Method for preparing rebaudioside I and use thereof

Abstract

Described herein are methods for making rebaudioside I, including highly purified rebaudioside I. These methods utilize biocatalysts that convert rebaudioside A to rebaudioside I. Also provided are compositions and consumables comprising rebaudioside I, including sweetener compositions and flavor enhancing compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Patent Application No. 62 / 039,344, filed on August 19, 2014, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a biocatalytic method for preparing rebaudioside I. The present invention also relates to the use of rebaudioside I as a sweetener, sweetness enhancer and / or flavor enhancer. Background of the Invention

[0004] High intensity sweeteners have a sweetness level that is many times higher than the sweetness level of sucrose. They are essentially caloric free and are commonly used in diet and low calorie products, including food and beverages. High intensity sweeteners do not trigger a glycemic response, making them suitable for use in products for diabetics and other populations interested in controlling their carbohydrate intake.

[0005] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain areas of South America. Their structure is characterized by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in the leaves of Stevia, constituting approximately 10%-20% of the total dry weight. On a dry basis, the four major glycosides found in the leaves of Stevia generally include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6%-1.0%), and dulcoside A (0.3%).

[0006] WO2010 / 03911 to Morita Kagaku Kogyo Co., Ltd discloses that small amounts of rebaudioside I are found in the leaves of certain stevia varieties. WO2010 / 03911 does not describe any organoleptic properties of isolated rebaudioside I or any composition containing high concentrations of rebaudioside I.

[0007] Therefore, there remains a need for simple, efficient and economical methods for preparing and purifying rebaudioside I, particularly if such methods are available on a commercial scale. Summary of the invention

[0008] The present invention provides a biocatalytic method for preparing a rebaudioside I composition, the method comprising contacting a starting composition comprising rebaudioside A with a biocatalyst capable of converting rebaudioside A into rebaudioside I.

[0009] In one embodiment, the biocatalyst is a UDP-glycosyltransferase (UGT). Suitable UGTs include, but are not limited to, UGT76G1 or a variant thereof, wherein the variant comprises at least about 75% amino acid sequence identity to UGT76G1.

[0010] In a specific embodiment, the UGT76G1 variant contains one or more point mutations that are found to increase the conversion of rebaudioside A to rebaudioside A and / or rebaudioside D to rebaudioside M by at least about 5% compared to using unmutated UGT76G1 under the same conditions. Suitable mutations include, but are not limited to, Q266E, P272A, R334K, G348P, L379G, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S 447A, S456L, I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R, K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P, V264C, E449D, and A352G.

[0011] In a preferred embodiment, the UGT76G1 variant is selected from the group consisting of UGT76G1-R1-F12, UGT76G1-R2-B9 and UGT76G1-R3-G3.

[0012] The biocatalyst may be provided in any form, such as, for example, a pure form, a crude lysate or a whole cell suspension. In another alternative embodiment, the biocatalyst is provided as a microorganism.

[0013] The starting composition can be a pure form of rebaudioside A, a steviol glycoside mixture, or a stevia extract. In one embodiment, the starting composition is a steviol glycoside mixture or a stevia extract containing at least about 1% rebaudioside A by weight.

[0014] The methods of the present invention provide a rebaudioside I composition comprising at least about 1% rebaudioside I by weight.

[0015] The method is typically performed in culture medium. Therefore, the method may further comprise separating the Rebaudioside I composition from the culture medium to provide an isolated Rebaudioside I composition. The method may further comprise a purification step, wherein the isolated Rebaudioside I composition is purified to provide a highly purified Rebaudioside I composition comprising at least about 80% by weight Rebaudioside I. In one embodiment, the isolated Rebaudioside I composition is purified to provide pure Rebaudioside I, i.e., >99% by weight.

[0016] The present invention also provides pure rebaudioside I.

[0017] The present invention also provides a composition comprising rebaudioside I. In one embodiment, the present invention is a composition comprising an amount of from about 1% to about 99% by weight of rebaudioside I. In one embodiment, the present invention is a highly purified rebaudioside A composition comprising at least about 80% by weight of rebaudioside I.

[0018] The present invention also includes methods of using rebaudioside I as a sweetener, flavor enhancer, or sweetness enhancer.

[0019] The present invention also extends to sweetener compositions, flavor enhancing compositions, and sweetness enhancing compositions comprising rebaudioside I.

[0020] Also provided are consumables comprising rebaudioside I and compositions comprising rebaudioside I. Exemplary consumables include pharmaceutical compositions, edible gel mixes or compositions, dental compositions, confections, flavoring compositions, chewing gum compositions, cereal compositions, baked goods, dairy products, and tabletop sweetener compositions, beverages, or beverage products.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are included to provide a further understanding of the present invention. These drawings illustrate embodiments of the present invention and together with the description serve to explain the principle of embodiments of the present invention.

[0023] Figure 1 Biocatalytic production of rebaudioside I is shown.

[0024] Figure 2 Shown is the biocatalytic production of rebaudioside A from stevioside using the enzyme UGT76G1 and the concomitant recycling of UDP to UDP-glucose by sucrose synthase.

[0025] Figure 3 Shown is the conversion of stevioside to rebaudioside A catalyzed by UGT76G1.

[0026] Figure 4The reaction characteristic curve of the conversion of rebaudioside A to rebaudioside I catalyzed by UGT76G1-R1-F12 is shown.

[0027] Figure 5 The activity of the UGT mutants for converting rebaudioside A to rebaudioside I is shown.

[0028] Figure 6 Shown is the HPLC trace of rebaudioside I before purification.

[0029] Figure 7 Shown are the sensory attributes of Rebaudioside I and Rebaudioside M at 400 ppm in water at 4°C. Detailed description

[0030] Method for preparing rebaudioside I

[0031] The present invention provides a biocatalytic method for preparing a rebaudioside I composition by contacting a starting composition comprising rebaudioside A with a biocatalyst capable of converting rebaudioside A to rebaudioside I. The rebaudioside I composition can optionally be further processed to provide highly purified rebaudioside I or even pure rebaudioside I.

[0032] As used herein, "starting composition" refers to any composition comprising rebaudioside A. Typically, the starting composition is an aqueous solution.

[0033] The starting composition can be purified rebaudioside A, a steviol glycoside mixture, or a stevia extract. A steviol glycoside mixture containing rebaudioside A, a stevia extract, and highly purified rebaudioside A are commercially available from various suppliers or can be easily prepared by methods provided in the literature. For example, U.S. Pat. No. 8,791,253 to Coca-Cola Company (the contents of which are incorporated herein by reference in their entirety) provides a method for obtaining rebaudioside A with a purity greater than 95%.

[0034] In one embodiment, the starting composition is purified Rebaudioside A, ie >99% by weight.

[0035] In another embodiment, the starting composition is a steviol glycoside mixture. The identity of the steviol glycoside mixture is not particularly limited and can include naturally occurring steviol glycosides, for example, stevioside, stevioside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O; synthetic steviol glycosides, such as enzymatically glycosylated steviol glycosides and combinations thereof.

[0036] In some embodiments, the starting composition is a steviol glycoside mixture or a stevia extract comprising rebaudioside A in an amount of about 1% or more by weight, such as, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more.

[0037] In a specific embodiment, the starting composition is a steviol glycoside mixture or a stevia extract comprising rebaudioside A in an amount of about 50% or more by weight.

[0038] In another specific embodiment, the starting composition is a steviol glycoside mixture or a stevia extract comprising rebaudioside A in an amount of about 80% or more by weight.

[0039] In yet another specific embodiment, the starting composition is a steviol glycoside mixture or a stevia extract comprising rebaudioside A in an amount of about 90% or more by weight.

[0040] In yet another specific embodiment, the starting composition is a steviol glycoside mixture or a Stevia extract comprising rebaudioside A in an amount of about 95% or higher, such as, for example, about 96%, about 97%, about 98% or about 99% by weight.

[0041] It is noteworthy that the starting composition can contain a certain amount of rebaudioside I. For example, the starting composition is a stevia extract or a steviol glycoside mixture. Contacting the biocatalyst with the starting composition produces a rebaudioside I composition that contains an increased amount of rebaudioside I compared to the amount of rebaudioside I present in the starting composition (if any). The increase in rebaudioside I is attributed to the action of the biocatalyst.

[0042] In a specific embodiment, the amount of rebaudioside I in the rebaudioside composition (i.e., the composition obtained by the method of the present invention) is about 0.5, about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 or about 65, about 70 or about 75% or more greater than the amount of rebaudioside I present in the starting composition.

[0043] The starting composition is contacted with the biocatalyst in an aqueous medium comprising water and, for example, various components selected from carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, bases, etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonate, bicarbonate. Nitrogen sources may include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

[0044] In a specific embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a specific embodiment, the medium comprises a phosphate buffer.

[0045] In one embodiment, the medium may further include an organic solvent, such as methanol, ethanol, propanol, and the like.

[0046] As used herein, "biocatalyst" refers to an enzyme capable of converting Rebaudioside A to Rebaudioside I. The enzyme can be a naturally occurring or recombinant protein. At least one biocatalyst is used in the method of the present invention for converting Rebaudioside A to Rebaudioside I. However, if necessary, multiple biocatalysts can be used. Therefore, in some embodiments, two or more biocatalysts are used, such as, for example, three or more biocatalysts, four or more biocatalysts, or five or more biocatalysts.

[0047] The biocatalyst can be provided as a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one embodiment, the biocatalyst is provided in a purified form, i.e., as a purified enzyme. In another embodiment, the biocatalyst is provided in the form of a crude lysate. In yet another embodiment, the biocatalyst is provided in the form of a whole cell suspension.

[0048] In another embodiment, the biocatalyst is provided in the form of one or more cells, ie, the biocatalyst is associated with one or more cells. The biocatalyst may be located on the cell surface, inside the cell, or both.

