CN112513059A

CN112513059A - High purity steviol glycosides

Info

Publication number: CN112513059A
Application number: CN201980032701.4A
Authority: CN
Inventors: A·马科斯雅恩; S·A/L·拉曼达赫; M·阿弗扎尔宾哈希姆; K·尼扎姆宾纳维; S·Y·周; S·普尔卡亚萨; M·派蒂特
Original assignee: PureCircle USA Inc
Current assignee: PureCircle USA Inc
Priority date: 2018-03-16
Filing date: 2019-03-15
Publication date: 2021-03-16
Also published as: EP3765472A4; JP2021518163A; BR112020018972A2; EP3765472A1; JP2024033006A; JP7432533B2; MX2020009635A; AU2024201079A1; WO2019178541A1; AU2019236279A1; AU2019236279B2; CA3094205A1; KR20200132940A

Abstract

Described are processes for the preparation of highly purified steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. The method comprises converting different starting compositions into the target steviol glycoside using an enzyme preparation and a recombinant microorganism. The highly purified rebaudiosides are used as non-caloric sweeteners, flavoring agents, sweetness enhancers, and foam inhibitors in comestible and chewable compositions, such as any beverages, confectioneries, baked goods, cookies, and chewing gums.

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 preparing them.

Background

High intensity sweeteners have sweetness levels many times greater than that of sucrose. They are largely non-caloric and are commonly used in dietary and low-calorie products, including foods and beverages. High intensity sweeteners do not elicit an ascending insulin response, making them suitable for use in products targeted to diabetes and other products where it is beneficial to control carbohydrate intake.

Steviol glycosides are a class of compounds found in the leaves of the perennial shrub Stevia (Stevia rebaudiana Bertoni) of the family Asteraceae (Compositae) of the earth-grown plants in certain areas of south america. They are structurally characterized by the single base steviol, which differs by the presence of saccharide residues at the C13 and C19 positions. They accumulate in Stevia (Stevia) leaves, accounting for approximately 10% -20% of the total dry weight. The 4 major glycosides found in stevia leaves typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%), and dulcoside A (0.3%) on a dry weight basis. Other known steviol glycosides include rebaudiosides B, C, D, E, F and M, steviolbioside and rubusoside.

Although methods of preparing steviol glycosides from stevia are known, many of these methods are not commercially applicable.

Thus, there remains a need for simple, efficient and economical processes for preparing steviol glycoside-containing compositions, including highly purified steviol glycoside compositions.

Summary of The Invention

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 an enzyme preparation, thereby producing a composition comprising the target steviol glycoside.

The starting composition may be any organic compound comprising at least one carbon atom. In one embodiment, the starting composition is selected from steviol glycosides, polyols or sugar alcohols, various sugars.

The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, rebaudioside AM or synthetic steviol glycosides.

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

In some preferred embodiments, an enzyme preparation comprising one or more enzymes or a microbial cell comprising one or more enzymes is used, which is capable of converting the starting composition into the target steviol glycoside. The enzyme may be located on the surface of the cell and/or within it. The enzyme preparation may be provided as a whole cell suspension, crude lysate or as purified enzyme. The enzyme preparation may be in free form or immobilized on a solid support made of inorganic or organic material.

In some embodiments, the microbial cells comprise the requisite enzymes and genes encoding them to convert the starting composition to the target steviol glycoside. Accordingly, the present invention also provides a process for preparing a composition comprising a target steviol glycoside by contacting a microbial cell, comprising an organic substrate, with a starting composition comprising at least one enzyme capable of converting the starting composition into the target steviol glycoside, thereby producing a medium comprising the at least one target steviol glycoside.

Enzymes essential for the conversion of the starting composition to the target steviol glycoside include steviol biosynthetic enzymes, UDP-glucosyltransferase (UGT) and/or UDP-recycling enzymes.

In one embodiment, the steviol biosynthetic enzyme comprises a Mevalonate (MVA) pathway enzyme.

In another embodiment, the steviol biosynthetic enzyme comprises a non-mevalonate 2-C-methyl-D-erythritol 4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the steviol biosynthetic enzyme is selected from: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase (copalyl diphosphate synthase), kaurene synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-cytosinyl diphosphate (diphosphocytidyl) -2-C-methyl-D-erythritol synthase (CMS), 4-cytosinyl diphosphate-2-C-methyl-D-erythritol kinase (CMK), 4-cytosinyl diphosphate synthase (MCS), l-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, and the like.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and/or steviol glycoside substrate to provide a target steviol glycoside.

As used below, unless otherwise specified, the term "SuSy _ AT" refers to a sucrose synthase having the amino acid sequence "SEQ ID 1" as described in example 1.

As used hereinafter, unless otherwise specified, the term "UGTSl 2" refers to a UDP-glucosyltransferase enzyme having the amino acid sequence "SEQ ID 2" as described in example 1.

As used hereinafter, the term "UGT 76G 1" refers to a UDP-glucosyltransferase having the amino acid sequence "SEQ ID 3" as described in example 1, unless otherwise specified.

In one embodiment, the steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, Escherichia coli (E.coli), Saccharomyces (Saccharomyces sp.), Aspergillus (Aspergillus sp.), Pichia (Pichia sp.), Bacillus (Bacillus sp.), Yarrowia (Yarrowia sp.), or the like. In another embodiment, a UDP-glucosyltransferase enzyme is synthesized.

In one embodiment, the UDP-glucosyltransferase is selected from: UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGT having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to these polypeptides; and isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, yarrowia.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or to any starting steviol glycoside having a-OH functionality at C13, resulting in a target steviol glycoside having a glycosidic bond of-O-glucose beta glucopyranoside at C13. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or to any starting steviol glycoside having a-COOH functional group at C19 to give the target steviol glycoside having a glycosidic bond of-COO-glucose β -glucopyranoside at C19. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to an existing glucose at C19 of any starting steviol glycoside, resulting in a target steviol glycoside having at least one additional glucose with at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to an existing glucose at C19 of any starting steviol glycoside, resulting in a target steviol glycoside having at least one additional glucose with at least one glycosidic bond of β 1 → 3 glucopyranoside on the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to an existing glucose at C13 of any starting steviol glycoside, resulting in a target steviol glycoside having at least one additional glucose with at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

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 another specific embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

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 another specific embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

Optionally, the process of the invention further comprises using more than one UGT on the starting composition to obtain the target steviol glycoside having more than one glucose unit than the starting composition. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2 or any UGT having > 85% amino acid sequence identity to UGT74G1, UGT85C2, UGT76G1, UGTSl2, EUGT11 and/or UGT91D2 or any combination thereof, which is capable of adding more than one glucose unit to a starting composition to obtain a steviol glycoside having more than one glucose unit compared to the starting composition.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding both glucose units to stevioside to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is selected from UGTSl2, EUGT11, UGT91D2, UGT76G1 or any UGT having > 85% amino acid sequence identity with UGTSl2, EUGT11, UGT91D2, UGT76G1 or any combination thereof. In another specific embodiment, the UDP-glucosyltransferase is UGTSl2 and UGT76G 1.

Optionally, the process of the invention further comprises recycling UDP to obtain UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycle catalyst and recycling the substrate such that catalytic amounts of UDP-glucosyltransferase and UDP-glucose are used to convert steviol and/or steviol glycoside substrate to the target steviol glycoside.

In one embodiment, the recycling catalyst is a sucrose synthase SuSy _ At or a sucrose synthase with > 85% amino acid sequence identity to SuSy _ At.

In one embodiment, the recycled substrate is sucrose.

Optionally, the method of the invention further comprises the use of a transglycosidase enzyme using an oligosaccharide or polysaccharide as a sugar donor to modify the acceptor target steviol glycoside molecule. Non-limiting examples include cyclodextrin glucosyltransferase (CGTase), fructofuranosidase, amylase, sucrase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl converting enzyme (fructosylinvertase), alkaline converting enzyme, acid converting enzyme, fructofuranosidase. In some embodiments, glucose and non-glucose sugars include, but are not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and conversion to the acceptor target steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside AM.

