glycosides from steviol.

FIG. 3 shows the biocatalytic production of rebaudioside AM from stevioside using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP glucose via sucrose synthase SuSyAt.

FIG. 4 shows the biocatalytic production of rebausioside AM from rebaudioside E using the enzyme UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSyAt.

FIG. 5 shows the HPLC chromatogram of stevioside. The peak with retention time of 25.992 minutes corresponds to stevioside.

FIG. 6 shows the HPLC chromatogram of the product of the biocatalytic production of rebaudioside AM from stevioside. The peak with retention time of 10.636 minutes corresponds to rebaudioside AM.

FIG. 7 shows the HPLC chromatogram of rebaudioside E. The peak with retention time of 10.835 minutes corresponds to rebaudioside E.

FIG. 8 shows the HPLC chromatogram of the product of the biocatalytic production of rebaudioside AM from rebaudioside E. The peaks with retention time of 10.936 and 11.442 minutes correspond to rebaudioside E and rebaudioside AM respectively.

FIG. 9 shows the HPLC chromatogram of rebaudioside AM after purification by methanol crystallization. The peak with retention time of 10.336 minutes corresponds to rebaudioside AM.

FIG. 10 shows the IH NMR spectrum of rebaudioside AM(500 MHz, pyridine-d5).

FIG. 11 shows the HSQC spectrum of rebaudioside AM(500 MHz, pyridine-d5).

FIG. 12 shows the H,H COSY spectrum of rebaudioside AM(500 MHz, pyridine d5).

FIG. 13 shows the HMBC spectrum of rebaudioside AM(500 MHz, pyridine-d5).

FIG. 14 shows the HSQC-TOCSY spectrum of rebaudioside AM(500 MHz, pyridine-d5).

FIG. 15a and FIG. 15b show the LC chromatogram and mass spectrum of rebaudioside AM respectively.

FIG. 16 is a graph showing the effect of Reb AM on the flavor modification of coconut water.

FIG. 17 is a graph showing the effect of Reb AM on the flavor modification of a chocolate protein shake.

DETAILED DESCRIPTION

The present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or enzyme preparation, thereby producing a composition comprising a target steviol glycoside.

One object of the invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or a synthetic steviol glycoside from various starting compositions.

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 including microorganisms, comprising one or more enzyme, capable of single or multiple step chemical transformations on organic compounds. Biocatalysis processes include fermentation, biosynthesis, bioconversion and biotransformation processes. Both isolated enzyme, and whole-cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

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 steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 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 still another embodiment, the starting composition steviol glycoside is rubusoside.

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

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

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

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

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

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

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

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 E3.

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 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM or other glycoside of steviol occurring in Stevia rebaudiana plant, 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 A.

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

In another embodiment, the target steviol glycoside is rubusoside.

In another embodiment, the target steviol glycoside is stevioside.

In another embodiment, the target steviol glycoside is stevioside A (rebaudioside KA).

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 E.

In another embodiment, the target steviol glycoside is rebaudioside E2.

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

In another embodiment, the target steviol glycoside is rebaudioside AM

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 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 rubusoside.

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 stevioside A (rebaudioside KA).

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 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 E3.

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

In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside AM from a starting composition comprising stevioside and UDP-glucose.

In another particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside AM from a starting composition comprising rebaudioside E 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 reb AM, the process described herein provides a composition having greater than about 90% reb AM content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is reb AM, the process described herein provides a composition comprising greater than about 95% reb AM 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 and UDP glucosyltransferases (UGTs). Optionally it may include UDP recycling enzyme(s).

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, EUGT11 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, 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 the existing glucose at C19 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 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 the existing glucose at C19 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 the existing glucose at Cl3 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 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 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 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 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 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 A to form rubusoside. 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 steviolbioside B to form stevioside C. 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 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.

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 another embodiment, the UDP-glucosyltransferase is any UDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside A (rebaudioside KA). 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 UJDP glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside.

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 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 A

(rebaudioside KA) 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 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 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 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 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 E to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G.

Optionally, the method of the present invention further comprises using more than one UGT on a starting composition, to give a target steviol glycoside(s) having more than one glucose unit than the starting composition. In a particular embodiment, the UDP glucosyltransferases are UGT74GI, UGT85C2, UGT76G1, UGTS12, EUGTll and/or UGT91D2 or any UGT having >85% amino-acid sequence identity with UGT74G1, UGT85C2, UGT76G1, UGTS2, EUGT I1and/or UGT91D2 or any combination thereof, capable of adding more than one glucose unit to a starting composition to give a steviol glycoside(s) having more than one glucose unit than the starting composition.

In one embodiment, the UDP-glucosyltransferases are any UDP glucosyltransferases capable of adding overall two glucose unit to stevioside to form rebaudioside AM In a particular embodiment, the UDP-glucosyltransferases are selected from UGTS2, EUGT1, UGT91D2, UGT76G1 or any UGT having >85% amino-acid sequence identity with UGTSl2, EUGTl, UGT91D2, UGT76G1 or any combination thereof. In another particular embodiment, the UDP-glucosyltransferases are UGTS12 and 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. The UDP recycling enzyme can be sucrose synthase SuSyAt or a sucrose synthase having >85% amino-acid sequence identity with SuSyAt and the recycling substrate can be sucrose.