[0049] In another embodiment, the biocatalyst is provided in the form of a microorganism, i.e., the biocatalyst is associated with a microorganism. The microorganism can be any microorganism having one or more necessary biocatalysts / enzymes for converting rebaudioside A to rebaudioside I. Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces species, Aspergillus species, Pichia species, Bacillus species, Yarrowia species, etc.

[0050] In one embodiment, the microorganism is free (ie, not immobilized) when contacted with the starting composition.

[0051] In another embodiment, the microorganism is fixed when contacted with the starting composition. For example, the microorganism can be fixed to a solid support made of an inorganic or organic material. Non-limiting examples of solid supports suitable for fixing microorganisms include derived cellulose or glass, ceramics, metal oxides or films. The microorganism can be fixed to the solid support, for example, by covalent attachment, adsorption, crosslinking, capture or encapsulation.

[0052] In yet another embodiment, the biocatalyst is secreted by the microorganism into the reaction medium.

[0053] Suitable biocatalysts for converting rebaudioside A to rebaudioside I include, but are not limited to, steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs).

[0054] In one embodiment, the biocatalyst is a steviol biosynthetic enzyme, such as a mevalonate (MVA) pathway enzyme.

[0055] In another embodiment, the biocatalyst is a steviol biosynthetic enzyme, such as a non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MFP / DOXP) enzyme.

[0056] In one embodiment, the biocatalyst is a steviol biosynthetic enzyme selected from the group consisting of geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidine-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidine-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidine-2-C- Methyl-D-erythritol kinase (CMK), 4-diphosphocytidine-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase and cytochrome P450 reductase.

[0057] In one embodiment, the biocatalyst is a UGT capable of adding at least one glucose unit to rebaudioside A to provide rebaudioside I. In one embodiment, the UGT is UGT76G1 or a variant thereof, wherein the variant comprises about 75% or greater amino acid sequence identity to UGT76G1. Exemplary UGT76G1 variants include, but are not limited to, UGTSL, UGTSL2, UGTLB, UGT91D2, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0058] In another embodiment, the biocatalyst is a UGT selected from the following list of GenInfo identification numbers, preferably from the group presented in Table 1, and more preferably from the group presented in Table 2.

[0059]

[0060]

[0061]

[0062]

[0063] Table 1

[0064]

[0065]

[0066]

[0067]

[0068] Table 2

[0069] GI number Accession Number origin Internal Standard 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 Ningxia wolfberry UGTLB 209954725 BAG80553.1 Ningxia wolfberry -

[0070] In one embodiment, the biocatalyst is a UGT76G1 variant containing one or more point mutations found to increase the conversion of rebaudioside A to rebaudioside I by at least about 5% compared to using unmutated UGT76G1 under the same conditions (wherein the results are normalized). In another embodiment, the biocatalyst is a UGT76G1 variant containing one or more point mutations found to increase the conversion of rebaudioside D to rebaudioside M by at least about 5% compared to using unmutated UGT76G1 under the same conditions (wherein the results are normalized).

[0071] In one embodiment, the biocatalyst is a purified UGT76G1 variant, i.e., a UGT76G1 provided as a purified enzyme. In another embodiment, the biocatalyst is a UGT76G1 variant provided as a crude lysate. In yet another specific embodiment, the biocatalyst is a UGT76G1 variant provided as a whole cell suspension.

[0072] In a specific embodiment, the biocatalyst is a purified UGT76G1 comprising one or more of the following point mutations: Q266E, P272A, R334K, G348P, and L379G. In a more specific embodiment, the biocatalyst is a UGT76G1 comprising all of the following mutations: Q266E, P272A, R334K, G348P, and L379G, i.e., the biocatalyst is UGT76G1-R1-F12. In a specific embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a purified enzyme. In another specific embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a crude lysate. In yet another specific embodiment, the biocatalyst is UGT76G1-R1-F12 provided in the form of a whole cell suspension.

[0073] In another specific embodiment, the biocatalyst is a UGT76G1 comprising one or more of the following point mutations: Q266E, P272A, R334K, G348P, L379G, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, and S456L. In a more specific embodiment, the biocatalyst is a UGT76G1 variant comprising all of the above mutations, i.e., the biocatalyst is UGT76G1-R2-B9. In a specific embodiment, the biocatalyst is purified UGT76G1-R2-B9, i.e., UGT76G1-R2-B9 provided in the form of a purified enzyme. In another specific embodiment, the biocatalyst is UGT76G1-R2-B9 provided in the form of a lysate. In yet another embodiment, the biocatalyst is UGT76G1-R2-B9 provided in the form of a whole cell suspension.

[0074] In yet another specific embodiment, the biocatalyst is a UGT76G1 variant comprising one or more of the following point mutations: Q266E, P272A, R334K, G348P, L379G, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N40 9R, Q425E, Q432E, S447A, S456L, I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R, K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P, V264C, E449D and A352G. In a more specific embodiment, the biocatalyst is a UGT76G1 variant comprising all of the above mutations, i.e., the biocatalyst is UGT76G1-R3-G3. In a specific embodiment, the biocatalyst is purified UGT76G1-R3-G3, i.e., UGT76G1-R3-G3 provided in the form of a purified enzyme. In another embodiment, the biocatalyst is UGT76G1-R3-G3 provided in the form of a lysate. In yet another embodiment, the biocatalyst is UGT76G1-R3-G3 provided in the form of a whole cell suspension.

[0075] The use of a UGT76G1 variant as a biocatalyst in the methods of the present invention results in an increase in the conversion of rebaudioside A to rebaudioside I by at least about 5% compared to the use of an unmutated UGT76G1 under the same conditions (wherein the results are normalized). In a preferred embodiment, the conversion rate is increased from about 5% to about 1,000,000%, such as, for example, from about 5% to about 100,000%, from about 5% to about 10,000%, from about 5% to about 1,000%, from about 5% to about 500%, from about 5% to about 250%, from about 5% to about 100%, from about 50%, from about 20% to about 50%, from about 30% to about 50% or from about 40% to about 50%.

[0076] Optionally, the methods of the present invention further comprise recycling UDP to provide UDP-glucose. Accordingly, these methods comprise concomitantly recycling UDP by providing a recycling catalyst (i.e., a catalyst capable of overproducing UDP-glucose) and a recycling substrate, thereby converting rebaudioside A to whole rebaudioside I using a catalytic amount of UDP-glucose syltransferase and UDP-glucose ( Figure 2 ).

[0077] In one embodiment, the UDP-glucose recycling catalyst is sucrose synthase and the recycled substrate is sucrose.

[0078] In a specific embodiment, the methods of the present invention provide a rebaudioside I composition comprising rebaudioside I in an amount of about 1% or more, such as about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more by weight.

[0079] In a specific embodiment, the method provides a composition comprising rebaudioside I in an amount of about 50% or more by weight.

[0080] In another specific embodiment, the method provides a composition comprising rebaudioside I in an amount of about 80% or more by weight.

[0081] In yet another specific embodiment, the method provides a composition comprising rebaudioside I in an amount of about 90% or more by weight.

[0082] In yet another specific embodiment, the method provides a composition comprising rebaudioside I in an amount of about 95% by weight or higher, such as, for example, about 96%, about 97%, about 98% or about 99% by weight.

[0083] Optionally, the method of the present invention further comprises separating Rebaudioside I from the culture medium. Any suitable separation method can be used, such as, for example, cracking, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, separation can be achieved by cracking and centrifugation.

[0084] In one embodiment, rebaudioside I is continuously removed from the culture medium while the conversion progresses. In another embodiment, rebaudioside I is separated and optionally purified from the culture medium after the reaction is complete.

[0085] Isolation from the culture medium may produce a composition having a lower than desired rebaudioside I content and / or the composition may contain additional components, such as undesirable steviol glycosides (in consistency or content) and / or residual reaction products. Therefore, the composition may be further purified to provide a highly purified rebaudioside I composition. As used herein, the term "highly purified" refers to a composition having greater than about 80% rebaudioside I by weight on a dry basis. In one embodiment, a highly purified rebaudioside I composition comprises greater than about 90% rebaudioside I by weight, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or about 99% rebaudioside I by weight. In an exemplary embodiment, the composition may be further purified to provide pure rebaudioside I, i.e., >99% rebaudioside I by weight on a dry basis.

[0086] Purification can be effected by any means known to those skilled in the art, including, but not limited to, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatography, HPLC (preparative or analytical), or a combination of such methods. In a specific embodiment, HPLC is used to purify rebaudioside I. In a more specific embodiment, semi-preparative HPLC is used to purify rebaudioside I.

[0087] In one embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I and (b) isolating rebaudioside I to provide a rebaudioside I composition (i.e., an isolated rebaudioside I composition).

[0088] In a more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity, and UDP-glucose to form a composition comprising rebaudioside I, and (b) isolating rebaudioside I to provide an isolated rebaudioside I composition. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3. Optionally, the method comprises concomitant UDP-glucose recycling by providing a sucrose synthase and sucrose in (a).

[0089] In another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a starting composition comprising rebaudioside A with at least UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity and UDP-glucose to form a composition comprising rebaudioside I, and (b) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3. Optionally, the method comprises recycling UDP-glucose concomitantly by providing a sucrose synthase and sucrose in (a).

[0090] In another embodiment, the present invention provides a method for preparing a rebaudioside I composition, which comprises (a) contacting a starting composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I, (b) isolating rebaudioside I to provide an isolated rebaudioside I composition, and (c) purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition.

[0091] In yet another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity, and UDP-glucose to form a composition comprising rebaudioside I, (b) isolating rebaudioside I to provide an isolated rebaudioside I composition, and (c) purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises recycling UDP-glucose concomitantly by providing a sucrose synthase and sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0092] In another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a starting composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity to provide a composition comprising rebaudioside I, (b) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition, and (c) purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises providing a sucrose synthase and concomitant UDP-glucose recycling of sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0093] In another embodiment, the present invention provides a method for preparing a highly purified rebaudioside I composition, the method comprising (a) contacting a starting composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I and b) purifying the composition comprising rebaudioside I to provide a highly purified rebaudioside I composition.