Optionally, the process of the present invention further comprises separating the target steviol glycoside from the medium to obtain a highly purified target steviol glycoside composition. The target steviol glycoside can be isolated by at least one suitable method, such as crystallization, separation by membrane separation, centrifugation, extraction, chromatography or a combination of such methods.

In one embodiment, the target steviol glycoside can be produced in a microorganism. In another embodiment, the steviol glycoside of interest may be secreted in a medium. In another embodiment, the released steviol glycosides may be continuously removed from the medium. In another embodiment, the target steviol glycoside is isolated after the conversion reaction is complete.

In one embodiment, the separation results in a composition comprising greater than about 80% by weight of the steviol glycoside of interest on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, the separating produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In a specific embodiment, the composition comprises greater than about 95% by weight of the target steviol glycoside. In further 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 a hydrate, a solvate, an anhydrate or a combination thereof.

The purified target steviol glycosides can be used in consumer products as sweeteners, flavor modifiers, flavors with modified properties and/or foam inhibitors. Suitable consumer products include, but are not limited to, food products, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

Brief Description of Drawings

Figure 1 shows the chemical structure of rebaudioside AM.

Figure 2 shows a pathway for the production of rebaudioside AM and various steviol glycosides from steviol.

Figure 3 shows the biocatalytic production of rebaudioside AM from steviol glycosides using the enzymes UGTSl2 and UGT76G1, with concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

Figure 4 shows the biocatalytic production of rebaudioside AM from rebaudioside E using the enzyme UGT76G1 with concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

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

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

Figure 7 shows an HPLC chromatogram of rebaudioside E. The peak with retention time of 10.835 min corresponds to rebaudioside E.

Figure 8 shows an HPLC chromatogram of the product of the biocatalytic production of rebaudioside E of rebaudioside AM. Peaks with retention times of 10.936 min and 11.442 min correspond to rebaudioside E and rebaudioside AM, respectively.

Figure 9 shows an 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 1H NMR spectrum (500MHz, pyridine-d 5) of rebaudioside AM.

FIG. 11 shows the HSQC spectrum (500MHz, pyridine-d 5) of rebaudioside AM.

FIG. 12 shows the H, H COSY spectrum (500MHz, pyridine-d 5) of rebaudioside AM.

Figure 13 shows the HMBC spectrum (500MHz, pyridine-d 5) of rebaudioside AM.

FIG. 14 shows the HSQC-TOCSY spectrum (500MHz, pyridine-d 5) of rebaudioside AM.

Figure 15a and figure 15b show the LC chromatogram of rebaudioside AM and the mass spectrum of rebaudioside AM, respectively.

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

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

Detailed Description

One object of the present invention is a biocatalytic process by preparation of a target steviol glycoside, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, rebaudioside AM or synthetic steviol glycosides from different starting compositions.

As used herein, the abbreviated term "reb" refers to "rebaudioside". Both terms have the same meaning and may be used interchangeably.

As used herein, "biocatalysis" or "biocatalysis" refers to the use of natural or genetically engineered biocatalysts, such as enzymes or cells (including microorganisms) comprising one or more enzymes, that are capable of performing a single or multiple step chemical transformation of organic compounds. Biocatalytic processes include fermentation, biosynthesis, bioconversion, and bioconversion processes. Isolated enzymes and whole cell biocatalysis are well known in the art. The biocatalyst protease may be a naturally occurring or recombinant protein.

As used herein, the term "steviol glycoside" refers to a glycoside of steviol, including, but not limited to: naturally occurring steviol glycosides, such as steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, rebaudioside AM, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides and combinations thereof.

Starting composition

As used herein, a "starting composition" refers to any composition (typically an aqueous solution) comprising one or more organic compounds containing at least one carbon atom.

In one embodiment, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols and various sugars.

The starting composition steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 or other glycosides of steviol present in the stevia plant, synthetic steviol glycosides, e.g. enzymatically glycosylated steviol glycosides and combinations thereof.

In one embodiment, the starting composition is steviol.

In another embodiment, the starting composition steviol glycoside is steviolmonoside.

In another embodiment, the starting composition steviol glycoside is steviolmonoside a.

In yet 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 rebaudioside B.

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

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

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

In yet 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 comprising more than one hydroxyl group. The polyol may be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. The polyol may also contain more than 4 hydroxyl groups, such as pentahydric, hexahydric, heptahydric, and the like, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, the polyol may also be a sugar alcohol, a polyol or a polyol which is a reduced form of a saccharide in which carbonyl groups (aldehydes or ketones, reducing sugars) have been reduced to primary or secondary hydroxyl groups. 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 hydrolysates, polyalditol (polyglycitol) and sugar alcohols or any other saccharide capable of being reduced.

The term "saccharide" refers to a compound of formula (CH)₂O)_n(wherein n is 3 to 30) aldehyde or ketone compounds substituted with a plurality of hydroxyl groups, and their oligomerizationAnd polymers. In addition, the saccharides of the present invention are substituted or deoxygenated at one or more positions. As used herein, saccharides include unmodified saccharides, saccharide derivatives, substituted saccharides and modified saccharides. As used herein, the phrases "saccharide derivative," "substituted saccharide," and "modified saccharide" are synonymous. Modified saccharide refers to any saccharide, or a combination thereof, in which at least one atom is added, removed, or substituted. Thus, derivatives of saccharides or substituted saccharides include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides and polysaccharides. The saccharide derivative or substituted saccharide is optionally deoxy at any corresponding C-position and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxy, acyl, acyloxy, amino, amido, carboxy derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfinyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinio, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamate, phospho, phosphonate, or any other functional group that can provide the saccharide derivative or substituted saccharide function to improve the sweet taste of the sweetener composition.

Examples of saccharides that can be used according to the present invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isohexose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, pullulan, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abicose, galactosamine, beet oligosaccharides, isomaltooligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, aspergillus niger oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, kestotetraose and the like), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the saccharides used herein may be in the D-configuration or the L-configuration.

The starting composition may be synthetic or purified (partial or complete), commercially available or prepared.

In one embodiment, the starting composition is glycerol.

In another embodiment, the starting composition is glucose.

In another embodiment, the starting composition is sucrose.

In another embodiment, the starting composition is starch.

In another embodiment, the starting composition is maltodextrin.

In another embodiment, the starting composition is cellulose.

In another embodiment, the starting composition is amylose.

The organic compounds of the starting composition are used as substrates for the production of the target steviol glycosides as described herein.

Target steviol glycosides

The steviol glycoside of the invention which is the object of the invention can be any steviol glycoside which 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 a, steviolbioside B, rubusoside, stevioside (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, rebaudioside AM, or other glycosides of steviol found in the stevia plant, synthetic steviol glycosides, e.g., enzymatically glycosylated 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 rebaudioside a.

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

In one embodiment, the invention is a biocatalytic process rebaudioside B for production.

In one embodiment, the present invention is a biocatalytic process for the production of rubusoside B.

In one embodiment, the invention is a biocatalytic method for producing stevioside.

In one embodiment, the invention is a biocatalytic method for the production of stevioside A (rebaudioside KA).

In one embodiment, the invention is a biocatalytic method for the production of stevioside B.

In one embodiment, the invention is a biocatalytic method for the production of stevioside C.

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

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E2.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E3.

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

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

In another specific embodiment, the invention provides a biocatalytic process for the production of rebaudioside AM from a starting composition comprising rebaudioside E and UDP-glucose.

Optionally, the process of the present invention further comprises separating the target steviol glycoside from the medium to obtain a highly purified target steviol glycoside composition. The target steviol glycoside can be isolated by any suitable method, e.g., crystallization, separation by membrane separation, centrifugation, extraction, chromatography or a combination of such methods.