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 GenInfo 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

Table 1

GI number Accession Origin 190692175 ACE87855.1 Stevia rebaudiana 41469452 AAS07253.1 Oryza saliva 62857204 BAD95881.1 Ipomoea nil 62857206 BAD95882.1 Ipomoeapurperea 56550539 BAD77944.1 Bellis perennis 115454819 NP 001051010.1 Oryza saliva 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 Piceasitchensis 122209731 Q2V6J9.1 Fragariax ananassa 125534461 EAY81009.1 Oryza sativa Indica Group 125559566 EAZ05102.1 Oryza salivaIndica Group 125588307 EAZ28971.1 Oryza sativaJaponicaGroup 148907340 ABR16806.1 Piceasitchensis 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 Arabidopsisthaliana 1685005 AAB36653.1 Nicotiana tabacum 183013903 ACC38471.1 Medicago truncatula 186478321 NP_172511.3 Arabidopsis thaliana 187373030 ACD03249.1 A vena strigosa 194701936 ACF85052.1 Zea mays 19743740 AAL92461.1 Solanum lycopersicum 212275846 NP_001131009.1 Zea mays 222619587 EEE55719.1 Oryza sativa JaponicaGroup 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 vinfera 225461556 XP_002285222 Vitis vinifera 225469540 XP_002270294.1 Vitis vinfera 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 Arabidopsis thaliana 296088529 CB137520.3 Vitis vinifera 297611791 NP_001067852.2 Oryza sativa JaponicaGroup 297795735 XP_002865752.1 Arabidopsislyrata subsp. lyrata 297798502 XP 002867135.1 Arabidopsislyrata subsp. lyrata

297820040 XP_002877903.1 Arabidopsislyratasubsp. 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 Arabidopsisthaliana 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 saliva JaponicaGroup 470104266 |XP004288529.1 Fragariavesca subsp. vesca 470142008 XP_004306714.1 Fragariavescasubsp. vesca 475432777 EMT01232.1 Aegilops tauschii 51090402 BAD35324.1 Oryza saliva JaponicaGroup

Table 2

GI number Accession Origin Internal reference 460409128 XP.004249992.1 Solanum lycopersicum UGTSl 460386018 XP.004238697.1 Solanum lycopersicum 460409134 XP.004249995.1 Solanum lycopersicum 460410132 XP.004250485.1 Solanum lycopersicum UGTS12

460410130 XP.004250484.1 Solanum /ycopersicum 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 5 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.

10 Suitable microoganisms include, but are not limited to, Ecoli, Saccharomyces sp., Aspergillus sp., Pichia sp., 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

5 Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc.

Highly purified target glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this invention may be employed as a sweetening compound as the sole sweetener, or it may be used together with at least one naturally occurring high intensity sweeteners such as rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside 0, rebaudioside 02, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2, stevioside F, 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 and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, siamenoside and combinations thereof.

In a particular embodiment, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AMcan be used in a sweetener composition comprising a compound selected from the group consisting of rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside 0, rebaudioside 02, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Ti, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2, stevioside F, NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be used in combination with synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.

Moreover, highly purified target steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be combined with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be mixed with umami 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with 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 combinationsthereof.

Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be combined with polyols or sugar alcohols. The term "polyol" refers to a molecule 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AMmay be combined with reduced calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L-arabinose and combinations thereof.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be combined with various carbohydrates. The term "carbohydrate" generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH2O), 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, 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.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as 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 A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to this 5 invention may be applied as a flavor modifier to produce zero calorie, reduced calorie or diabetic beverages and food products with modified flavor. When used as a flavor modifier, or a flavor with modifying properties (FMP), the highly purified target steviol glycoside is used in a consumable product below the detection level of the flavor modifier or FMP. The flavor modifier or FMP therefore does not impart a detectable taste or flavor 10 of its own to the consumable product, but instead serves to modify the consumer's detection of tastes and/or flavors of other ingredients in the consumable product. One example of taste and flavor modification is sweetness enhancement, in which the flavor modifier or FMP itself does not contribute to the sweetness of the consumable product, but enhances the quality of the sweetness tasted by the consumer.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used as 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; instantjuices; 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.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used as 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.

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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. Accordingly, the present invention provides a beverage comprising a sweetener and steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM as a sweetness enhancer, wherein steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 0.5% to about 12% by weight, such as, for example, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 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 0.5% of about 10%, such as for example, from about 2% to about 8%, from about 3% to about 7% or from about 4% to about 6% by weight. In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.5% to about 8% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount from about 2% to about 8% by weight.

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

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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM or a combination thereof, wherein steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM are present in a concentration at or below their sweetness recognition thresholds.

Addition of steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM 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 A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. 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.