[0094] In yet another specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, comprising (a) contacting a starting composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity and UDP glucose to form a composition comprising rebaudioside I, and (b) purifying the composition comprising rebaudioside I to provide a highly purified rebaudioside I composition. Optionally, the method comprises providing a sucrose synthase and concomitant UDP-glucose recycling of sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0095] In yet another specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a starting composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity and UDP-glucose to form a composition comprising rebaudioside I, and (b) purifying the composition comprising rebaudioside I to provide a highly purified rebaudioside I composition. Optionally, the method comprises providing a sucrose synthase and concomitant UDP-glucose recycling of sucrose in (a). Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0096] Fermentation can also be used to synthesize rebaudioside I de novo. For example, a method of producing a rebaudioside I composition comprises (a) contacting glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside I to provide a rebaudioside I composition, and (b) isolating rebaudioside I to provide an isolated rebaudioside I composition. Optionally, the method further comprises purifying rebaudioside I to provide highly purified rebaudioside I.

[0097] The fermentation and biocatalysis steps can be used sequentially. For example, a composition comprising glucose can be first fermented with a microorganism containing at least one enzyme capable of converting glucose into a target steviol glycoside (e.g., rebaudioside A). The target steviol glycoside (e.g., rebaudioside A), which now becomes the starting material for the next bioconversion purpose, can then be contacted with a biocatalyst capable of converting it into the next target steviol glycoside (e.g., rebaudioside I).

[0098] Between each transformation, the target steviol glycoside can optionally be isolated from the culture medium before contacting with the next biocatalyst.

[0099] In one embodiment, the rebaudioside A of the starting composition of the present method is prepared by contacting stevioside with an enzyme capable of converting stevioside to rebaudioside A. In a specific embodiment, the enzyme is any UDP-glucosyltransferase capable of adding at least one glucose unit thereto to produce rebaudioside A. The UDP-glucosyltransferase can be, for example, UGT76G1.

[0100] Therefore, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising stevioside with an enzyme capable of converting stevioside to rebaudioside A to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A, (c) contacting a composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I.

[0101] In a more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A, (c) contacting a composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in one or both contacting steps. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0102] In another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A from the culture medium, (c) contacting a culture medium containing a composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in one or both contacting steps. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0103] In another embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside A to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A, (c) contacting the composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition.

[0104] In a more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside A to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A, (c) contacting the composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in one or both contacting steps. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0105] In another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside A to provide a composition comprising rebaudioside A, (b) isolating rebaudioside A from the culture medium, (c) contacting a culture medium containing a composition comprising rebaudioside A with UGT76G1 or a variant having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I from the culture medium to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in one or both contacting steps. Exemplary UGT variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0106] In one embodiment, stevioside is prepared by contacting rubusoside with an enzyme capable of converting rubusoside into stevioside. In one embodiment, the enzyme is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside. The UDP-glucosyltransferase can be, for example, UGT91D2 or a variant thereof having about 75% or higher amino acid sequence identity. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL2.

[0107] Therefore, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising rubusoside with an enzyme capable of converting rubusoside to stevioside, (b) isolating stevioside, (c) contacting a composition comprising stevioside with an enzyme capable of converting stevioside to rebaudioside A to provide a composition comprising rebaudioside A, (d) isolating rebaudioside A, (e) contacting a composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I, and (d) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I.

[0108] In a more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising rubusoside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising stevioside, (b) isolating stevioside, (c) contacting the composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (d) isolating rebaudioside A, (e) contacting a composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity and UDP-glucose to provide a composition comprising rebaudioside I, and (f) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide a highly purified rebaudioside I composition. Optionally, the method comprises concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in any or all contacting steps. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0109] In another more specific embodiment, the present invention provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a composition comprising rubusoside with UGT91D2 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to provide a composition comprising stevioside, (b) isolating stevioside from the culture medium, (c) contacting a culture medium containing a composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (d) isolating rebaudioside A from the culture medium, (e) contacting a culture medium containing a composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or more amino acid sequence identity, and UDP-glucose to provide a composition comprising rebaudioside I, and (f) isolating rebaudioside I from the culture medium. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method includes recycling of UDP-glucose by providing a sucrose synthase and concomitant sucrose in any or all contacting steps. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0110] The present invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism capable of converting glucose into rubusoside, (b) isolating rubusoside, (c) contacting the composition comprising rubusoside with an enzyme capable of converting rubusoside into stevioside to provide a composition comprising stevioside, (d) isolating stevioside, (e) contacting the composition comprising stevioside with an enzyme capable of converting stevioside into rebaudioside A to provide a composition comprising rebaudioside A, (f) isolating rebaudioside A, (g) contacting the composition comprising rebaudioside A with an enzyme capable of converting rebaudioside A into rebaudioside I to provide a composition comprising rebaudioside I, and (h) isolating rebaudioside I. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I.

[0111] In a more specific embodiment, the present invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a composition comprising glucose with a microorganism capable of converting glucose into rubusoside, (b) isolating rubusoside, (c) contacting the composition comprising rubusoside with UGT91D2 or a variant thereof having an amino acid sequence identity of about 75% or more, and UDP-glucose to provide a composition comprising stevioside, (d) isolating stevioside, (e) contacting the composition comprising stevioside with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (f) isolating rebaudioside A, (g) contacting the composition comprising rebaudioside A with UGT76G1 or a variant thereof having an amino acid sequence identity of about 75% or more, and UDP-glucose to provide a composition comprising rebaudioside I, and (h) isolating rebaudioside I to provide an isolated rebaudioside I composition. The method can further include purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method includes concomitant UDP-glucose recycling by providing sucrose synthase and sucrose in one, two or all contacting steps. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0112] In another more specific embodiment, the present invention also provides a method for preparing a rebaudioside I composition, the method comprising (a) contacting a culture medium containing a composition comprising glucose with a microorganism capable of converting glucose into rubusoside, (b) isolating rubusoside from the culture medium, (c) contacting a culture medium containing a composition comprising rubusoside with UGT91D2 or a variant thereof having an amino acid sequence identity of about 75% or more and UDP-glucose to provide a composition comprising stevioside, (d) isolating rubusoside from the culture medium, The method further comprises the steps of: (i) contacting a medium containing a composition comprising steviol glycosides with UGT76G1 and UDP-glucose to provide a composition comprising rebaudioside A, (ii) contacting a medium containing a composition comprising rebaudioside A with UGT76G1 or a variant thereof having about 75% or greater amino acid sequence identity, and UDP-glucose to provide a composition comprising rebaudioside I, and (iii) isolating rebaudioside I from the medium to provide an isolated rebaudioside I composition. The method may further comprise purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I. Optionally, the method comprises recycling UDP-glucose concomitantly by providing a sucrose synthase and sucrose in any or all contacting steps. Exemplary UGT91D2 variants include, but are not limited to, UGTSL and UGTSL2. Exemplary UGT76G1 variants include, but are not limited to, UGT76G1-R1-F12, UGT76G1-R2-B9, and UGT76G1-R3-G3.

[0113] Compounds and compositions

[0114] The present invention provides rebaudioside I having the following formula:

[0115]

[0116] 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kauri-16-ene-19-oic acid-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (Rebaudioside I)

[0117] In exemplary embodiments, rebaudioside I can be isolated and purified (ie, >99% rebaudioside I by weight on a dry basis) or isolated and highly purified (ie, greater than about 80% by weight on a dry basis).

[0118] The present invention includes compositions, particularly consumer products, comprising rebaudioside I.

[0119] In one embodiment, the composition comprises rebaudioside I provided as part of a mixture. In a specific embodiment, the mixture is selected from the group consisting of a mixture of steviol glycosides, stevia extracts, byproducts of other steviol glycoside separation and purification methods, or any combination thereof. In one embodiment, the mixture comprises rebaudioside I in an amount ranging from about 1% to about 99% by weight on a dry basis, such as, for example, from about 2% to about 99%, from about 3% to about 99%, from about 4% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 20% to about 99%, from about 30% to about 99%, from about 40% to about 99%, from about 50% to about 99%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99% and from about 90% to about 99%. In specific embodiments, the mixture comprises rebaudioside I in an amount greater than about 90% by weight on a dry basis, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, and greater than about 99%.

[0120] In one embodiment, the composition comprises rebaudioside I provided in the form of a stevia extract. The stevia extract comprises one or more additional steviol glycosides, including but not limited to naturally occurring steviol glycosides, such as steviol monoside, steviol bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides such as enzymatically glucosylated steviol glycosides, and combinations thereof.

[0121] In yet another embodiment, the present invention provides rebaudioside I as a pure compound (ie, >99% purity on a dry basis).

[0122] When the composition is added to a consumer product, rebaudioside I can be present in the composition in an amount effective to provide a concentration of from about 1 ppm to about 10,000 ppm, such as, for example, from about 5 ppm to about 10,000 ppm, from about 10 ppm to about 10,000 ppm, from about 15 ppm to about 10,000 ppm, from about 20 ppm to about 10,000 ppm, from about 25 ppm to about 10,000 ppm, from about 50 ppm to about 10,000 ppm, from about 100 ppm to about 10,000 ppm, from about 200 ppm to about 10,000 ppm, from about 300 ppm to about 10,000 ppm. ppm, from about 400 ppm to about 10,000 ppm, from about 500 ppm to about 10,000 ppm, from about 600 ppm to about 10,000 ppm, from about 700 ppm to about 10,000 ppm, from about 800 to about 10,000 ppm, from about 900 ppm to about 10,000 ppm, from about 1,000 ppm to about 10,000 ppm, from about 2,000 ppm to about 10,000 ppm, from about 3,000 ppm to about 10,000 ppm, from about 4,000 ppm to about 10,000 ppm, and from about 5,000 ppm to about 10,000 ppm.