In particular embodiments, the methods described herein produce highly purified target steviol glycoside compositions. As used herein, the term "highly purified" refers to a composition having greater than about 80% by weight of a steviol glycoside of interest, on an anhydrous (dry) basis. In one embodiment, the highly purified target steviol glycoside composition comprises greater than about 90% by weight of the target steviol glycoside on an anhydrous (dry) basis, e.g., a target steviol glycoside content of 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% on a dry weight basis.

In one embodiment, when the steviol glycoside of interest is reb AM, the methods described herein provide a composition having a reb AM content of greater than about 90% on a dry weight basis. In another specific embodiment, when the steviol glycoside of interest is reb AM, the methods described herein provide a composition comprising a reb AM content of greater than about 95% on a dry weight basis.

Microbial and enzyme preparations

In one embodiment of the present invention, a microorganism (microbial cell) and/or an enzyme preparation is contacted with a medium comprising a starting composition to produce a target steviol glycoside.

The enzyme is provided as a whole cell suspension, crude lysate, purified enzyme, or a combination thereof. In one embodiment, the biocatalyst is a purified enzyme capable of converting a starting composition into a target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising at least one enzyme capable of converting a starting composition into a target steviol glycoside. In another embodiment, the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting a starting composition into a target steviol glycoside.

In another embodiment, the biocatalyst is one or more microbial cells comprising an enzyme capable of converting a starting composition into a target steviol glycoside. The enzyme is located on the cell surface, within the cell or both on the cell surface and within the cell.

Suitable enzymes for converting the starting composition to the target steviol glycoside include, but are not limited to, steviol biosynthetic enzymes and UDP-glucosyltransferase (UGT). Optionally, it may comprise a UDP-recycling enzyme.

In one embodiment, the steviol biosynthetic enzyme is selected from: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), cytosinyl-2-C-methyl-D-erythritol synthase (CMS), cytosinyl-2-C-methyl-D-erythritol kinase 4-diphosphate (CMK), cytosinyl-2-C-methyl-D-erythritol kinase 4-diphosphate (MCS), l-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, and the like.

In one embodiment, the steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, yarrowia, or the like. In another embodiment, the UDP-glucosyltransferase is synthetic.

In one embodiment, the UDP-glucosyltransferase is selected from: UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGTs having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to these polypeptides and isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus and yarrowia.

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 a functional group of-OH at C13 to give the target steviol glycoside having the glycosidic bond of-O-glucose beta glucopyranoside at C13. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

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 the target steviol glycoside having the glycosidic bond of-COO-glucose β -glucopyranoside at C19. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to an existing glucose at C19 of any starting steviol glycoside, resulting in a target steviol glycoside having at least one additional glucose with at least one glycosidic bond of β 1 → 3 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside a. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

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 another specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

Optionally, the process of the invention further comprises recycling UDP to obtain UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycle catalyst and a recycle substrate to biologically convert steviol and/or a steviol glycoside substrate to a target steviol glycoside using a catalytic amount of UDP-glucosyltransferase and UDP-glucose. The UDP recycling enzyme may be a sucrose synthase SuSy _ At or a sucrose synthase with > 85% amino acid sequence identity to SuSy _ At, and the substrate in circulation may be sucrose.

Optionally, the method of the invention further comprises the application of a transglycosidase enzyme using an oligosaccharide or polysaccharide as a sugar donor in order to modify the acceptor target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, sucrase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl converting enzyme, alkaline converting enzyme, acid converting enzyme, fructofuranosidase. In some embodiments, glucose and non-glucose sugars include, but are not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and conversion to the acceptor target steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside AM.

In another embodiment, the UDP-glucosyltransferase enzyme capable of adding at least one glucose unit to a steviol glycoside of the starting composition has > 85% amino acid sequence identity with a UGT selected from the list GenInfo identifier below, preferably from tables 1 and 2.

TABLE 1

TABLE 2

GI numbering	Accession number	Source	Internal reference
				460409128	XP.004249992.1	Tomato	UGTSl
460386018	XP.004238697.1	Tomato	-
				460409134	XP.004249995.1	Tomato	-
460410132	XP.004250485.1	Tomato	UGTSl2
				460410130	XP.004250484.1	Tomato	-
460410128	XP.004250483.1	Tomato	-
				460378310	XP.004234916.1	Tomato	-
209954733	BAG80557.1	Matrimony vine (Lycium barbarum)	UGTLB
				209954725	BAG80553.1	Chinese wolfberry	-

One embodiment of the invention is a microbial cell comprising an enzyme, i.e. an enzyme capable of converting a starting composition into a target steviol glycoside. Thus, some embodiments of the present methods comprise contacting the microorganism with a medium comprising the starting composition to obtain a medium comprising at least one steviol glycoside of interest.

The microorganism may be any microorganism having the requisite enzymes to convert the starting composition to the target steviol glycoside. These enzymes are encoded within the microbial genome.

Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, and yarrowia, among others.

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 microorganisms may be immobilized on a solid support made of inorganic or organic materials. Non-limiting examples of solid supports include derivatized cellulose or glass, metal oxides or membranes. For example, the microorganisms may be immobilized on the solid support by covalent binding, adsorption, cross-linking, capture or encapsulation.

In another embodiment, the enzyme capable of converting the starting composition to the target steviol glycoside is secreted from the microorganism and into the reaction medium.

Optionally purifying the target steviol glycoside. Purification of the target steviol glycoside from the reaction medium can be carried out by at least one suitable method to obtain a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.

Use of

The highly purified target glycosides obtained according to the present invention, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used "as such" or in combination with additional sweeteners, flavors, food ingredients and combinations thereof.

Non-limiting examples of flavorants include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, almond, cola, cinnamon, sugar, raffinose, vanilla, and combinations thereof.

Non-limiting examples of additional food ingredients include, but are not limited to, acidulants, organic and amino acids, colorants, fillers, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof.

Highly purified target glycosides obtained according to the present invention, particularly steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be prepared in various polymorphic forms including, but not limited to, hydrates, solvates, anhydrates, amorphous forms and combinations thereof.

Highly purified target glycosides obtained according to the present invention, particularly steviolmonoside, steviolmonoside a, steviolbioside, rubusoside, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be incorporated as high-intensity natural sweeteners in foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tabletop products (tabletop products), cereals, dairy products, toothpastes and other oral compositions, and the like.

The highly purified target glycosides obtained according to the invention, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as sole sweetening agents or can be used in combination with at least one naturally occurring high intensity sweetener, such as rebaudioside a, rebaudioside a2, rebaudioside A3, rebaudioside B, rebaudioside C2, rebaudioside D2, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L, rebaudioside M2, rebaudioside N, rebaudioside O2, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T1, rebaudioside U2, rebaudioside V, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E2, stevioside F, mogroside, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and salts thereof, thaumatin, tansine, pindolamin (pernaultidin), sesquiterpenes (rebaudioside K, rebaudioside I), rebaudioside I, rebaudioside K, rebaudioside E2, rebaudioside F, mogroside, rebaudioside K, rebaudioside I, rebaudioside K, rebaudioside I, rebaudioside K, rebaudioside K, rebaudioside, abrin, brazilian glycyrrhizin, fleshy hemsleyasaponin (carnosiflorides), cyclocarin, secodammarane-type triterpene glycosides (pterocaryosides), polypodoside a, brazilein, hernandulcin (hernandulcin), fexodulcin (philoducin), sarsasaponin, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastin (neoastibin), trans-cinnamaldehyde, monatin and salts thereof, hederanin a (selliguein a), hematoxylin, monellin, caritin, pterocaryosin a (pterocaryoside a), pterocaryosin B, mabinlin, pertactin (pentadin), modified taste glycoprotein, curculin, neonaulin (neonaulin), chlorogenic acid, cerulin, mogroside V, mogroside and combinations thereof.