EXAMPLES

EXAMPLE 1

Protein sequences of engineered enzymes used in the biocatalytic process SEQID1: >SuSyAt, variant PM-54-2-E05 (engineered sucrose synthase; source of WT gene: Arabidopsis thaliana) MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKI QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL DMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCG ERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVEL SKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDI YWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHT AFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEI EELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL RELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMD RVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPA EIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIE EKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQ AVPLAQDD

SEQID2: >UGTS2 variant 0234 (engineered glucosyltransferase; source of WT gene: Solanum lycopersicum) MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN FSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSF RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENS TVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP EGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVP IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGE TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3: >UGT76G variant 0042 (engineered glucosyltransferase; source of WT gene: Stevia rebaudiana) MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSC LITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQAS GFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAP

SFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLV DSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWN STLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEG EYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

EXAMPLE 2

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 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 (0.2 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 (OD600)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with distilled water, resulting in an enzymatic active preparation. For enzymatic active preparations of SuSy.At, 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, 3 mM MgC2, and 15 mM uridine diphosphate (UDP).

EXAMPLE 3

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 pLE1A17 (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 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 pm 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 UGTS12 turns over 1 nmol of rebaudioside A (RebA) 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 RebA at to, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSyAt.

EXAMPLE 4

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 pLE1A17 (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 (OD600)) 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 RebA at to, 500 mM sucrose, 3mM MgC2,

0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSyAt.

EXAMPLE 5

Synthesis of rebaudioside AM from stevioside in a one-pot reaction, adding UGTS12, SuSyAt and UGT76G1 at the same time Rebaudioside AM (reb AM) was synthesized directly from stevioside in a one-pot reaction (Fig. 3), utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4): UGTSl2 (variant of SEQ ID 2), SuSyAt-(variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 105 U/L UGTS2, 405 U/L SuSyAt, 3 U/L UGT76G1, 5 mM stevioside, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl2 and potassium phosphate buffer (pH 6.6). First, 207 mL of distilled water were mixed with 0.24 g MgCl2.6H20, 103g sucrose, 9.9 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 15g stevioside. After dissolving the components, the temperature was adjusted to 45°C and UGTS12, 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 8hrs and 18hours. The content of reb AM, reb E, stevioside, reb M, reb B, steviolbioside and reb Iat several time points was analyzed by HPLC. For analysis, biotransformation samples were inactivated by adjusting the reaction mixture to pH5.5 using 17% H3PO4 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 pm 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 tL. 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 stevioside and the reaction mixture at 24 hours are shown in Fig. 5 and Fig. 6, 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 AM Time, % conversion from stevioside hrs Reb E Reb AM Reb M Reb I Stevioside Reb B Steviolbioside 0 0 0 0 0 100 0 0 6 1.9 35.9 1.3 1.7 58.7 0.0 0.4 18 0.9 96.7 1.3 0.6 0.0 0.0 0.4 24 0.3 96.4 2.1 0.7 0.0 0.2 0.4

EXAMPLE 6

Synthesis of rebaudioside AM from rebaudioside E in a one-pot reaction, SuSyAt and UGT76G1 at the same time

Rebaudioside AM(reb AM) was synthesized directly from rebaudioside E (reb E) in a one-pot reaction (Fig. 4), utilizing the two enzymes (see EXAMPLES 1, 2 and 4): SuSyAt-(variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 405 U/L SuSyAt, 3 U/L UGT76G1, 5 mM reb E, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl 2 .6H20 and potassium phosphate buffer (pH 6.6). First, 37 mL of distilled water were mixed with 40.3 mgMgCl 2 , 17.12g sucrose, 1.65 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 5.04 g reb E. After dissolving the components, the temperature was adjusted to 45°C and SuSyAt, UGT76G1 and 6.5 mg UDP were added. The reaction mixture was incubated at 45°C shaker for 24 hrs. Additional 6.5 mg UDP was added at 8hrs and 18hours. The content of reb AM, reb E, stevioside, reb A, reb M, reb B, and steviolbioside at several time points was analyzed by HPLC.

For analysis, biotransformation samples were inactivated by adjusting the reaction mixture to pH5.5 using 17% H3PO4 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 tm 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 IL. Rebaudioside species were detected by UV at 210 nm.

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

Table 4 Biotransformation of reb E to reb AM Time, % conversion from Reb E hrs RebE RebAM Reb M Reb A Stevioside Reb B Steviolbioside 0 99.46 0 0 0.54 0 0 0 4 40.75 57.92 0 0.59 0 0.73 0 7 24.79 73.92 0 0.58 0 0.71 0 24 4.38 94.33 0 0.59 0 0.70 0

EXAMPLE 7

Purification of rebaudioside AM The reaction mixture of EXAMPLE 5, after 24 hrs, was inactivated by adjusting the pH to pH 5.5 with H 3PO4 and then boiled for 10 minutes. After boiling the reaction mixture was filtered and diluted with RO water to 5% solids content. The diluted solution was passed through 1 L column packed with YWD03 macroporous adsorption resin (Cangzhou Yuanwei, China). Adsorbed steviol glycosides were eluted with 5L 70% ethanol. The obtained eluate was evaporated until dryness to yield 16 g of dry powder which was dissolved in 80 mL of 70% methanol. The solution was crystallized at 20°C for 3 days. The crystals were separated by filtration and dried in vacuum oven at 80°C for 18 hours to yield 10.4 g of pure reb AM crystals with 95.92% purity, determined by HPLC assay. The chromatogram of reb AM is shown in Fig. 9. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.