[0123] In another embodiment, when the composition is added to a consumer product, Rebaudioside I is present in the composition in an amount effective to provide a concentration of from about 10 ppm to about 1,000 ppm, such as, for example, from about 10 ppm to about 800 ppm, from about 50 ppm to about 800 ppm, from about 50 ppm to about 600 ppm, or from about 200 ppm to about 250 ppm. In a specific embodiment, when the composition is added to a consumer product, Rebaudioside I is present in the composition in an amount effective to provide a concentration of from about 300 ppm to about 600 ppm.

[0124] In a particular embodiment, when the composition is added to a consumer product, rebaudioside I is present in the composition in an amount effective to provide a concentration from about 50 ppm to about 800 ppm, such as, for example, from about 50 ppm to about 100 ppm, about 100 ppm to about 150 ppm, about 200 ppm to about 250 ppm, about 250 ppm to about 300 ppm, from about 300 ppm to about 350 ppm, about 350 ppm to about 400 ppm, from about 400 ppm to about 450 ppm, about 450 ppm to about 500 ppm, about 500 ppm to about 550 ppm, about 550 ppm to about 600 ppm, about 600 ppm to about 650 ppm, about 650 ppm to about 700 ppm, about 700 ppm to about 750 ppm or about 750 ppm to about 800 ppm.

[0125] In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of between about 200 ppm and about 300 ppm when the composition is added to a beverage.

[0126] In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of between about 500 ppm and about 600 ppm when the composition is added to a beverage.

[0127] In another specific embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 50 ppm, about 100 ppm, about 150 ppm, about 200 ppm, about 250 ppm, about 300 ppm, about 350 ppm, about 400 ppm, about 450 ppm, about 500 ppm, about 550 ppm, about 600 ppm, about 650 ppm, about 700 ppm, about 750 ppm or about 800 ppm when the composition is added to a consumer product.

[0128] In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 200 ppm when the composition is added to a beverage.

[0129] In one exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 275 ppm when the composition is added to a beverage.

[0130] In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 550 ppm when the composition is added to a beverage.

[0131] In an exemplary embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 600 ppm when the composition is added to a beverage.

[0132] In an exemplary embodiment, the isolated and purified rebaudioside I or a composition comprising rebaudioside I exhibits a sweetness lingering intensity that is reduced compared to rebaudioside M. In a specific embodiment, the isolated and purified rebaudioside I or a composition comprising rebaudioside I exhibits a sweetness lingering intensity that is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% compared to rebaudioside M.

[0133] Sweetener composition

[0134] In one embodiment, the present invention is a sweetener composition comprising rebaudioside I. In a more specific embodiment, the present invention is a sweetener composition comprising highly purified or pure rebaudioside I.

[0135] As used herein, a "sweetener composition" refers to a composition that can be used to sweeten a sweetenable composition (ie, a composition that can be sweetened) that contains at least one sweet component in combination with at least one other substance.

[0136] In one embodiment, rebaudioside I is the sole sweetener in the sweetener composition, i.e., rebaudioside I is the only compound present in the sweetener composition that provides detectable sweetness. In another embodiment, the sweetener composition comprises a compound of rebaudioside I in combination with one or more sweetener compounds.

[0137] In an exemplary embodiment, the sweetener composition comprises rebaudioside I and a compound selected from the group consisting of rebaudioside A, B, C, D, E, M, N, O, M2, D2, glycosylated steviol glycosides, mogroside V, erythritol, psicose, stevia extract, monk fruit extract, and combinations thereof.

[0138] The amount of rebaudioside I in the sweetener composition can vary. In one embodiment, rebaudioside I is present in the sweetener composition in any amount that imparts a desired sweet taste when the sweetener composition is added to a sweetenable composition or a sweetenable consumable.

[0139] The sweetness of a non-sucrose sweetener can also be measured relative to a sucrose reference by determining the sucrose equivalence of the non-sucrose sweetener. Typically, trained tasters detect the sweetness of reference sucrose solutions containing between 1%-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percentage of the sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times more potent than sucrose.

[0140] In one embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide greater than about 10% (w / v) sucrose equivalents when added to a sweetener composition or sweetened consumable, such as, for example, greater than about 11%, greater than about 12%, greater than about 13% or greater than about 14%.

[0141] The amount of sucrose in a reference solution, and therefore another measure of sweetness, can be described in degrees Brix (°Bx). One degree Brix is ​​1 gram of sucrose in 100 grams of solution, and represents the strength of the solution as a weight percentage (% w / w) (strictly speaking, by mass). In one embodiment, a sweetener composition contains rebaudioside I effective in providing an amount equivalent to from about 0.50 to 14 degrees Brix when present in a sweetened composition, such as, for example, from about 5 to about 11 degrees Brix, from about 4 to about 7 degrees Brix, or about 5 degrees Brix. In yet another embodiment, the composition comprising rebaudioside I and at least one other sweetener are present in an amount effective to provide any one of the sweetness equivalents listed above.

[0142] In one embodiment, when the sweetener composition is added to a consumable (e.g., a beverage), rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of from about 1 ppm to about 10,000 ppm, such as, for example, from about 5 ppm to about 10,000 ppm, from about 10 ppm to about 10,000 ppm, from about 15 ppm to about 10,000 ppm, from about 20 ppm to about 10,000 ppm, from about 25 ppm to about 10,000 ppm, from about 50 ppm to about 10,000 ppm, from about 100 ppm to about 10,000 ppm, from about 200 ppm to about 10,000 ppm, from about 300 ppm to about 10,000 ppm m to about 10,000 ppm, from about 400 ppm to about 10,000 ppm, from about 500 ppm to about 10,000 ppm, from about 600 ppm to about 10,000 ppm, from about 700 ppm to about 10,000 ppm, from about 800 to about 10,000 ppm, from about 900 ppm to about 10,000 ppm, from about 1,000 ppm to about 10,000 ppm, from about 2,000 ppm to about 10,000 ppm, from about 3,000 ppm to about 10,000 ppm, from about 4,000 ppm to about 10,000 ppm, and from about 5,000 ppm to about 10,000 ppm. In another embodiment, when the sweetener composition is added to a consumable, Rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of from about 10 ppm to about 1,000 ppm, such as, for example, from about 10 ppm to about 800 ppm, from about 50 ppm to about 800 ppm, from about 50 ppm to about 600 ppm, or from about 200 ppm to about 250 ppm. In a specific embodiment, when the sweetener composition is added to a consumable, Rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of from about 300 ppm to about 600 ppm.

[0143] In one embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of between about 400 to about 800 ppm when the sweetener composition is added to a consumable, such as, for example, from about 50 ppm to about 100 ppm, about 100 ppm to about 150 ppm, about 200 ppm to about 250 ppm, about 250 ppm to about 300 ppm, about 300 ppm to about 350 ppm, about 350 ppm to about 400 ppm, about 400 ppm to about 450 ppm, about 450 ppm to about 500 ppm, about 500 ppm to about 550 ppm, about 550 ppm to about 600 ppm, about 600 ppm to about 650 ppm, about 650 ppm to about 700 ppm, about 700 ppm to about 750 ppm or about 750 ppm to about 800 ppm.

[0144] In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of between about 400 to about 800 ppm when the sweetener composition is added to a beverage.

[0145] In an exemplary embodiment, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of between about 500 to about 600 ppm when the sweetener composition is added to a beverage.

[0146] When the sweetener composition includes rebaudioside I in combination with one or more sweetener compounds, the amount of the sweetener compound can vary. In one embodiment, when the sweetener compound is added to the consumable, the sweetener composition is present in the sweetener composition in an amount effective to provide a concentration between about 1% and about 20%, such as between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, or more specifically, about 1%, about 2%, about 3%, 4% or about 5%.

[0147] In an exemplary embodiment, the present invention provides a sweetener composition comprising (i) rebaudioside I in an amount effective to provide a concentration between about 500 and about 600 ppm when the sweetener composition is added to a beverage; and (ii) a compound selected from the group consisting of rebaudioside A, B, C, D, E, M, N, O, M2, D2, glycosylated steviol glycosides, mogroside V, erythritol, psicose, stevia extract, monk fruit extract, and combinations thereof.

[0148] In an exemplary embodiment, the present invention provides a sweetener composition comprising (i) rebaudioside I in an amount effective to provide a concentration between about 500 and about 600 ppm when the sweetener composition is added to a beverage; and (ii) psicose in an amount effective to provide a concentration between about 1% and about 5% when the sweetener composition is added to a beverage.

[0149] In another embodiment, the amount present in the sweetener composition is effective to provide a concentration of about 50 ppm, about 100 ppm, about 150 ppm, about 200 ppm, about 250 ppm, about 300 ppm, about 350 ppm, about 400 ppm, about 450 ppm, about 500 ppm, about 550 ppm, about 600 ppm, about 650 ppm, about 700 ppm, about 750 ppm or about 800 ppm when the sweetener composition is added to a consumable.

[0150] In an exemplary embodiment, when the sweetener composition is added to a beverage, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 550 ppm. Optionally, the sweetener composition further includes, for example, between about 1% and about 5%, or more particularly about 3.5% psicose.

[0151] In an exemplary embodiment, when the sweetener composition is added to a beverage, rebaudioside I is present in the composition in an amount effective to provide a concentration of about 600 ppm. Optionally, the sweetener composition further includes, for example, between about 1% and about 5%, or more particularly about 3.5% psicose.

[0152] In an exemplary embodiment, a sweetener composition comprising rebaudioside I exhibits a sweetness lingering intensity that is reduced compared to rebaudioside M or a sweetener composition comprising it. In a specific embodiment, the isolated and purified rebaudioside I or a composition comprising rebaudioside I exhibits a sweetness lingering intensity that is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% compared to rebaudioside M or a sweetener composition comprising it.

[0153] In some embodiments, rebaudioside I is present in the sweetener composition in an amount effective to provide a concentration of the compound above, at, or below its threshold sweetener recognition level when the sweetener composition is added to a consumable (e.g., a beverage).