In a particular embodiment, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be used in a sweetener composition comprising a compound selected from the group consisting of: rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C2, rebaudioside D2, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K2, rebaudioside L, rebaudioside M2, rebaudioside N, rebaudioside O2, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T1, rebaudioside U632, rebaudioside U, rebaudioside V, rebaudioside W638, rebaudioside W2, rebaudioside W638, rebaudioside Z2, rebaudioside Z638, rebaudioside D, rebaudioside Z8, rebaudioside D638, rebaudioside D, rebaudioside Z, rebaudioside D638, rebaudioside D, rebaudioside W638, rebaudioside W638, rebaudioside D, rebaudioside K368, rebaudioside K368, rebaudioside D, rebaudioside K638, rebaudioside D, rebaudioside K638, rebaudioside W, rebaudioside, stevioside E, stevioside E2, stevioside F, NSF-02, mogroside V, Lo Han Guo, psicose, allose, D-tagatose, erythritol, and combinations thereof.

Highly purified glycosides of interest, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can also be used in combination with synthetic high intensity sweeteners, such as sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, sulosan advantAMe and salts and combinations thereof.

Furthermore, highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with natural sweetener inhibitors, such as gymnemic acid, dulcin, ziziphin, sweetening essence (lactisola) and the like. Steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be used in combination with various umami enhancers. Steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be mixed with umami and sweet amino acids, such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamic acid, lysine, tryptophan, and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with one or more additives selected from the group consisting of sugars, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts, including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavor and flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with a polyol or alditol. The term "polyol" refers to a molecule comprising more than one hydroxyl group. The polyol may be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. The polyols may also contain more than four hydroxyl groups, such as pentahydric, hexahydric, heptahydric, and the like, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, the polyol may also be a sugar alcohol, a polyol or a polyol which is a reduced form of a sugar in which carbonyl groups (aldehydes or ketones, reducing sugars) have been reduced to primary or secondary hydroxyl groups. Examples of polyols include, but are not limited to erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomaltulose, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycols and sugar alcohols or any other sugar capable of reduction, which do not adversely affect the taste of the sweetener composition.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in combination with a low calorie sweetener, such as D-tagatose, L-sugar, L-sorbose, L-arabinose and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, rubusoside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and rebaudioside E3And/or rebaudioside AM may also be used in combination with various sugars. The term "saccharide" is generally meant to have the formula (CH)₂O)_n(wherein n is 3 to 30) aldehyde or ketone compounds substituted with a plurality of hydroxyl groups, and oligomers and polymers thereof. In addition, the saccharides of the present invention may be substituted or deoxygenated at one or more positions. As used herein, saccharides include unmodified saccharides, saccharide derivatives, substituted saccharides and modified saccharides. As used herein, the phrases "saccharide derivative," "substituted saccharide," and "modified saccharide" are synonymous. Modified saccharide means any saccharide in which at least one atom or a combination thereof has been added, removed or substituted. Thus, saccharide derivatives or substituted saccharides include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides and polysaccharides. The carbohydrate derivative or substituted carbohydrate may be optionally deoxygenated at any corresponding C-position and/or substituted with one or more moieties (e.g., hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imino, sulfonyl, sulfinyl, sulfoxy, sulfamoyl, alkoxycarbonyl, carboxamide, phosphono, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamoyl, phosphorous, phosphonate, or any other viable functional group) so long as the carbohydrate derivative or substituted carbohydrate acts to improve the sweetness of the sweetener composition.

Examples of saccharides for use according to the invention include, but are not limited to: psicose, melibiose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isohydralose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abicose, galactosamine, oligosaccharose, isomaltooligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, aspergillus niger oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, kestotetraose and the like), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the saccharides used herein may be in the D-configuration or the L-configuration.

The highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to the invention can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients are generally classified into, for example, the following categories: carotenoids, dietary fibers, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, phytosterols and stanols (phytosterols and phytostanols); a polyol; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/mercaptans; an amino acid; a protein; vitamins and minerals. Functional ingredients may also be classified based on their health benefits (e.g., cardiovascular, cholesterol lowering and anti-inflammatory). Exemplary functional ingredients are provided in WO2013/096420 (the contents of which are hereby incorporated by reference).

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to the invention can be used as high intensity sweeteners in order to produce zero-calorie, low-calorie or diabetic beverages and foodstuffs with improved taste characteristics. It can also be used in beverages, foods, pharmaceuticals and other products where sugar is not used. In addition, highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as sweeteners not only for beverages, foods and other products intended for human consumption, but also for animal feeds and forage with improved properties.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to the invention can be used as flavor modifiers to produce zero-calorie, low-calorie or diabetic beverages and foodstuffs with modified flavor. When used as a flavor modifier or a flavoring agent (FMP) with improved properties, highly purified steviol glycosides of interest are used in consumer products at detection levels below the flavor modifier or FMP. Thus, the flavor modifier or FMP does not impart its own detectable taste or flavor to the consumable product, but rather serves to improve the consumer's detection of the taste and/or flavor of other ingredients in the consumable product. An example of taste and flavor modification is sweetness enhancement, where the flavor modifier or FMP itself does not contribute to the sweetness of the consumer product, but can enhance the quality of the sweetness perceived by the consumer.

Examples of consumer products in which highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as flavor modifiers or flavoring agents with modified properties include, but are not limited to, alcoholic beverages such as vodka, wine, beer, white spirit, sake and the like; natural juice; refreshing beverage; a carbonated soft drink; a sugar-free beverage; a zero-calorie beverage; reduced-calorie beverages and foods; a yogurt drink; instant juice; instant coffee; a powdered instant beverage; can products; syrup; fermenting the soybean paste; soy sauce; vinegar; a seasoning; mayonnaise; tomato sauce; curry; soup; an instant bouillon; soy sauce powder; vinegar powder; various types of biscuits; rice biscuits; a thin crisp biscuit; bread; chocolate; caramel; a candy; a chewing gum; jelly; pudding; preserved fruit and vegetable; fresh cream; jam; citrus pulp; flower sauce; milk powder; ice cream; a snowflake; bottling vegetables and fruits; canning and boiling beans; cooking meat and food with sweet juice; agricultural vegetable food; seafood; ham; sausages; fish ham; fish sausages; fish paste; a fried fish product; drying marine products; freezing the food; pickling the seaweed; pickling meat; tobacco; a pharmaceutical product; and many other food products. Which in principle can be used in an unlimited number of applications.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to the invention can be used as antifoaming agents to produce zero-calorie, low-calorie or diabetic beverages and foodstuffs.

Examples of consumer products in which highly purified target steviol glycosides, in particular steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as sweet compounds include, but are not limited to: alcoholic beverages such as vodka, wine, beer, white spirit and sake and the like; natural juice; refreshing beverage; a carbonated soft drink; a sugar-free beverage; a zero-calorie beverage; reduced-calorie beverages and foods; a yogurt drink; instant juice; instant coffee; a powdered instant beverage; can products; syrup; fermenting the soybean paste; soy sauce; vinegar; a seasoning; mayonnaise; tomato sauce; curry; soup; an instant bouillon; soy sauce powder; vinegar powder; various types of biscuits; rice biscuits; a thin crisp biscuit; bread; chocolate; caramel; a candy; a chewing gum; jelly; pudding; preserved fruit and vegetable; fresh cream; jam; citrus pulp; flower sauce; milk powder; ice cream; a snowflake; bottling vegetables and fruits; canning and boiling beans; cooking meat and food with sweet juice; agricultural vegetable food; seafood; ham; sausages; fish ham; fish sausages; fish paste; a fried fish product; drying marine products; freezing the food; pickling the seaweed; pickling meat; tobacco; a pharmaceutical product; and many other food products.

In the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, table top products and chewing gum, conventional methods such as mixing, kneading, dissolution, acid leaching, osmosis, percolation, spraying, atomising, infusing and other methods may be used.

Furthermore, the highly purified target steviol glycoside, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to the invention can be used in dry or liquid form.

Highly purified target steviol glycosides can be added before or after heat treatment of the food product. The amount of highly purified target steviol glycosides, in particular steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM depends on the purpose of use. As mentioned above, it can be used alone or in combination with other compounds.