EXAMPLE 8

Structure elucidation of rebaudioside AM 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. IH-NMR-spectrum of rebaudioside AM in pyridine-ds reveal the excellent quality of the sample (see Fig. 10). The HSQC (see Fig. 11) 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. 12). Other deep-fielded signals of the quaternary carbons (C-13, C-16 and C-19) are detected by the HMBC (Fig. 13). Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure: 12 13 OR1 9 14 17 2

15 34 1 15 8

R20 1 0

Correlation of HSQC and HMBC signals reveal five anomeric signals. The coupling constant of the anomeric protons of about 8 Hz and the broad signals of their sugar linkage allows the identification of these five sugars as p-D-glucopyranosides.

The observation of the anomeric protons in combination with HSQC and HMBC reveal the sugar linkage and the correlation to the aglycone. The assignment of the sugar sequence was confirmed by using the combination of HSQC-TOCSY (Fig. 14) and HSQC. The NMR experiments above were applied to assign the chemical shifts of the protons and carbons, main coupling constants and main HMBC correlations (see Table 5).

Table 5 Chemical shifts of rebaudioside AM Position 8C [ppm 8H [ppm] J [Hz] HMBC (H -C Aglycone moiety 0.68 M 1 39.9t .68m 14 m 1.39 m 2 19.4t .39m 1.05m 3 37.4t 1.0m 2.80 m 4 44.2 s 5 5 7.3 d 0.95 m 1.90 M 6 21.7t 19 1.26 m 7 41.0t 1.26 8 41.9 s 9 5 3.3d 0.85m 10 39.2 s 1.59 m 11 20.1t 1.61m 161 m 36.9t 1.65m 12 1.92m 13 85.9s 1.78 d 11.0 14 43.8t 2.52 d 11.0 2.00 d 16.0 15 47.4 2.06d 16.0 7,8,9, 14 16 154.6 s 5.03 brs 17 104.3 t 5.brs 13, 15, 16 5.71 br s 18 28.5 q 1.40 s 3,4,5,19 19 175.2 s 20 16.2q 1.06 s 1,5,9, 10

Table 5 (continued) Chemical shifts of rebaudioside AM

Position I c [ppm] 8H [ppm] J [Hz] HMBC (H->C Sugar moiety Sugar I: /-D-Glucopyranoside P97.5 d 5.13 d 7.T1 2 84.0 d 4.14 m 3 77.6 d 4.20 m 41 71.3 d 4.19 M 5 77.6 d 3.70m 4.23 m 6 62.0t 4.2 mm 4.32 Sugar II:/-D-Glucopyranoside 106.3 d 5.26 d 8.0 2 2 76.8 d 4.13m 311 77.3 d 4.21m 411 71.6 d 4.18 m 5" 77.9 d 3.91 m 4.29 4.41 m m 6 62.4t 4.41 m Sugar IIl: I §D-Glucopyranoside 1iii 92.9 d 6.20 d 8.1 19 2 1 77.0 d 4.46 m 3" 88.1 d 4.24 m 4 111 69.0 d 4.12 m 5 78.4 d 3.82 m 61 61.3 t4.20 m 4.33 m Sugar IV:/-D-Glucopyranoside liV 103.4 d 5.73 d 7.7 21 21" 75.4 d 3.98 m 3jV 78.1 d 4.09 m 4iv 72.6 d 4.08 m 5v 77.4 d 3.92 m 62.9 4.32 m 61V 4.51 m Sugar V:f3-D-Glucopyranoside 1" 104.4 d 5.29 d 8.1 3 2v 75.1 d 4.00 m 3v 7 8.2 d 4.24 m 4v 71.4 d 4.27m 5 78.2 d 3.99 m 4.27 m 4.48 m

Correlation of all NMR data indicates rebaudioside AM having five p-D glucopyranoses attached to a steviol aglycone, as depicted with the following chemical structure:

OH OH HO HOVOH

12 O H 11 3 HO HO 120 17

HONOHO O 4 7 4 HH6

OH 0

HO . HO OH

The chemical formula of rebaudioside AM is C5oH 80 0 2 8, which corresponds to a calculated monoisotopic molecular mass of 1128.5. For LCMS analysis, rebaudioside AM was dissolved in methanol and analyzed using Shimadzu Nexera 2020 UFLC LCMS instrument on a Cortecs UPLC C18 1.6ptm, 50 x 2.1 mm column. The observed LCMS (negative ESI mode) result of 1127.3 (see Fig. 15a and Fig. 15b respectively) is consistent with rebaudioside AM and corresponds to the ion (M-H)-.

Solubility, Sweetness and Flavor Modification Properties of Reb AM

EXAMPLE 9 Reb AM was evaluated for it solubility and solution stability properties. Tables 6a and 6b, below, show the composition of the test sample, with the total steviol glycoside (TSG) percentage shown in the final column of Table 6b.