[0154] Flavor enhancing compositions

[0155] In one aspect, the present invention is a flavor enhancement composition comprising rebaudioside I. In a specific embodiment, the present invention is a flavor enhancement composition comprising isolated and purified rebaudioside I.

[0156] As used herein, a "flavor enhancer composition" refers to a composition that is capable of enhancing or intensifying the perception of a particular flavor in a consumable. The term "flavor enhancing composition" or "flavor enhancer" is synonymous with the terms "flavor synergist," "flavor amplifier," and "flavor intensifier." Generally, the flavor enhancing compositions provided herein can improve or enhance the taste of flavor ingredients, i.e., any substance that provides sweetness, sourness, saltiness, fragrance, bitterness, metallic taste, astringency, a persistent sweet aftertaste, sweetness onset, etc. Without being bound by any theory, the flavor enhancing composition may not provide any noticeable taste to the consumable to which it is added because rebaudioside I is present in the consumable at a concentration at or below its flavor recognition threshold concentration.

[0157] As used herein, "flavor recognition threshold concentration" refers to the lowest concentration at which a particular flavor or aftertaste of a component (e.g., a compound) is perceptible in a consumable. The flavor recognition threshold concentration varies for different compounds and can vary relative to an individual or specific consumable that perceives the flavor.

[0158] In one embodiment, the flavor enhancing composition comprises rebaudioside I in an amount effective to provide a concentration at or below a threshold flavor recognition concentration of rebaudioside I when the flavor enhancing composition is added to a consumable.

[0159] In a particular embodiment, rebaudioside I is present in the flavor enhancing composition in an amount effective to provide a concentration below a threshold flavor recognition concentration of rebaudioside I when the flavor enhancing composition is added to a consumable.

[0160] In certain embodiments, when the flavor enhancing composition is added to a consumable, rebaudioside I is present in the flavor enhancing composition in an amount effective to provide a concentration at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% or more below the threshold flavor recognition concentration.

[0161] In some embodiments, when added to a consumable (e.g., a beverage), rebaudioside I is present in the flavor enhancing composition in an amount providing a concentration ranging from 1 ppm to about 10,000 ppm, such as, for example, from about 5 ppm to about 10,000 ppm, from about 10 ppm to about 10,000 ppm, from about 15 ppm to about 10,000 ppm, from about 20 ppm to about 10,000 ppm, from about 25 ppm to about 10,000 ppm, from about 50 ppm to about 10,000 ppm, from about 100 ppm to about 10,000 ppm ppm, from about 200 ppm to about 10,000 ppm, from about 300 ppm to about 10,000 ppm, from about 400 ppm to about 10,000 ppm, from about 500 ppm to about 10,000 ppm, from about 600 ppm to about 10,000 ppm, from about 700 ppm to about 10,000 ppm, from about 800 to about 10,000 ppm, from about 900 ppm to about 10,000 ppm, from about 1,000 ppm to about 10,000 ppm, from about 2,000 ppm to about 10,000 ppm, from about 3,000 ppm to about 10,000 ppm, from about 4,000 ppm to about 10,000 ppm, and from about 5,000 ppm to about 10,000 ppm. In some embodiments, rebaudioside I is present in the flavor enhancing composition in an amount to provide a concentration ranging from 1 ppm to about 1,000 ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5 ppm to about 1,000 ppm, from about 10 ppm to about 1,000 ppm, from about 20 ppm to about 1,000 ppm, from about 30 ppm to about 1,000 ppm, from about 40 ppm to about 1,000 ppm, from about 50 ppm to about 1000 ppm, from about 100 ppm to about 1,000 ppm, from about 200 ppm to about 1,000 ppm, from about 300 ppm to about 1,000 ppm, from about 400 ppm to about 1,000 ppm, and from about 500 ppm to about 1,000 ppm.

[0162] A person of ordinary skill in the art will be able to select the concentration of rebaudioside I in the flavor enhancing composition so that it can impart an enhanced flavor to a consumable product comprising at least one flavor ingredient. For example, a skilled artisan can select the concentration of rebaudioside I in the flavor enhancing composition so that when the flavor enhancing composition is added to a consumable product, the flavor enhancing composition and / or the rebaudioside I do not impart any perceptible flavor to the consumable product.

[0163] In one embodiment, the addition of the flavor enhancing composition increases the detected flavor of at least one flavoring ingredient in the consumable compared to the detected flavor of the same ingredient in the consumable in the absence of the flavor enhancer.

[0164] Suitable flavoring ingredients include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, melaleuca, almond, menthol (including mint-free menthol), grape skin extract, and grape seed extract. "Flavoring" and "flavoring ingredient" are synonymous and may include natural or synthetic substances or combinations thereof. Flavoring also includes any other substance that imparts flavor and may include natural or non-natural (synthetic) substances that are safe for humans or animals when used in a generally accepted range. Non-limiting examples of specialty flavorings include Natural flavor sweetener K14323( Darmstadt, Germany), sweeteners 161453 and 164126 Symrise TM Natural flavoring mask (Symrise TM , Holzminden, Germany (Gcrmany)), Natural Advantagc TM Bitterness Blockers 1, 2, 9 and 10 (Natural Advantagc TM , Freehold, New Jersey, USA), and Sucramask TM (Creative Research Management, Stockton, California, USA).

[0165] In another embodiment, a flavor enhancer composition comprising rebaudioside I enhances flavor (either individual flavor or overall flavor) when added to the consumer's product. Alternatively, rebaudioside I can be added directly to the consumer's product, i.e., not provided in the form of a composition, to increase flavor. In this embodiment, rebaudioside I is a flavor enhancer and is added to the consumer's product at a concentration at or below its threshold flavor recognition concentration.

[0166] In a specific embodiment, the flavor enhancing composition is a sweet enhancing composition. As used herein, a "sweet enhancing composition" refers to a composition that can enhance or strengthen the sweet taste sensation of a consumer's product (e.g., beverage). The term "sweet enhancer" is synonymous with the term "sweet taste enhancer," "sweet taste enhancer," "sweet taste amplifier," and "sweetness intensifier."

[0167] As used herein, "sweetness recognition threshold concentration" is the lowest known concentration of a sweet compound that is perceptible by the human palate. Typically, the sweetness enhancing compositions of the present invention can improve or enhance the sweet taste of a consumable without providing any noticeable sweetness of its own, because the concentration of rebaudioside I in the sweetness enhancing composition is at or below its sweetness recognition threshold concentration, in the sweetness enhancing composition, in the consumable after the sweetness enhancing composition is added, or in both. The sweetness recognition threshold concentration is specific to a particular compound and can vary based on temperature, substrate, ingredients, and / or flavor systems.

[0168] In one embodiment, the sweet taste enhancing composition comprises rebaudioside I in an amount effective to provide a concentration at or below the threshold sweet taste recognition concentration of rebaudioside I when the sweet taste enhancing composition is added to a consumable.

[0169] In a particular embodiment, the sweet taste enhancing composition comprises rebaudioside I in an amount effective to provide a concentration below the threshold sweet taste recognition concentration of rebaudioside I when the sweet taste enhancing composition is added to a consumable.

[0170] In certain embodiments, rebaudioside I is present in the sweet taste enhancing composition in an amount effective to provide a concentration at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% or more below a threshold sweetness recognition concentration when the sweet taste enhancing composition is added to a consumable.

[0171] In some embodiments, rebaudioside I is present in the sweet taste enhancing composition in an amount to provide a concentration from about 1 ppm to about 10,000 ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5 ppm to about 10,000 ppm, from about 10 ppm to about 10,000 ppm, from about 15 ppm to about 10,000 ppm, from about 20 ppm to about 10,000 ppm, from about 25 ppm to about 10,000 ppm, from about 50 ppm to about 10,000 ppm, from about 100 ppm to about 10,000 ppm, from about 200 ppm to about 10,000 ppm, from about 300 ppm to about 10,000 ppm. ,000ppm, from about 400ppm to about 10,000ppm, from about 500ppm to about 10,000ppm, from about 600ppm to about 10,000ppm, from about 700ppm to about 10,000ppm, from about 800 to about 10,000ppm, from about 900ppm to about 10,000ppm, from about 1,000ppm to about 10,000ppm, from about 2,000ppm to about 10,000ppm, from about 3,000ppm to about 10,000ppm, from about 4,000ppm to about 10,000ppm, and from about 5,000ppm to about 10,000ppm. In some embodiments, rebaudioside I is present in the sweetness enhancing composition in an amount to provide a concentration ranging from 1 ppm to about 1,000 ppm when added to a consumable (e.g., a beverage), such as, for example, from about 5 ppm to about 1,000 ppm, from about 10 ppm to about 1,000 ppm, from about 20 ppm to about 1,000 ppm, from about 30 ppm to about 1,000 ppm, from about 40 ppm to about 1,000 ppm, from about 50 ppm to about 1000 ppm, from about 100 ppm to about 1,000 ppm, from about 200 ppm to about 1,000 ppm, from about 300 ppm to about 1,000 ppm, from about 400 ppm to about 1,000 ppm, and from about 500 ppm to about 1,000 ppm.

[0172] Alternatively, rebaudioside I can be added directly to the consumable, i.e., not provided in the form of a composition, to increase sweetness. In this embodiment, rebaudioside I is a sweetness enhancer, and it is added to the consumable at a concentration at or below its sweetness recognition threshold concentration.

[0173] The sweetness of a given composition is typically measured with reference to a sucrose solution. See generally "A Systematic Study of Concentration-Response Relationships of Sweeteners," GE DuBois, DE Walters, SS Schiffman, ZS Warwick, BJ Booth, SD Pecore, K. Gibes, BT Carr, and LM Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, ed. DE Walters, FT Orthoefer and GE DuBois, American Chemical Society, Washington, DC (1991), pp. 261-276.