The present invention also relates to a method of sweetness enhancement in a beverage using as sweetness enhancer the following compounds: steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, and/or rebaudioside AM. Accordingly, the present invention provides a beverage comprising a sweetener and, as a sweetness enhancer, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM, wherein steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM are present at a concentration at or below their respective sweetness recognition thresholds.

As used herein, the term "sweetness enhancer" refers to a compound that is capable of enhancing or potentiating the perception of sweetness in a composition, such as a beverage. The term "sweet taste enhancer (sweet taste enhancer)" is synonymous with the terms "sweet taste enhancer (sweet taste promoter)", "sweet taste potentiator (sweet taste potentiator)", "sweet taste potentiator (sweet taste enhancer)" and "sweet taste enhancer (sweet taste enhancer)".

As generally used herein, the term "sweet taste recognition threshold concentration" is the lowest known concentration of sweet compounds that can be perceived by human taste, typically about 1.0% sucrose equivalent (1.0% SE). Generally, a sweetness enhancer, when present at or below the sweetness recognition threshold concentration for a given sweetness enhancer, can enhance or potentiate the sweetness of the sweetener without providing any noticeable sweetness by itself; however, the sweetness enhancer itself may provide sweetness at a concentration above its sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific to a particular enhancer and may vary depending on the beverage matrix. The sweetness recognition threshold concentration can be readily determined by taste testing by increasing the concentration of a given enhancer until a sucrose equivalent greater than 1.0% is detected in a given beverage base. A concentration that provides about 1.0% sucrose equivalent is considered a sweet taste recognition threshold.

In some embodiments, the sweetener is present in the beverage in an amount from about 0.5% to about 12% by weight, e.g., 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% to about 10%, for example from about 2% to about 8%, from about 3% to about 4%, 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 specific 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, high fructose corn syrup and high fructose corn syrup.

In another embodiment, the sweetener is erythritol.

In another embodiment, the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose, D-turanose, D-leucrose, and combinations thereof.

It is contemplated that the sweetener may be used alone or in combination with other sweeteners.

In one embodiment, the rare sugar is D-allose. In a more specific embodiment, D-allose is present in the beverage in an amount of from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is D-psicose. In a more specific embodiment, the D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is D-ribose. In a more specific embodiment, the D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is D-tagatose. In a more specific embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is L-glucose. In a more specific embodiment, the L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In one embodiment, the rare sugar is L-fucose. In a more specific embodiment, L-fucose is present in the beverage in an amount from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is L-arabinose. In a more specific embodiment, the L-arabinose is present in the beverage in an amount of from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is D-turanose. In a more specific embodiment, D-turanose is present in the beverage in an amount from about 0.5% to about 10% by weight, e.g., from about 2% to about 8%.

In another embodiment, the rare sugar is D-leucrose. In a more specific embodiment, D-leuconostoc disaccharide is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

Adding the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalent for a beverage comprising the sweetener and sweetness enhancer as compared to a corresponding beverage in the absence of the sweetness enhancer. Furthermore, the sweetness increase may be greater than the detectable sweetness of a solution comprising the same concentration of the at least one sweetness enhancer without any sweetener.

Accordingly, the present invention also provides a method for enhancing the sweetness of a beverage comprising a sweetener, the method comprising providing a beverage comprising a sweetener, and adding a sweetness enhancer selected from the group consisting of steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM or combinations thereof, wherein steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM are present at a concentration at or below its sweetness recognition threshold.

Addition of steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, and/or rebaudioside AM to a beverage comprising a sweetener at a concentration at or below the sweetness recognition threshold can increase the sucrose equivalent detected by about 1.0% to about 5.0%, e.g., 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 embodiments illustrate preferred embodiments of the invention for preparing highly purified target steviol glycosides, in particular steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. It is to be understood that this invention is not limited to the materials, proportions, conditions and methods recited in the examples, which are exemplary only.

Examples

Example 1

Protein sequences of engineered enzymes for use in biocatalytic processes

SEQ ID 1：

(> SuSy _ At, variant PM1-54-2-E05 (engineered sucrose synthase; WT gene source: Arabidopsis)

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQAVPLAQDD

SEQ ID 2:

' UGTSl2 variant 0234 (engineered glucosyltransferase; WT gene source: tomato)

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENSTVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALPEGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3：

UGT76G1 variant 0042 (engineered glucosyltransferase; WT gene source: stevia rebaudiana Bertoni)

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLVDSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWNSTLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

Example 2

Expression and formulation of the SuSy _ At variant of SEQ ID 1

The gene encoding the SuSy _ At variant of SEQ ID 1 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were cultured at 37 ℃ in ZYM505 medium (F. WilliaM student, Protein Expression and Purification 41 (2005)) supplemented with kanamycin (50mg/l) 207-234. IPTG (0.2mM) induced gene expression in log phase and was carried out at 30 ℃ and 200rpm for 16-18 hours.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH7.0; 2mM MgCl)₂DNA nuclease 20U/mL, lysozyme 0.5mg/mL) was resuspended at an optical density of 200 (at 600nm (OD)₆₀₀) Measured at (b). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted with distilled water at 50:50 to obtain an enzyme activity preparation.

For the enzymatically active preparation of SuSy _ At, the unit activity is defined as follows: 1mU of SuSy _ At converted 1nmol of sucrose to fructose in 1 minute. The reaction conditions for this assay were 30 ℃, 50mM potassium phosphate buffer pH7.0, t₀400mM sucrose, 3mM MgCl₂And 15mM Uridine Diphosphate (UDP).

Example 3

Expression and formulation of UGTSl2 variant of SEQ ID 2

The gene encoding the UGTSl2 variant of SEQ ID 2 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were cultured at 37 ℃ in ZYM505 medium (F. WilliaM student, Protein Expression and Purification 41 (2005)) supplemented with kanamycin (50mg/l) 207-234. Gene expression was induced in log phase with IPTG (0.1mM) and at 30 ℃ and 200rpm for 16-18 hours.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mMMgCl₂DNA nuclease 20U/mL, lysozyme 0.5mg/mL) was resuspended to an optical density of 200 (measured at 600nm (OD)₆₀₀)). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μ M filter and diluted 50:50 with 1M sucrose solution to give an enzyme active preparation.

For the enzyme activity preparation of UGTSl2, the unit activity is defined as follows: 1mU UGTSl2 converted 1nmol of rebaudioside A (RebA) to rebaudioside D (Reb D) in 1 minute. The reaction conditions for this assay were 30 ℃, 50mM potassium phosphate buffer pH7.0, t₀10mM of RebA, 500mM of sucrose, 3mM of MgCl₂And 0.25mM Uridine Diphosphate (UDP) and 3U/mL of SuSy _ At.

Example 4

Expression and formulation of UGT76G1 variant of SEQ ID 3

The gene encoding the UGT76G1 variant of SEQ ID 3 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl)₂DNA nuclease 20U/mL, lysozyme 0.5mg/mL) was resuspended to an optical density of 200 (measured at 600nm (OD)₆₀₀)). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μ M filter and diluted 50:50 with 1M sucrose solution to give an enzyme active preparation.

For the enzyme activity preparation of UGT76G1, the unit activity is defined as follows: 1mU UGT76G1 converted 1nmol of rebaudioside D (Reb D) to rebaudioside M (Reb M) in 1 minute. The reaction conditions for this assay were 30 ℃ with 50mM potassium phosphate bufferpH 7.0，t₀10mM of RebA, 500mM of sucrose, 3mM of MgCl₂And 0.25mM Uridine Diphosphate (UDP) and 3U/mL of SuSy _ At.