Table 6a: Composition of Test Sample:

Assay, %(as dried)

Sample Reb Reb Reg Reb Reb Reb Reb Reb Reb ID E AM 0 D N M H I A

Reb AM 0.23 99.30 0.00 0.00 0.00 0.00 0.00 0.05 0.00 Sample 1

Table 6b Assay, %(as dried)

Sample Stev Reb F Reb C Dul. Rubu Reb B Sbio TSG ID A Reb AM 0.00 0.00 0.00 0.00 0.00 0.00 0.26 99.84 Sample 1

Table 7: Physical Properties of Reb AM:

Physical Material& Reb AM Results Description Method

Form Visual Evaluation Powder

Appearance Visual Evaluation Very Fine

Odor Olfactory Odorless Evaluation

Color Visual Evaluation White

Moisture Content

Solution Stability:

Solubility characteristics were measured as follows. Prepare the following solutions in water and stir at 700 rpm for each. Add heat if necessary at 2 minutes and 30 seconds of stirring. Using a stopwatch, determine how long it takes all powder to dissolve completely and record the temperature at which it dissolves. The following table summarizes the solubility characteristics of Rebaudiosides D, M, and AM. Surprisingly, Reb AM shows significantly higher solubility than other minor and major steviol glycosides.

Table 8: Comparison of Solubility Characteristics

Product Test Dissolution Dissolution in Solution Solution Comment Conc. water water after 24 hrs after 48 on (Ambient) (Heated) hrs solubility

Reb D 0.05% Added heat at 2 8 min 30 sec Clear Clear Easily Soluble minutes/30 temp: 39 deg. C seconds.

Reb D 0.1% Added heat at 2 15 min 4 sec Clear Clear Easily Soluble minutes/30 temp: 72 deg. C seconds.

Reb D 0.3% Added heat at 2 20 min 27 Precipitate in Requires minutes/30 seconds temp: less than 24 dispersion seconds. 78.5 deg. C hrs agent

Reb M 0.1% 12 minutes of No heat needed Clear Clear Easily Soluble agitation

Reb M 0.3% Added heat at 2 Heated to 99 deg Clear Slight Moderately min 30 seconds. C with agitation precipitation Soluble

Reb M 0.5% Added heat at 2 Heated to 87 Moderate - Requires min 30 sec deg. C with Precipitation dispersion agitation. 16 min in 2 hours agent 12seconds

Reb AM 1% Stirred for 3 min No heat needed Clear Clear Easily Soluble 28sec

Reb AM 5% Added heat at 2 15 min 32 sec at Clear Clear Easily Soluble minutes/30 temperature: seconds. 45C

Reb AM 10% Added heat at 2 10 min 2 sec Clear Slight Moderately minutes/30 Temperature: precipitation soluble seconds. 54C

Table 9: Summary of Solution Stability of Major and Minor steviol glycosides:

SG/Property Reb A* Stevioside* Reb AM Reb D Reb M Solubility <0.7% <0.7% 10% .0.1% 0.3% * Solubility of Stevioside was slightly lower than Reb A in aqueous solution. Ref: Celaya et al (2016). Int. J. of Food Studies, V.5, p 158-166

EXAMPLE 10

Reb AM was evaluated for its sensory attributes.

Sensory Attributes

Steviol glycoside molecules are known for their varied sweetness profiles, which are a function of the sugar moieties present in their structures. Since steviol glycosides contain hydrophobic (steviol) and hydrophilic (sugar moieties), they can display flavor modification at a certain dosage level without contributing any significant detectable sweetness perception.

Isosweet Determination of Reb AM and other Steviol glycosides:

• Five concentration levels of Test sweetener were identified to match 2.5%, 5%, 7.5% and 10% sucrose-equivalent in acidified water (pH of 3.2), for which a panel of 40 participants was recruited to conduct two alternate forced choice (2-AFC) test at each concentration level.

• Samples were evaluated and isosweet point was determined at a point in which 50% of the panelist selected sucrose sample as sweeter and 50% selected stevia sample as sweeter

• A Beidler model was used to fit the concentration-response relationship using the four isosweet concentrations and their corresponding target sweetness values as the data.

• Sweetness potency is calculated as a ratio of sugar concentration to sweetness equivalent. As an example, Reb AM was evaluated.

Table 10: Iso-sweet concentration (ppm) and Sweetness Potency (x sugar equivalent) of Reb AM and other steviol glycosides

Sweetness Equivalent (ppm) in Water (sweetened to Sweetness Potency in Water (sweetened to achieve designated % SE @ pH = 3.2) achieve x % SE @ pH =3.2) sugar concentration 2.5% 5.0% 7.5% 10.0% 2.5% 1 5.0% 7.5% 10.0% Reb A 94 299 NA NA 266 167 NA NA Delta ( Reb D) 62 212 500 926 403 236 150 108

PCS-4000 ( Reb M) 84 209 418 832 298 239 179 90 Reb AM (Reb AM) 150 365 869 1750 167 137 86 57

Effect of Reb AM on Taste & Flavor Profiles of Food and Beverage Applications

A series of experiments were performed to evaluate the effect of Reb AM on taste and flavor profile. The sweetness and taste/flavor modification can influence each other in food and beverage applications. To determine the influence of the taste and flavor modification in different applications, the FEMA (Flavor and Extract Manufacturing Association) prescribes a sensory method that determines the sweetness perception threshold determination presented in Experiment 1, which is discussed below.

Experiment 1 provides the estimate of Reb AM concentration in water that barely contributes to sweetness perception. The sweetness perception threshold concentration provides significantly less sweetness than 1.5% sugar aqueous solution. The summary of sweetness perception threshold for selected steviol glycosides is below in Table 11.