[0174] It is contemplated that the sweet taste enhancing composition may include one or more sweet taste enhancers in addition to Rebaudioside I. In one embodiment, the sweet taste enhancing composition may include a kind of other sweet taste enhancer. In other embodiments, the sweet taste enhancing composition may include two or more other sweet taste enhancers. In the embodiment utilizing two or more sweet taste enhancers, each sweet taste enhancer should exist below its respective sweet taste recognition threshold concentration.

[0175] Suitable sweetness enhancers include, but are not limited to, the group consisting of 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, FEMA GRAS enhancer 4469, FEMA GRAS enhancer 4701, FEMA GRAS enhancer 4720, FEMA GRAS enhancer 4774, FEMA GRAS enhancer 4708, FEMA GRAS enhancer 4728, FEMA GRAS enhancer 4601, and combinations thereof.

[0176] In one embodiment, the addition of the one or more sweetness enhancers increases the detected sucrose equivalence of the at least one sweetener in the consumable as compared to the sucrose equivalence in the same consumable in the absence of the sweetness enhancer.

[0177] More specifically, the use of rebaudioside I and optionally one or more other sweetness enhancers (alone or in the form of a composition) in a consumable (e.g., a beverage) provides a detected sucrose equivalent that is at least about 0.5% greater than the sucrose equivalent of the corresponding consumable (e.g., a beverage) in the absence of rebaudioside I and optionally one or more other sweetness enhancers. For example, the detected sucrose equivalent of a consumable (e.g., a beverage) comprising rebaudioside I and optionally one or more other sweetness enhancers (alone or in the form of a composition) can be at least about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.5%, about 5.0%, or about 5.5% or more greater than the sucrose equivalent of the corresponding consumable in the absence of rebaudioside I and optionally one or more other sweetness enhancers.

[0178] Suitable sweeteners include, but are not limited to, sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheptulose, octulose, fucose, rhamnose, arabinose, turanose, sialic sugars, rebaudioside A, rebaudioside B, rebaudioside C ... C, rebaudioside C, rebaudioside C, rebaudioside C, rebaudi D, rebaudioside E, rebaudioside F, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, monk fruit, simonoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin ), brazzein, hernandulcin, phylloside, phloridzin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, stevioside and cyclocaryoside I, sugar alcohols such as erythritol, sucralose, acesulfame potassium, acesulfame acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamates, cyclamic acid and its salts, neotame, advantame, glycosylated steviol glycosides (GSG), and combinations thereof.

[0179] In one embodiment, the sweetener is a caloric sweetener or a mixture of caloric sweeteners. In another embodiment, the caloric sweetener is selected from sucrose, fructose, glucose, high fructose corn / starch syrup, beet sugar, sucrose, and combinations thereof.

[0180] In another embodiment, the sweetener is a rare sugar selected from D-psicose, D-allose, L-ribose, D-tagatose, L-glucose, L-trehalose, L-arabinose, turanose, and combinations thereof.

[0181] In another embodiment, the sweetener is a mixture of a non-caloric sweetener or a non-caloric sweetener. In an example, the non-caloric sweetener is a natural high-efficiency sweetener. As used herein, phrase "natural high-efficiency sweetener" refers to any composition that is not naturally found in nature and characteristically has a sweetness effect greater than sucrose, fructose, or glucose, and has less calories. Natural high-efficiency sweeteners can be provided as a pure compound or alternatively as a part of an extract.

[0182] In yet another example, the non-caloric sweetener is a synthetic high-potency sweetener. As used herein, the phrase "synthetic sweetener" refers to any composition that is not naturally found in nature and characteristically has a sweetening potency greater than sucrose, fructose or glucose, but has fewer calories.

[0183] In a specific embodiment, the consumable is a beverage. The beverage comprises rebaudioside I and at least one sweetener, wherein rebaudioside I is present at a concentration at or below its sweetness recognition threshold. Rebaudioside I and at least one sweetener can be provided separately or in the form of a sweetness enhancing composition. In a specific embodiment, the detected sucrose equivalent increases from, for example, about 0.2% to about 5.0%, for example, about 1%, about 2%, about 3%, about 4% or about 5%.

[0184] The sweetener can be any natural or synthetic sweetener provided herein. In a specific embodiment, the sweetener is a carbohydrate sweetener providing calories. Therefore, the amount of the carbohydrate sweetener providing calories that must be used in a given consumer's product is reduced in conjunction with the sweet taste enhancer, allowing the preparation of low-calorie consumer's products.

[0185] Said composition can be customized to provide desired caloric content.For example, these compositions can be "rich in heat", so that they give desired sweetness and have every 8 ounces of about 120 calories when added to a kind of consumer's goods (such as, beverage).Alternately, these compositions can be "median value heat", so that they give desired sweetness and have every 8 ounces of less than about 60 calories when added to a kind of consumer's goods (such as, beverage).In other embodiments, these compositions can be "low-calorie", so that they give desired sweetness and have every 8 ounces of less than 40 calories when added to a kind of consumer's goods (such as, beverage).In other embodiments, these compositions can be "zero heat", so that they give desired sweetness and have every 8 ounces of less than 5 calories when added to a kind of consumer's goods (such as, beverage).

[0186] additive

[0187] Compositions, such as sweetener compositions and flavor enhancement compositions, may also include one or more additives in addition to rebaudioside I, as described in detail below. In some embodiments, the composition includes additives, including but not limited to carbohydrates, 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, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighting agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, these additives are used to improve the temporal profile and flavor profile of the sweetener to provide a sweetener composition having a taste similar to sucrose.

[0188] In one embodiment, the composition further comprises one or more polyols. As used herein, the term "polyol" refers to a molecule containing more than one hydroxyl group. Polyols can be diols, triols or tetraols containing 2, 3 and 4 hydroxyls respectively. Polyols can also contain more than 4 hydroxyls, for example, pentahydric alcohols, hexahydric alcohols, heptahydric alcohols etc. containing 5, 6 or 7 hydroxyls respectively. In addition, a polyol can also be a sugar alcohol, a polyhydric alcohol or a polyol (polyalcohol) in the reduced form of a carbohydrate, wherein the carbonyl (aldehyde or ketone, reducing sugar) has been reduced to a primary hydroxyl group or a secondary hydroxyl group.

[0189] In some embodiments, non-limiting examples of polyols include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerine), threitol, dulcitol, palatinose, reduced isomalt-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, and sugar alcohols or any other carbohydrates that can be reduced that do not adversely affect the taste of the composition.

[0190] In certain embodiments, when present in a consumer product (e.g., such as a beverage), the polyol is present in the composition in an amount effective to provide a concentration from about 100 ppm to about 250,000 ppm. In other embodiments, when present in a consumer product (e.g., such as a beverage), the polyol is present in the composition in an amount effective to provide a concentration from about 400 ppm to about 80,000 ppm, such as, for example, from about 5,000 ppm to about 40,000 ppm.

[0191] In other embodiments, rebaudioside I and the polyol are present in the composition in a weight ratio from about 1:1 to about 1:800, such as, for example, from about 1:4 to about 1:800, from about 1:20 to about 1:600, from about 1:50 to about 1:300 or from about 1:75 to about 1:150.

[0192] Suitable amino acid additives include, but are not limited to, aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, arabinose, trans-4-hydroxyproline, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (α, β, and / or δ-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and salt forms thereof such as sodium salts or potassium salts or acid salts. These amino acid additives can also be in the D-configuration or L-configuration and be in the mono-, di- or ternary-form of the same or different amino acids. In addition, if appropriate, these amino acids can be α-, β-, γ- and / or δ-isomers. In certain embodiments, the combination of the above amino acids and their corresponding salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts or other alkali metal salts or alkaline earth metal salts, or acid salts) is also suitable. The amino acid of modification can be natural or synthetic. These amino acids can also be modified. The amino acid of modification refers to any amino acid (for example, N-alkyl amino acid, N-acyl amino acid or N-methyl amino acid) wherein at least one atom has been added, removed, replaced or its combination. The limiting examples of the amino acid of modification include amino acid derivatives, such as trimethylglycine, N-methyl-glycine and N-methyl-alanine. As used herein, the amino acid of modification encompasses both the amino acid of modification and the amino acid of unmodified. As used herein, the amino acid of modification also encompasses both peptides and polypeptides (for example, dipeptides, tripeptides, tetrapeptides and pentapeptides), such as glutathione and L-alanyl-L-glutamine. Suitable polyamino acid additives include poly-L-aspartic acid, poly-L-lysine (e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g., poly-La-ornithine or poly-L-ε-ornithine), poly-L-arginine, other polymer forms of amino acids, and salt forms thereof (e.g., calcium salts, potassium salts, sodium salts or magnesium salts, such as monosodium L-glutamate). The polyamino acid additive may also be in the D-configuration or the L-configuration. In addition, if appropriate, the polyamino acid may be an α-, β-, γ-, δ-, and ε-isomer. In some embodiments, a combination of the above polyamino acids and their corresponding salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts or other alkali metal salts or alkaline earth metal salts or acid salts) is also suitable. The polyamino acids described herein may also include copolymers of different amino acids. These polyamino acids may be natural or synthetic. The polyamino acid may also be modified so that at least one atom is added, removed, substituted, or a combination thereof (e.g., N-alkyl polyamino acids or N-acyl polyamino acids). As used herein, polyamino acids encompass both modified polyamino acids and unmodified polyamino acids.For example, modified polyamino acids include, but are not limited to, polyamino acids having different molecular weights (MW), such as poly-L-α-lysine having a MW of 1,500, a MW of 6,000, a MW of 25,200, a MW of 63,000, a MW of 83,000, or a MW of 300,000.

[0193] In a specific embodiment, when present in a consumable (such as, for example, a beverage), the amino acid is present in the composition in an amount effective to provide a concentration from about 10 ppm to about 50,000 ppm. In another embodiment, when present in a consumable, the amino acid is present in the composition in an amount effective to provide a concentration from about 1,000 ppm to about 10,000 ppm, such as, for example, from about 2,500 ppm to about 5,000 ppm or from about 250 ppm to about 7,500 ppm.