Example 5

Synthesis of rebaudioside AM from stevioside in a one pot reaction with addition of UGTSl2, SuSy _ At and UGT76G1

Rebaudioside am (reb am) was synthesized directly from stevioside in a one-pot reaction using 3 enzymes (see examples 1, 2, 3 and 4) (see fig. 3): UGTSl2 (variant of SEQ ID 2), SuSy _ At- (variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 105U/L UGTSl2, 405U/L SuSy _ At, 3U/L UGT76G1, 5mM stevioside, 0.25mM Uridine Diphosphate (UDP), 1M sucrose, 4mM MgCl₂And potassium phosphate buffer (pH 6.6). First, 207mL of distilled water was mixed with 0.24g of MgCl₂·6H₂O, 103g sucrose, 9.9mL 1.5M potassium phosphate buffer (pH 6.6) and 15g stevioside. After dissolving the components, the temperature was adjusted to 45 ℃ and UGTSl2, SuSy _ At, UGT76G1 and 39mg UDP were added. The reaction mixture was incubated in a 45 ℃ shake flask for 24 hr. An additional 39mg of UDP was added at 8 and 18 hours. The content of reb AM, reb E, stevioside, reb M, reb B, steviolbioside and reb I was analyzed by HPLC at several time points.

For analysis, 17% H was used₃PO₄The biotransformation sample was inactivated by adjusting the reaction mixture to ph5.5 and then boiled for 10 minutes. The resulting sample was filtered, and the filtrate was diluted 10-fold for HPLC analysis. HPLC analysis was performed on an Agilent HP 1200HPLC system consisting of a pump, column thermostat, autosampler, UV detector capable of background correction and data acquisition system. The analytes were isolated using an Agilent Poroshell 120 SB-C18, 4.6mm x 150mm, 2.7 μm at 40 ℃. The mobile phase consisted of two premixes:

premix 1 comprising 75% 10mM phosphate buffer (ph2.6) and 25% acetonitrile; and

premix 2 containing 68% 10mM phosphate buffer (ph2.6) and 32% acetonitrile.

The elution gradient started from premix 1, becoming premix 2 to 50% at 12.5 minutes and premix 2 to 100% at 13 minutes. The total run time was 45 minutes. The column temperature was maintained at 40 ℃. The amount of sample was 5. mu.L. The rebaudioside species were detected by UV at 210 nm.

Table 3 shows the process of converting stevioside to the identified rebaudioside species (area percentage) at each time point. FIGS. 5 and 6 show chromatograms of stevioside and the reaction mixture at 24 hours, respectively. One skilled in the art will appreciate that retention times can sometimes vary with solvent and/or equipment.

TABLE 3

Bioconversion of stevioside to reb AM

Example 6

Synthesis of rebaudioside AM from rebaudioside E in a one-pot reaction, with addition of SuSy _ At and UGT76G1

Rebaudioside am (reb am) was synthesized directly from rebaudioside e (reb e) using 2 enzymes in a one-pot reaction (see examples 1, 2 and 4) (see figure 4): SuSy _ At- (variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final reaction solution contained 405U/L UGTSl2, 405U/L SuSy _ At, 3U/L UGT76G1, 5mM reb E, 0.25mM Uridine Diphosphate (UDP), 1M sucrose, 4mM MgCl₂.6H₂O and potassium phosphate buffer (pH 6.6). First, 37mL of distilled water was mixed with 40.3mg of MgCl₂17.12g of sucrose, 1.65mL of 1.5M potassium phosphate buffer (pH 6.6) and 5.04g of reb E were mixed. After dissolving the components, the temperature was adjusted to 45 ℃, and SuSy _ At, UGT76G1 and 6.5mg UDP were added. The reaction mixture was incubated in a 45 ℃ shake flask for 24 hr. An additional 6.5mg of UDP was added at 8 and 18 hours. The content of reb AM, reb E, stevioside, reb a, reb M, reb B and steviolbioside was analyzed by HPLC at several time points.

For analysis, 17% H was used₃PO₄The biotransformation sample was inactivated by adjusting the reaction mixture to ph5.5 and then boiled for 10 minutes. The resulting sample was filtered, and the filtrate was diluted 10-fold for HPLC analysis. In an Agilent HP 1200HPLC systemHPLC analysis was performed and the system consisted of a pump, column thermostat, autosampler, UV detector with background correction and data acquisition system. The analytes were isolated using an Agilent Poroshell 120 SB-C18, 4.6mm x 150mm, 2.7 μm at 40 ℃. The mobile phase consisted of two premixes:

premix 1 comprising 75% 10mM phosphate buffer (ph2.6) and 25% acetonitrile; and

premix 2 containing 68% 10mM phosphate buffer (ph2.6) and 32% acetonitrile.

Table 4 shows the process of converting rebaudioside E to the identified rebaudioside species (area percentage) at each time point. Figures 7 and 8 show the chromatograms of rebaudioside E and the reaction mixture at 24 hours, respectively. One skilled in the art will appreciate that retention times can sometimes vary with solvent and/or equipment.

TABLE 4

Biotransformation of reb E into reb AM

Example 7

Purification of rebaudioside AM

The reaction mixture of example 5 (after 24 hr) was washed with H₃PO₄The pH was adjusted to pH5.5 to inactivate it and then boiled for 10 minutes. After boiling, the reaction mixture was filtered and diluted to 5% solids with RO water. The diluted solution was passed through a 1L column (Cangzhou Yuanwei, China) packed with YWD03 macroporous adsorbent resin. The adsorbed steviol glycosides were eluted with 5L 70% ethanol. The resulting eluate was evaporated to dryness to give 16g of dry powder, which was dissolved in 80mL of 70% methanol. The solution was crystallized at 20 ℃ for 3 days. The crystals were isolated by filtration and dried in a vacuum oven at 80 deg.C18 hours, 10.4g of pure reb AM crystals with a purity of 95.92% are obtained, determined by HPLC assay. The chromatogram for reb AM is shown in FIG. 9. One skilled in the art will appreciate that retention times can sometimes vary with solvent and/or equipment.

Example 8

Structural elucidation of rebaudioside AM

NMR experiments were performed using a Bruker 500MHz spectrometer, where the samples were dissolved in pyridine-d 5. Observed from delta_C123.5, 135.5, 149.9ppm and delta_HSignals from 7.19, 7.55, 8.71ppm pyridine-d 5 and from the sample.

In pyridine-d₅Of rebaudioside AM¹The H-NMR spectrum showed excellent quality of the sample (see FIG. 10). HSQC (see FIG. 12b) shows the presence of an exo-methylene group in the sugar domain with long-range coupling to C-15, which can be observed in H, H-COSY (FIG. 12). The deep field signals of the other quaternary carbons (C-13, C-16 and C-19) were detected by HMBC (FIG. 13). The correlation of the signals in HSQC, HMBC and H, H-COSY reveals the presence of steviol glycosides with the following aglycone structure:

correlation of the HSQC and HMBC signals revealed five anomeric signals. The coupling constant of the anomeric proton is about 8Hz and the broad signal of its sugar bond enables the identification of these 5 sugars as β -D-glucopyranosides.

The observation that the anomeric proton was bound to HSQC and HMBC indicates the sugar bond and the association with aglycon. Assignment of sugar sequences was determined by using a combination of HSQC-TOCSY (FIG. 14) and HSQC.

The above NMR experiments were used to specify the chemical shifts, major coupling constants and major HMBC correlations of protons and carbons (see table 5).

TABLE 5

Chemical shifts of rebaudioside AM

TABLE 5 (continuation)

Chemical shifts of rebaudioside AM

Correlation of all NMR data shows rebaudioside AM with five β -D-glucopyranosides linked to the stevioside aglycone, as shown in the following chemical structure:

rebaudioside AM has the formula C₅₀H₈₀O₂₈Corresponding to a calculated monoisotopic molecular weight of 1128.5. For LCMS analysis, rebaudioside AM was dissolved in methanol and analyzed on a Cortecs UPLC 181.6 μm, 50x 2.1mm column using a Shimadzu Nexera 2020 UFLC LCMS instrument. The observed LCMS (negative ESI mode) result was 1127.3 (see fig. 15a and 15b, respectively), consistent with rebaudioside AM and corresponding to ion (M-H) -.

Solubility, sweetness and flavor modifying properties of Reb AM

Example 9

Solubility and solution stability of Reb AM were evaluated. Tables 6a and 6b below show the composition of the test samples, with the Total Steviol Glycoside (TSG) percentages shown in the last column of table 6 b.