Table 11 Steviol Sweetness Perception FEMA FEMA GRAS Publication Glycosides Threshold GRAS Reference (FEMA Website) Concentration No Reb A 30 ppm 4601 GRAS Flavoring Substances 24 (2008) Reb D 32.5 ppm 4921 GRAS Flavoring Substances29 (2018) Reb M 24 ppm 4922 GRAS Flavoring Substances 29 (2018) Reb AM 50 ppm NA NA

Experiment 2, which is further discussed below, explores the effect of Reb AM on the flavor profile of a non-alcoholic beverage. A commercial Raspberry Watermelon Coconut Water sample was used without (control) and with Reb AM (test) to determine the effect of Reb AM on different taste attributes of the beverage. The results indicated the test sample having Reb AM had significantly higher mango peach flavor, coconut water flavor, and overall liking compared to the control samples (at 95% confidence).

Experiment 3, which is further discussed below, explores the effect Reb AM on taste

& flavor profile of a sweetened dairy product. A sensory panel tested samples of stevia (Reb A) sweetened, no-sugar-added chocolate flavored dairy protein shake without (control) and with Reb AM. The panel found the test sample containing 50 ppm of Reb AM to be significantly lower bitterness, metallic note, whey protein and lower bitter aftertaste than the control (at 95% confidence) and higher in cocoa flavor, dairy notes, vanilla flavor, and overall liking (at 95% confidence).

A group of trained and experienced taste panel members evaluated no-calorie Lemon-lime carbonated soft drink (CSD) sweetened with 500 ppm of Reb AM, Reb D, or Reb M samples. The panel members found the CSD with Reb AM is less sweet but has significantly less bitterness and sweetness lingering compared to other samples, especially the CSD sweetened with Reb M.

EXPERIMENT 1 OF EXAMPLE 10: Sweetness Perception Threshold With Reb AM Application: Neutral Water

The sweetness perception of 1.5% sugar solution and different solutions of Reb AM were tested with a sensory panel and found that 50 ppm of Reb AM solution in water provided sweetness perception significantly lower than that of 1.5% sugar solution. Therefore we selected 50 ppm of Reb AM as the recognition threshold concentration.

METHODOLOGY

Table 12 * Nature of Participants: Trained panel • Number of Sessions 1 • Number of Participants: 30 • Test Design: 2- AFC, Balanced, randomized within pair. Blind • Sensory Test Method: Intensity ratings Environmental Standard booth lighting Conditions

• Attributes and Scales: Which sample is sweeter? • Statistical Analysis: Paired comparison Test • Sample Size ~1.5 oz. in a clear capped plastic cup • Serving Temperature Room temperature (-70°F) • Serving/Panelists Samples served simultaneously. Panelists instructed to read Instruction: ingredient statement, evaluate each sample.

The following table (Table 13) shows an evaluation of the recognition threshold concentration to follow the methodology outlined in section 1.4.2 of the "Guidance for the Sensory Testing of Flavorings with Modifying Properties within the FEMA GRASTM Program", issued by FEMA (Flavor and Extract Manufacturers Association https://www.femaflavor.org/).

Table 13

DATA: n=30 Two-Tailed Analysis Table Report for Result Reb AM Binomial Distribution Probability

Percent 1.5% 30 ppm Frequency Sucrose Reb AM Sample 1 P-value Sig PC 29 1 96.7% 0.0001 % Frequency 96.7% 3.3%

Percent 1.5% 50 ppm Frequency Sucrose Reb AM Sample 1 P-value Sig PC 23 7 76.7% 0.01 % Frequency 76.7% 23.3%

DATA: n=30

Two-Tailed Analysis Table Report for Result IS03026A Binomial Distribution Probability

Percent 1.5% 70 ppm of Frequency Sucrose Reb AM Sample 1 P-value Sig PC 9 21 30.0% 0.05 % Frequency 30% 70% DATA: n=30 Two-Tailed Analysis Table Report for Result Reb AM Binomial Distribution Probability

Percent 1.5% 100 ppm Frequency Sucrose Reb AM Sample 1 P-value Sig PC 3 27 10% 0.0001 % Frequency 10% 90%

EXPERIMENT 2 OF EXAMPLE 10:

Raspberry Watermelon Coconut Water With Reb AM

Application: Non-alcoholic Beverage

SUMMARY

Thirty panel members evaluated two samples of raspberry watermelon flavored coconut water for overall acceptance and attribute intensities (sweetness, Raspberry flavor, watermelon flavor, coconut water flavor, saltiness, bitterness, and sweet aftertaste, bitter aftertaste) in two sessions. In session one, the two samples included: 1) store-bought Raspberry Watermelon Coconut Water control sample and 2) store-bought Raspberry Watermelon Coconut Water test sample containing Reb AM. The objective of the test was to determine if the addition of Reb AM affects the flavor profile of a non-alcoholic beverage. The results indicated the test sample Reb AM had significantly higher mango peach flavor, coconut water flavor, and overall liking compared to the control samples (at 95% confidence).

OBJECTIVE

The project objective is to assess if the addition of stevia extract solids has an effect on

key flavor attributes in various beverage applications.

TEST OBJECTIVE

The test objective is to determine if the flavor profile and overall acceptance of a Control

sample of flavored coconut water differs from a Test sample of the same beverage

containing Reb AM.