[0194] Suitable sugar acid additives include, but are not limited to, aldonic acid, uronic acid, aldaric acid, alginic acid, gluconic acid, glucuronic acid, glucaric acid, galactaric acid, galacturonic acid, and salts thereof (e.g., sodium salt, potassium salt, calcium salt, magnesium salt or other physiologically acceptable salts), and combinations thereof.

[0195] Suitable nucleotide additives include, but are not limited to, inosine monophosphate ("IMP"), guanosine monophosphate ("GMP"), adenosine monophosphate ("AMP"), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate, alkali metal salts or alkaline earth metal salts thereof, and combinations thereof. The nucleotides described herein may also contain nucleotide-related additives, such as nucleosides or nucleic acid bases (e.g., guanine, cytosine, adenine, thymine, uracil).

[0196] When present in a consumable, such as, for example, a beverage, the nucleotide is present in the composition in an amount effective to provide a concentration from about 5 ppm to about 1,000 ppm.

[0197] Suitable organic acid additives include any compound containing a -COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxy C2-C30 carboxylic acids, butyric acid (ethyl ester), substituted butyric acid (ethyl ester), benzoic acid, substituted benzoic acids (e.g., 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxy acids, substituted hydroxybenzoic acids, anisic acid substituted cyclohexyl carboxylic acids, tannic acid, aconitic acid, lactic acid, tartaric acid, citric acid, isocitric acid, gluconic acid, glucoheptonic acid, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic acid, fumaric acid, and tartaric acid), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acid, acetic acid, ascorbic acid, alginic acid, isoascorbic acid, polyglutamic acid, glucono delta lactone, and alkali metal or alkaline earth metal salt derivatives thereof. Additionally, the organic acid additive may also be in the D-configuration or the L-configuration.

[0198] Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids, such as citrate, malate, tartrate, fumarate, lactate (e.g., sodium lactate), alginates (e.g., sodium alginate), ascorbate (e.g., sodium ascorbate), benzoate (e.g., sodium benzoate or potassium benzoate), sorbate, and adipate. The example of the organic acid additive can be optionally substituted by at least one group selected from the following: hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imine, sulfonyl, alkylthio, sulfinyl, sulfamoyl, carboxyalkoxy, carbonamide (carboxamido), phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, anhydride, oxime, hydrazine, carbamoyl, phosphorus or phosphonate. In a specific embodiment, when present in a consumer product (such as, beverage), the organic acid additive is present in the composition in an amount effective to provide a concentration of from about 10ppm to about 5,000ppm.

[0199] Suitable inorganic acid additives include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogen phosphate, and alkali metal or alkaline earth metal salts thereof (eg, phytate Mg / Ca).

[0200] When present in a consumable product, such as, for example, a beverage, the inorganic acid additive is present in the composition in an amount effective to provide a concentration from about 25 ppm to about 25,000 ppm.

[0201] Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, quassia, and salts thereof.

[0202] When present in a consumable, such as, for example, a beverage, the bitter compound is present in the composition in an amount effective to provide a concentration from about 25 ppm to about 25,000 ppm.

[0203] Suitable flavoring agents and flavoring ingredients include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, viridiflorol, almonds, menthol (including mint-free menthol), grape skin extract, and grape seed extract. "Flavoring agent" and "flavoring ingredient" are synonymous and may include natural or synthetic substances or combinations thereof. Flavoring agents also include any other substances that impart flavor and may include natural or non-natural (synthetic) substances that are safe for humans or animals when used in a generally accepted range. Non-limiting examples of specialized flavoring agents include Natural flavor sweetener K14323( Darmstadt, Germany), sweeteners 161453 and 164126 Symrise TM Natural flavoring mask (Symrise TM , Holzminden, Germany), Natural Advantage TM Bitter Blockers 1, 2, 9 and 10 (Natural Advantage TM , Freehold, New Jersey, USA), and Sucramask TM (Creative Research Management, Stockton, California, USA).

[0204] When present in a consumable product such as, for example, a beverage, the flavoring agent is present in the composition in an amount effective to provide a concentration from about 0.1 ppm to about 4,000 ppm.

[0205] Suitable polymer additives include, but are not limited to, chitin, pectin, pectinic acid, polyuronic acid, polygalacturonic acid, starch, food hydrocolloids or crude extracts thereof (e.g., Senegalese gum (Fibergum TM), gum arabic, carrageenan), poly-L-lysine (e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g., poly-L-α-ornithine or poly-L-ε-ornithine), polypropylene glycol, polyethylene glycol, poly(ethylene glycol methyl ether), polyarginine, polyaspartic acid, polyglutamic acid, polyethyleneimine, alginic acid, sodium alginate, propylene glycol alginate, and sodium polyethylene glycol alginate, sodium hexametaphosphate and its salts, and other cationic polymers and anionic polymers.

[0206] When present in a consumable product, such as, for example, a beverage, the polymer is present in the composition in an amount effective to provide a concentration of from about 30 ppm to about 2,000 ppm.

[0207] Suitable protein or protein hydrolysate additives include, but are not limited to, bovine serum albumin (BSA), whey protein (including fractions or concentrates such as 90% instant whey protein isolate, 34% whey protein, 50% hydrolyzed whey protein, and 80% whey protein concentrate), soluble rice protein, soy protein, protein isolate, protein hydrolysate, reaction products of protein hydrolysate, glycoprotein and / or proteoglycans containing amino acids (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, arginine, valine, isoleucine, leucine, norvaline, methionine, proline, tyrosine, hydroxyproline, etc.), collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., hydrolyzed fish collagen), and collagen hydrolysate (e.g., porcine collagen hydrolysate).

[0208] When present in a consumable product, such as, for example, a beverage, the protein hydrolysate is present in the composition in an amount effective to provide a concentration from about 200 ppm to about 50,000 ppm.

[0209] Suitable surfactant additives include, but are not limited to, polysorbates (e.g., polyoxyethylene sorbitan monooleate (polysorbate 80), polysorbate 20, polysorbate 60), sodium dodecylbenzene sulfonate, dioctyl sulfosuccinate or sodium dioctyl sulfosuccinate, sodium lauryl sulfate, cetylpyridinium chloride (cetylpyridinium chloride), cetyltrimethylammonium bromide, sodium cholate, carbamoyl, choline chloride, sodium glycocholate, sodium taurodeoxycholate, lauroyl arginine ester, sodium stearoyl lactylate, sodium taurocholate, lecithin, sucrose oleate, sucrose stearate, sucrose palmitate, sucrose laurate and other emulsifiers.

[0210] When present in a consumable product such as, for example, a beverage, the surfactant additive is present in the composition in an amount effective to provide a concentration from about 30 ppm to about 2,000 ppm.

[0211] Suitable flavonoid additives are classified as flavonols, flavones, flavanones, flavan-3-ols, isoflavones or anthocyanidins. Non-limiting examples of flavonoid additives include, but are not limited to, catechins (e.g., green tea extracts such as Polyphenon TM 60. Polyphenon TM 30 and Polyphenon TM 25 (MitsuiNorin Co., Ltd., Japan), polyphenols, rutin (e.g., enzyme-modified rutin Sanmelin TM AO (San-fi Gen FFI, Inc., Osaka, Japan), neohesperidin, naringin, neohesperidin dihydrochalcone, etc.

[0212] When present in a consumable product, such as, for example, a beverage, the flavonoid additive is present in the composition in an amount effective to provide a concentration from about 0.1 ppm to about 1,000 ppm.

[0213] Suitable alcohol additives include, but are not limited to, ethanol. In particular embodiments, when present in a consumable product such as, for example, a beverage, the alcohol additive is present in the composition in an amount effective to provide a concentration from about 625 ppm to about 10,000 ppm.

[0214] Suitable astringent compounds include, but are not limited to, tannic acid, europium chloride (EuCl3), gadolinium chloride (GdCl3), terbium chloride (TbCl3), alum, tannic acid, and polyphenols (e.g., tea polyphenols). When present in a consumer product (e.g., such as a beverage), the astringent additive is present in the composition in an amount effective to provide a concentration of from about 10 ppm to about 5,000 ppm.

[0215] Functional Ingredients

[0216] The compositions provided herein can also contain one or more functional ingredients that provide actual or perceived health benefits to the compositions. Functional ingredients include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

[0217] Saponin

[0218] In certain embodiments, the functional ingredient is at least one saponin. As used herein, the at least one saponin can include a single saponin or multiple saponins as a functional ingredient of the composition provided herein. Usually, according to a particular embodiment of the present invention, the at least one saponin is present in the composition in an amount that is enough to promote health and health care.

[0219] Saponins are glycoside natural plant products containing a glycoside ring structure and one or more sugar moieties. The combination of the nonpolar aglycone and the water-soluble sugar moieties gives saponins surfactant properties that allow them to form foams when shaken in aqueous solutions.

[0220] These saponins are grouped together based on several common characteristics. Specifically, saponin is a surfactant that exhibits hemolytic activity and forms a complex with cholesterol. Although saponins have these characteristics in common, they are different in structure. The type of aglycone ring structure that forms a ring structure in saponin can change significantly. Non-limiting examples of these aglycone ring structure types in the saponins used in the specific embodiments of the present invention include steroids, triterpenes and steroidal alkaloids. Non-limiting examples of specific aglycone ring structures used in the specific embodiments of the present invention include soyasapogenol A, soyasapogenol B and soyasapogenol E. The number and type of sugar moieties connected to the aglycone ring structure can also vary greatly. Non-limiting examples of sugar moieties used in the specific embodiments of the present invention include glucose, galactose, glucuronic acid, xylose, rhamnose and methylpentose moieties. Non-limiting examples of specific saponins used in the specific embodiments of the present invention include A group acetyl saponin, B group acetyl saponin, and E group acetyl saponin.