Table 6 a: composition of the test samples:

TABLE 6b

Table 7: physical properties of Reb AM:

solution stability:

the solubility characteristics were measured as follows. The following solutions were prepared in water and stirred at 700rpm, respectively. If necessary, heat was applied while stirring for 2 minutes and 30 seconds. The time required for all the powders to dissolve completely was determined using a stopwatch and the dissolution temperature was recorded. The following table summarizes the solubility characteristics of rebaudioside D, M and AM. Surprisingly, the solubility of Reb AM is significantly higher than other minor and major steviol glycosides.

Table 8: comparison of solubility characteristics

Table 9: summary of the stability of the primary and secondary steviol glycoside solutions:

SG/characteristics	Reb A*	Stevioside	Reb AM	Reb D	Reb M
						Solubility in water	<0.7％	<0.7％	10％	0.1％	0.3％

Stevioside has a slightly lower solubility in aqueous solution than Reb a. Ref: celaya et al (2016) int.J. of Food studios, V.5, p 158-

Example 10

Reb AM was evaluated for sensory attributes.

Sensory attributes

Steviol glycoside molecules are known for their altered sweetness characteristics, which are a function of the sugar moiety present in their structure. Because steviol glycosides comprise both hydrophobic (steviol) and hydrophilic (sugar moieties), they can exhibit flavor modification at a dosage level without producing any noticeable perception of sweetness.

Isocaloric assay of Reb AM and other steviol glycosides:

concentration levels of five test sweeteners were determined to match 2.5%, 5%, 7.5% and 10% sucrose equivalents in acidified water (pH 3.2), for which 40 panelists were enrolled to conduct a two-out forced choice (2-AFC) test at each concentration level.

Evaluate the samples and determine the desserts at which point 50% of the panelists chose the sucrose sample to be sweeter and 50% chose the stevia sample to be sweeter.

Using four equal sweetness concentrations and their corresponding target sweetness values as data, a Beidler model was used to fit the concentration-response relationship.

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

Table 10: equivalent sweetness concentration (ppm) and sweetness (x sugar equivalent) of Reb AM and other steviol glycosides

Effect of Reb AM on taste and flavor characteristics of food and beverage applications

A series of experiments were performed to evaluate the effect of Reb AM on taste and flavor. Sweet taste and taste/flavor changes can interact in food and beverage applications. To determine the impact of taste and flavor modification in different applications, a sensory method was specified by fema (flavor and Extract Manufacturing association) that determines the sweetness perception threshold determination introduced in experiment 1, as will be discussed below.

Experiment 1 provided an estimate of the concentration of Reb AM in water that had little effect on sweetness perception. The sweetness perception threshold concentration provides a significantly lower sweetness than a 1.5% sugar water solution. A summary of the sweetness perception thresholds for selected steviol glycosides is provided in table 11 below.

TABLE 11

Experiment 2 (discussed further below) discusses the effect of Reb AM on the flavor profile of non-alcoholic beverages. The effect of Reb AM on different taste attributes of beverages was determined using commercially available samples of raspberry watermelon coconut water, without (control) and with Reb AM (test). The results show that the test sample with Reb AM has significantly higher mango peach flavor, coconut water aroma, and overall preference compared to the control sample (95% confidence).

Experiment 3, discussed further below, discusses the effect of Reb AM on the taste and flavor characteristics of sweet dairy products. The sensory panel tested samples of stevia rebaudiana (Reb a), a sweet, sugarless chocolate flavored milk protein milkshake, without (control) and with Reb AM. The panel found that the test sample containing 50ppm Reb AM had significantly lower bitterness, metallic notes, whey protein and bitter aftertaste, and higher cocoa, dairy, vanilla and overall liking (95% confidence) than the control (95% confidence).

A panel of trained and experienced taste panellists evaluated non-caloric lemon-lime Carbonated Soft Drinks (CSD) sweetened with 500ppm Reb AM, Reb D or Reb M samples. Panelists found that CSD with Reb AM was less sweet than the other samples, but had significantly less bitter and lingering sweetness, especially CSD sweetened with Reb M.

Experiment 1 of example 10:

sweetness perception threshold for Reb AM

The application comprises the following steps: neutral water

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

Method of producing a composite material

TABLE 12

The following Table (Table 13) shows "guide for the sensing Testing of deflection with Modifying Properties with the FEMA GRAS issued according to FEMA^TMEvaluation of recognition threshold concentrations in the method outlined in section 1.4.2 of Programm "(flavour and Extract Manufacturers Association https:// www.femaflavor.org /). .

Watch 13

Experiment 2 of example 10:

rubus coreanus watermelon coconut water with Reb AM

The application comprises the following steps: alcohol-free beverage

Summary of the invention

The overall acceptability and strength of attributes of the two raspberry watermelon flavored coconut water samples were evaluated by 30 panelists in two links (sweet, raspberry, watermelon, coconut water, salty, bitter, and sweet, bitter aftertaste). In the first segment, the two samples included: 1) a control sample of shop purchased raspberry watermelon coconut water, and 2) a test sample of shop purchased raspberry watermelon coconut water containing Reb AM. The purpose of this test was to determine if the addition of Reb AM would affect the flavor profile of the non-alcoholic beverage. The results show that the test sample Reb AM has significantly higher mango and peach aroma, coconut water aroma and overall preference compared to the control sample (95% confidence).

Purpose(s) to

The purpose of this project was to assess whether the addition of stevia extract solids would have an impact on key flavor attributes in various beverage applications.

Purpose of testing

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

Method of producing a composite material

TABLE 14

Sample (I)

TABLE 15 beverage type I, non-Alcoholic

	Reference device	Reb AM
			Coconut juice, raspberry juice and watermelon juice	100	99.995
Reb AM		0.005
			In total (g)	100	100

Vitea Coco store brand

Results

Table 16 (below) summarizes the overall acceptance and average property intensity results for each sample.

Table 16: average fraction Rubi fructus watermelon coconut water containing 50ppm Reb AM

*＝80％CI，**＝90％CI，***＝95％CI

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

Conclusion

The overall acceptability and strength of attributes of the two raspberry watermelon flavored coconut water samples were evaluated by 30 panelists in two links (sweetness, watermelon flavor, raspberry flavor, coconut water flavor, astringency, artificial/chemical notes, bitterness, and sweetness aftertaste, bitterness aftertaste). In the first segment, the two samples included: 1) a control sample of shop purchased raspberry watermelon coconut water, and 2) a test sample of shop purchased raspberry watermelon coconut water containing Reb AM. The purpose of this test was to determine if the addition of Reb AM would affect the flavor profile of the non-alcoholic beverage. The results show that the test sample Reb AM has significantly higher watermelon aroma and overall preference compared to the control sample (95% confidence). A graph of the results is shown in fig. 16.

The test sample Reb AM had significantly lower sweetness aftertaste intensity (90% confidence) compared to the control sample.

Experiment 3 of example 10:

chocolate protein milkshake with Reb AM

The application comprises the following steps: milk/dairy product

Summary of the invention

The overall acceptability and attribute intensity (cocoa flavour, dairy flavour, whey protein, vanilla, metallic, sweet, bitter and aftertaste) of the two chocolate flavored dairy protein milkshake samples was evaluated by 30 trained panelists. These two samples included: 1) a "control" sample without added sugar comprising 300ppm purecruclle Reb a; and 2) a "test" sample without added sugar comprising 300ppm pureCircle Reb A and 50ppm Reb AM. The purpose of this test was to determine if the addition of Reb AM would affect the flavour profile of a dairy product. The panel found that the test sample containing 50ppm Reb AM had significantly lower bitterness, metallic notes, whey protein and lower bitter aftertaste, and had higher cocoa, dairy notes, vanilla notes and overall liking, compared to the control sample (95% confidence). Furthermore, there was no significant effect on sweetness intensity.