METHODOLOGY

Table 14 " Nature of Participants: Trained panel • Number of Sessions 1 • Number of Participants: 30 • Test Design: Balanced, randomized within pair. Blind • Sensory Test Method: Intensity and acceptance ratings • Environmental Condition Standard booth lighting

• Attributes and Scales: • Overall Acceptance on a 10-pt hedonic scale where 10 = Extremely Like and 0= Extremely Dislike • Overall liking, sweetness, raspberry flavor, watermelon flavor, coconut water flavor, astringency, artificial chemical note, bitterness, and sweet aftertaste, bitter aftertaste. 10-pt continuous intensity scale where 0 = Imperceptible and 10 = Extremely Pronounced • Statistical Analysis: ANOVA (by Block) with Post Hoc Duncan's Test " Sample Size -1.5 oz. in a clear capped plastic cup • Serving Temperature Refrigerated temperature (-45°F) • Serving/Panelists Samples served simultaneously. Panelists instructed to read Instruction: ingredient statement, evaluate each sample.

SAMPLES

Table 15

Beverage Type I, Non-alcoholic

Reference Reb AM *Coconut water raspberry watermelon juice 100 99.995 Reb AM 0.005 Total (g) 100 100 *Vita Coco store brand

RESULTS Table 16 (below) summarizes the overall acceptance and mean attribute intensity results for each sample.

5 Table 16: Mean Scores Raspberry Watermelon Coconut Water with 50 ppm Reb AM Summary of Mean-Scores, P-Values, and Significance Test Result Code: Coconut Water (raspberry/watermelon flavor) Reb AM at 50 ppm This test was performed on 30 panelists.

Coconut water Coconut water Attribute control with 50 ppm of P-Value Sig Reb AM Sweet Intensity 4.38 4.44 0.6990 NS

Bitter Intensity 0.32 0.24 0.4267 NS

Astringency 1.04 1.10 0.4942 NS

Coconut Flavor 4.89 5.11 0.4372 NS

b a Watermelon Flavor 3.85 4.41 0.0221

Raspberry Flavor 0.68 0.95 0.2423 NS

Artificial/Chemical Note 2.94 2.55 0.2583 NS

a b Sweet Aftertaste 1.60 1.33 0.0905 **

Bitter Aftertaste 0.36 0.29 0.5409 NS

b a Overall Liking 4.49 5.04 0.0710 ** * 80% CI,**= 90% CI, ***= 95%CI

The results indicate the test sample Reb AM had significantly higher watermelon flavor and overall liking compared to the control samples (at 95% confidence). Test sample Reb AM had significantly lower sweet aftertaste intensity compared to the control samples (at 90% confidence).

CONCLUSION

Thirty panelists evaluated two samples of Raspberry Watermelon flavored coconut water for overall acceptance and attribute intensities (sweetness, watermelon flavor, raspberry flavor, coconut water flavor, astringency, artificial/chemical note, bitterness, and sweet aftertaste, bitter aftertaste) in two sessions. In session one, the two samples included: 1) store-bought Raspberry Watermelon Coconut Water control sample and 2) store-bought Raspberry Watermelon Coconut Water test sample containing Reb AM. The objective of the test was to determine if the addition of Reb AM affects the flavor profile of a non alcoholic beverage. The results indicated the test sample Reb AM had significantly higher watermelon flavor and overall liking compared to the control samples (at 95% confidence). A graph of the results is shown in FIG. 16.

Test sample Reb AM had significantly lower sweet aftertaste intensity compared to the control samples (at 90% confidence).

EXPERIMENT 3 OF EXAMPLE 10:

Chocolate Protein Shake With Reb AM

Application: Milk/Dairy Product

SUMMARY

Thirty trained panelists evaluated two samples of chocolate flavored dairy protein shake for overall acceptance and attribute intensities (cocoa flavor, dairy note, whey protein, vanilla, metallic, sweetness, bitterness and aftertaste). The two samples included: 1) no sugar added "Control" sample containing 300 ppm PureCircle Reb A and 2) no sugar added "Test" sample containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. The objective of the test was to determine if the addition of Reb AM affects the flavor profile of a milk product. The panel found the test sample containing 50 ppm of Reb AM to be significantly lower bitterness, metallic note, whey protein and lower bitter aftertaste than the control (at 95% confidence) and higher in cocoa flavor, dairy notes, vanilla flavor, and overall liking (at 95% confidence). Further, there was no significant impact on sweetness intensity.

OBJECTIVE

The project objective is to assess if the addition of stevia extract solids has an effect on key flavor attributes in various beverage applications.

TEST OBJECTIVE

The test objective is to determine if the flavor profile and overall acceptance of a control sample of dairy beverage application differs from a Test sample of the same beverage containing Reb AM.

METHODOLOGY

10 Table 17 • Nature of Participants: Trained panel " Number of Sessions 1 * Number of Participants: 30 • Test Design: Balanced, randomized within pair. Blind • Sensory Test Method: Intensity and acceptance ratings • Environmental Condition Standard booth lighting " Attributes and Scales: • Overall Acceptance on a 10-pt hedonic scale where 10 = Extremely Like and 0 Extremely Dislike • Overall Liking, sweetness, bitterness, cocoa flavor, dairy notes, chocolate, whey protein notes, metallic note, vanilla note, and Aftertaste. 10-pt continuous intensity scale where 0= Imperceptible and 10 = Extremely Pronounced • Statistical Analysis: ANOVA (by Block) with Post Hoc Duncan's Test • Sample Size -1.5 oz. in a clear capped plastic cup • Serving Temperature Refrigerated temperature (-45°F) • Serving/Panelists Samples served simultaneously. Panelists instructed to read Instruction: ingredient statement, evaluate each sample.