Purpose(s) to

Purpose of testing

The purpose of the test was to determine whether the flavor profile and overall acceptability of a control sample for dairy beverage applications was different from the test sample of the same beverage comprising Reb AM.

Method of producing a composite material

TABLE 17

Sample (I)

Watch 18

Ingredient table	Sugar references	50ppm Reb AM
			Milk, 2%	86.47	86.465
Whey protein 90 instant-non-GMO (Prod: 18618)	6.8250	6.8250
			Defatted milk powder	4.6269	4.6269
Maltrin QD M585	1.1066	1.1066
			Vitamin mixture-	0.0063	0.0063
Xanthan gum (Cold soluble)	0.0359	0.0359
			Forbes 10/12 cocoa powder 7113	0.7194	0.7194
Vanilla-flavored powder	0.1799	0.1799
			Reb A	0.0300	0.0300
Reb AM		0.0050
			Total of	100	100

Sugar contribution (g) per 100 g	Sugar references	165ppm Reb AM
			Milk, 2%	4.08	4.15
Defatted milk powder	2.41	2.41
			Maltrin QD M585	0.08	0.08
Total of	8.07	6.64

Calculation by Genesis R & D version 11.4

Table 19: effect of Reb AM on flavor modification of chocolate protein milkshakes

*＝80％CI，**＝90％CI，***＝95％CI

The panel found that the test sample containing 50ppm Reb AM had significantly lower bitterness, metallic notes, whey protein and lower bitter aftertaste (95% confidence) compared to the control.

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

Conclusion

The overall acceptability and attribute intensity (cocoa flavour, dairy flavour, whey protein, vanilla, metallic, sweet, bitter and aftertaste) of the two chocolate flavored dairy protein milkshake samples was evaluated by 30 panellists. These two samples included: 1) a "control" sample without added sugar comprising 300ppm purecruclle Reb a; and 2) a "test" sample without added sugar comprising 300ppm pureCircle Reb A and 50ppm Reb AM. The purpose of this test was to determine if the addition of Reb AM would affect the flavour profile of a dairy product. The panel found that the test sample containing 50ppm Reb AM had significantly lower bitterness, metallic notes, whey protein and lower bitter aftertaste, and had higher cocoa, dairy notes, vanilla notes and overall liking, compared to the control sample (95% confidence). Furthermore, there was no significant effect on sweetness intensity. A graph of the results is shown in fig. 16.

Although the present 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 present 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 present invention, 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 present invention.

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Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Glu Trp Leu Asp Gln Gln Ala

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Gly Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Lys Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Pro Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Ser Phe Gly Gly Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Arg Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Val Val Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu

450 455

Claims

1. A method for enhancing flavor in a consumable product comprising adding highly purified rebaudioside AM to the product at a level below the sweetness detection level of rebaudioside AM, wherein rebaudioside AM has the formula:

2. a process for the production of highly purified rebaudioside AM according to claim 1, comprising the steps of:

a. providing a starting composition comprising an organic compound having at least one carbon atom;

b. providing a biocatalyst selected from an enzyme preparation, a cell or a microorganism; the biocatalyst comprises at least one enzyme capable of converting the starting composition into rebaudioside AM;

c. contacting the biocatalyst with a medium comprising the starting composition to produce a medium comprising rebaudioside AM.

3. The method of claim 2, further comprising the steps of:

d. separating rebaudioside AM from the medium to provide a highly purified rebaudioside AM composition.

4. The process of claim 2, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside a, steviolbioside a, steviolbioside B, rubusoside, stevioside a (rebaudioside KA), stevioside B, stevioside C, rebaudioside E2, rebaudioside E3, other steviolbiosides, polyols, sugars, and combinations thereof.

5. The method of claim 2, wherein the microorganism is selected from the group consisting of Escherichia coli (E.coli), Saccharomyces (Saccharomyces sp.), Aspergillus (Aspergillus sp.), Pichia (Pichia sp.), Bacillus (Bacillus sp.) and Yarrowia (Yarrowia sp.).

6. The method of claim 2, wherein the enzyme is selected from the group consisting of: steviol biosynthetic enzymes, UDP glucosyltransferase, UDP glucose recycling enzyme, Mevalonate (MVA) pathway enzymes, 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), cytosinyl-2-C-methyl-D-erythritol 4-diphosphate synthase (CMS), cytosinyl-2-C-methyl-D-erythritol 4-diphosphate kinase (CMK), 4-diphosphocymidyl-2-C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MCS), l-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, UGT74G1, UGT85C2, UGT91D2, EUGT11, UGL 2, UGT76G1 or a variant thereof having > 85% amino acid sequence identity, > 86% amino acid sequence identity, > 87% amino acid sequence identity, > 88% amino acid sequence identity, a mutant variant of > 89% amino acid sequence identity, > 90% amino acid sequence identity, > 91% amino acid sequence identity, > 92% amino acid sequence identity, > 93% amino acid sequence identity, > 94% amino acid sequence identity, > 95% amino acid sequence identity, > 96% amino acid sequence identity, > 97% amino acid sequence identity, > 98% amino acid sequence identity, > 99% amino acid sequence identity; and combinations thereof.

7. The method of claim 3, wherein the rebaudioside AM content of the highly purified rebaudioside AM composition is greater than about 95% by weight on a dry weight basis.

8. A consumable product prepared by the process of claim 1, wherein the product is selected from the group consisting of a food product, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition.

9. The consumable product of claim 8, further comprising at least one additive selected from the group consisting of sugars, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavor and flavor ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

10. The consumable product of claim 8, further comprising at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, body weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

11. The consumer product of claim 8, further comprising a compound selected from the group consisting of: rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C2, rebaudioside D2, rebaudioside E2, rebaudioside E3, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside N, rebaudioside O2, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T638, rebaudioside U68692, rebaudioside W2, rebaudioside W68692, rebaudioside W638, rebaudioside W, rebaudioside D638, rebaudioside D6348, rebaudioside W6328, rebaudioside K63K, rebaudioside K, rebaudioside L, rebaudioside K638, rebaudioside D, rebaudioside O, rebaudioside W638, rebaudioside W2, rebaudioside W2, rebaudioside D, rebaudioside W, rebaudioside D, rebaudioside W638, rebaudioside W, rebaudioside D, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, rubusoside, steviolbioside A, steviolbioside B, steviolmonoside A, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E2, stevioside F, NSF-02, mogroside V, Lo Han Guo, psicose, D-allose, D-tagatose, erythritol, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and salts thereof, thaumatin, perillartine, pilferin, sappan-dulcin, sapindololoside, rubusoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, Brazilian glycyrrhizin, hemsleyasaponin, cyclotamideane-type triterpene, polyboroside A, brazilin, hernandulcin, phenazine, sarsasaponin, phlorizin, trilobatin, flavanonol, dihydroquercetin-3-acetate, neoastin, trans-cinnamaldehyde, monatin and salts thereof, mullein A, hematoxylin, monellin, stachyoside, pterocarosin A, pterocaryosin B, mabinlin, pethidine, amethystine, curculin, neocurculin, chlorogenic acid, cynarin, siamenoside, sucralose, acesulfame potassium, aspartame, alite, saccharin, cyclamate, neotame, dulcin, sulosan advantame, gymnemic acid, dulcitol, ziphin, nordulcin, 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 hydrolysates, polyglucitol, sugar alcohols, L-sugars, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isohexhalulose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, talose, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannosamine, glucuronic acid, gluconic acid, gluconolactone, abiocose, galactosamine, beet oligosaccharide, isomaltooligosaccharide (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharide (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharide, gentiooligosaccharide (gentiobiose, gentiotriose, gentiotetraose, etc.), aspergillus niger oligosaccharide, palatinose oligosaccharide, fructooligosaccharide (kestose, nystotetraose, etc.), maltotetraol, maltotriose, maltooligosaccharide (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin oligosaccharide, lactulose, melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, soybean oligosaccharides, D-psicose, D-ribose, L-glucose, L-fucose, D-turanose, D-leucrose.