SAMPLES

Table 18 Ingredient list Sugar 50 ppm Reb Reference AM Milk, 2% 86.47 86.465 Whey Protein 90 Instant - Non GMO (Prod: 18618) 6.8250 6.8250 Non-Fat Dry Milk 4.6269 4.6269 Maltrin QD M585 1.1066 1.1066 Vitamin Blend - 0.0063 0.0063 Xanthan Gum (Cold dissolve) 0.0359 0.0359 Forbes 10/12 Cocoa powder 7113 0.7194 0.7194 Vanilla Flavor Powder 0.1799 0.1799 Reb A 0.0300 0.0300 Reb AM 0.0050 TOTAL 100 100

Sugar Contribution (grams) per 100 grams* Sugar 165 ppm Reference Reb AM Milk, 2% 4.08 4.15 Non-Fat Dry Milk 2.41 2.41 Maltrin QD M585 0.08 0. 08 TOTAL 8.07 6.64 * Calculated with Genesis R&D version 11.4

Table 19: Effect Reb AM on flavor modification of Chocolate Protein shake

Summary of Mean-Scores, P-Values, and Significance Test Result Code: PROTEIN6 Test Description: Chocolate Vanilla Protein Dairy Shake: 50 ppm Reb AM This test was performed on 30 panelists.

Control - NSA Test - NSA Protein Attribute Protein Shake w/ Shake w Reb A & P-Value Sig Reb A 50 ppm Reb AM

Sweet Intensity 6.04 5.98 0.7329 NS

a b Bitterness 1.98 1.46 0.0138 a b Metallic Note 1.93 1.48 0.0311 b a Cocoa Flavor 4.06 4.55 0.0409 b a Dairy Note 4.10 4.59 0.0515 ** a b Whey Protein Note 4.79 4.32 0.0460 b a Vanilla Note 2.10 2.52 0.0174

Sweet Aftertaste 1.82 1.65 0.2130 NS

a b Bitter Aftertaste 1.03 0.77 0.0495 b a Overall Liking 4.80 5.59 0.0001

80% Cl,**= 90% C, 95%Cl 5*= The panel found the test sample containing 50 ppm of Reb AM to be significantly lower bitterness, metallic note, whey protein and lower bitter aftertaste than the control (at 95% confidence). The panel found the test sample containing 50 ppm of Reb AM to be significantly higher in cocoa flavor, dairy notes, vanilla flavor, and overall liking (at 95% confidence).

CONCLUSION

Thirty panelists evaluated two samples of chocolate flavored dairy protein shake for overall acceptance and attribute intensities (cocoa flavor, dairy note, whey protein, vanilla, metallic, sweetness, bitterness and aftertaste). The two samples included: 1) no sugar added "Control" sample containing 300 ppm PureCircle Reb A and 2) no sugar added "Test" sample containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. The objective of the test was to determine if the addition of Reb AM affects the flavor profile of a milk product. The panel found the test sample containing 50 ppm of Reb AM to be significantly lower bitterness, metallic note, whey protein and lower bitter aftertaste than the control (at 95% confidence) and higher in cocoa flavor, dairy notes, vanilla flavor, and overall liking (at 95% confidence), Further, there was no significant impact on sweetness intensity. A graph of the results is shown in FIG. 17.

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 to be 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 to be 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 (1).txt PC_77WO_Final_ST25 (1) 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 <213> Arabidopsis thaliana

<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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) 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 (1).txt PC_77WO_Final_ST25 (1) txt

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 (1).txt PC_77WO_Final_ST25 (1) 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

CLAIMS We claim:

1. A method for enhancing flavor in a consumable product, comprising adding highly purified Rebaudioside AM having greater than about 90% Rebaudioside AM content by weight on a dried basis to the product at a concentration below a sweetness recognition threshold concentration of Rebaudioside AM, wherein Rebaudioside AM has the formula:

OH OH HO

HO OH

O OH O 0

HO HO

HO HO H

-0 H HO 0 OH 0 0

HO O HO OH

2. The method 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.

3. The method of claim 2, wherein the product further comprises at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

4. The method of claim 2 or claim 3, wherein the product further comprises at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

5. The method of any one of claims 2 to 4, wherein the product further comprises a compound selected from the group consisting of rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside N, rebaudioside 0, rebaudioside 02, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Ti, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zi, rebaudioside Z2, dulcoside A, dulcoside C, rubusoside, steviolbioside, steviolbioside A, steviolbioside B, steviolmonoside, steviolmonoside A, stevioside, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E, stevioside E2, stevioside F, NSF-02, Mogroside V, Luo Han Guo, allulose, D-allose, D-tagatose, erythritol, 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, hemandulcin, 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.

PureCircle USA Inc.

Patent Attorneys for the Applicant/Nominated Person

SPRUSON&FERGUSON