[0221] Saponins can be found in a wide variety of plants and plant products, and are particularly prevalent in plant skins and barks, where they form a waxy protective coating. Several common sources of saponins include soybeans, soapwort plants (Saponaria, whose roots have been used as soap in history), and alfalfa, aloe, asparagus, grapes, chickpeas, yucca, and various other beans and weeds, which have about 5% saponin content by dry weight. Saponins can be obtained from these sources using extraction techniques known to those of ordinary skill in the art. A description of conventional extraction techniques is found in U.S. Patent Application No. 2005 / 0123662, the disclosure of which is expressly incorporated by reference.

[0222] Antioxidants

[0223] In certain embodiments, the functional ingredient is at least one antioxidant. As used herein, the at least one antioxidant can include a single antioxidant or multiple antioxidants as a functional ingredient of the composition provided herein. Typically, according to a specific embodiment of the invention, the at least one antioxidant is present in the composition in an amount sufficient to promote health and health care.

[0224] As used herein, "antioxidant" refers to any substance that inhibits, suppresses or reduces oxidative damage to cells and biomolecules. Without being bound by theory, it is believed that antioxidants prevent, suppress or reduce oxidative damage to cells or biomolecules by stabilizing free radicals before they can cause harmful reactions. In this way, antioxidants can prevent or delay the onset of some degenerative diseases.

[0225] Examples of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, vitamins, vitamin cofactors, minerals, hormones, carotenoids, carotenoid terpenes, non-carotenoid terpenes, flavonoids, flavonoid polyphenols (such as bioflavonoids), flavonols, flavonoids, phenols, polyphenols, phenolic esters, polyphenolic esters, non-flavonoid phenols, isothiocyanates, and combinations thereof. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, ubiquinone, mineral selenium, manganese, melatonin, alpha-carotene, beta-carotene, lycopene, lutein, zeanthin, crypoxanthin, reservatol, eugenol, quercetin, catechins, gossypol, hesperidin, curcumin, ferulic acid, thymol, hydroxytyrosol, turmeric, thyme, olive oil, lipoic acid, glutathione, gutamine, oxalic acid, tocopherol derivative compounds, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, acetic acid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxanthin, soap glucosides, limonene, kaempfedrol, myricetin, isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin, tangeritin, hesperidin, naringenin, erodictyol, flavan-3-ols (e.g., anthocyanidins), epigallocatechin gallate, epicatechin and its gallate forms, epigallocatechin and its gallate forms (ECGC), theaflavins and its gallic acid ester forms, thearubin, isoflavone phytoestrogens, genistein, daidzein, glycitein, anythocyanin, cyaniding, delphinidin, malvaccin, malvidin, malvidin, methylanthocyanidin, petunidin, ellagic acid, gallic acid, salicylic acid, rosmarinic acid, cinnamic acid and its derivatives (e.g., ferulic acid), chlorogenic acid, chicoric acid, gallic tannins, ellagitannins, anthocyanins, beta-cyanins and other plant pigments, silymarin, citric acid, lignans, antinutrients, bilirubin, uric acid, R-alpha-lipoic acid, N-acetylcysteine, emblicanin, apple extract, apple peel extract (apple polyphenols), rooibos extract red, rooibos extract green,green), hawthorn fruit extract, raspberry extract, green coffee antioxidant (GCA), wild aronia extract 20%, grape seed extract (VinOseed), cocoa bean extract, hops extract, mangosteen extract, mangosteen shell extract, cranberry extract, pomegranate extract, pomegranate peel extract, pomegranate seed extract, hawthorn berry extract, pomegranate extract, cinnamon bark extract, grape skin extract, bilberry extract, pine bark extract, pycnogenol, elderberry extract, mulberry root extract, wolfberry (gogi) extract, blackberry extract, blueberry extract, blueberry leaf extract, raspberry extract, turmeric extract, citrus bioflavonoids, black currant, ginger, acai powder, green coffee bean extract, green tea extract, and phytic acid, or a combination thereof. In alternative embodiments, the antioxidant is a synthetic antioxidant, such as, for example, butylated hydroxytoluene or butylated hydroxyanisole. Other sources of suitable antioxidants for use in embodiments of the present invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, organ meats from livestock, yeast, whole grains, or cereal grains.

[0226] Specific antioxidants belong to the class of phytonutrients known as polyphenols (also known as "polyphenols"), which are a group of chemicals found in plants characterized by the presence of more than one phenolic group per molecule. A variety of health benefits can be derived from polyphenols, including, for example, prevention of cancer, heart disease, and chronic inflammatory diseases, and improved mental and physical strength. Suitable polyphenols for use in embodiments of the present invention include catechins, proanthocyanidins, proanthocyanidins, anthocyanidins, quercetin, rutin, resveratrol, isoflavones, curcumin, punicalagins, ellagitannins, hesperidin, naringin, citrus flavonoids, chlorogenic acid, other similar materials, and combinations thereof.

[0227] In specific embodiments, the antioxidant is a catechin, such as, for example, epigallocatechin gallate (EGCG). Suitable sources of catechins for embodiments of the present invention include, but are not limited to, green tea, white tea, black tea, oolong tea, chocolate, cocoa, red wine, grape seeds, red grape skins, purple grape skins, red grape juice, purple grape juice, berries, pycnogenol, and red apple peels.

[0228] In some embodiments, the antioxidant is selected from proanthocyanidins, proanthocyanidins, or a combination thereof. Suitable sources of proanthocyanidins and proanthocyanidins for embodiments of the present invention include, but are not limited to, red grapes, purple grapes, cocoa, chocolate, grape seeds, red wine, cocoa beans, cranberries, apple peels, plums, blueberries, black currants, choke berries, green tea, sorghum, cinnamon, barley, red kidney beans, black pinto beans, hops, almonds, hazelnuts, pecans, pistachios, pycnogenol, and colored berries.

[0229] In a specific embodiment, the antioxidant is an anthocyanidin. Suitable sources of anthocyanidins for embodiments of the present invention include, but are not limited to, red berries, blueberries, bilberries, cranberries, raspberries, cherries, pomegranates, strawberries, elderberries, rowan berries, red grape skins, purple grape skins, grape seeds, red wine, black currants, red currants, cocoa, plums, apple skins, peaches, red pears, red cabbage, red onions, red oranges, and blackberries.

[0230] In some embodiments, the antioxidant is selected from quercetin, rutin or a combination thereof. Suitable sources of quercetin and rutin for embodiments of the present invention include, but are not limited to, red apples, onions, kale, blueberries, bilberries, rowan berries, cranberries, blackberries, blueberries, strawberries, raspberries, blackcurrants, green tea, black tea, plums, apricots, parsley, leeks, broccoli, red peppers, berry wine, and ginkgo.

[0231] In some embodiments, the antioxidant is resveratrol. Suitable sources of resveratrol for use in embodiments of the present invention include, but are not limited to, red grapes, peanuts, cranberries, blueberries, bilberries, mulberries, Japanese Itadori tea, and red wine.

[0232] In specific embodiments, the antioxidant is an isoflavone. Suitable sources of isoflavones for embodiments of the present invention include, but are not limited to, soybeans, soybean products, legumes, alfalfa spouts, chickpeas, peanuts, and red clover.

[0233] In some embodiments, the antioxidant is curcumin. Suitable sources of curcumin for embodiments of the present invention include, but are not limited to, turmeric and mustard.

[0234] In a specific embodiment, the antioxidant is selected from quercetin, ellagitannins or a combination thereof. Suitable sources of quercetin and ellagitannins for embodiments of the present invention include, but are not limited to, pomegranate, raspberry, strawberry, walnut, and old red wine.

[0235] In some embodiments, the antioxidant is a citrus flavonoid, such as hesperidin or naringin. Suitable sources of citrus flavonoids such as hesperidin or naringin for embodiments of the present invention include, but are not limited to, orange, grapefruit, and citrus juice.

[0236] In a specific embodiment, the antioxidant is chlorogenic acid. Suitable sources of chlorogenic acid for embodiments of the present invention include, but are not limited to, green coffee, yerba mate, red wine, grape seeds, red grape skins, purple grape skins, red grape juice, purple grape juice, apple juice, cranberries, pomegranates, blueberries, strawberries, sunflowers, echinacea, pycnogenol, and apple peels.

[0237] Dietary fiber

[0238] In certain embodiments, the functional ingredient is at least one dietary fiber source. As used herein, the at least one dietary fiber source can include a single dietary fiber source or multiple dietary fiber sources as a functional ingredient of the composition provided herein. Usually, according to a specific embodiment of the present invention, the at least one dietary fiber source is present in the composition in an amount that is enough to promote health and health care.

[0239] A variety of polymeric carbohydrates having significantly different structures in both composition and connection fall within the definition of dietary fiber. Such compounds are well known to those skilled in the art, and their non-limiting examples include non-starch polysaccharides, lignin, cellulose, methylcellulose, hemicellulose, beta-glucan, pectin, gum, mucilage, wax, inulin, oligosaccharides, oligofructose, cyclodextrin, chitin, and combinations thereof.

[0240] Polysaccharides are complex carbohydrates composed of monosaccharides linked by glycosidic bonds. Non-starch polysaccharides are linked with β bonds, and humans cannot digest them due to the lack of an enzyme that breaks β bonds. In contrast, digestible starch polysaccharides usually contain α (1-4) bonds.

[0241] Lignin is a large, highly branched and cross-linked polymer based on oxidized phenylpropane units. Cellulose is a linear polymer of glucose molecules linked by a β (1-4) bond, which cannot be hydrolyzed by mammalian amylases. Methylcellulose is a methyl ester of cellulose commonly used as a thickener and emulsifier in foods. It is commercially available (e.g., Citrucel sold by GlaxoSmithKline, Celevac sold by Shire Pharmaceuticals). Hemicellulose is a highly branched polymer composed mainly of glucuronic acid- and 4-0-methylglucuronic acid xylans. Beta glucans are mixed-linkage (1-3), (1-4) β-D-glucose polymers found primarily in cereals such as oats and barley. Pectins such as beta pectin are a group of polysaccharides composed primarily of D-galacturonic acid, which is methoxylated to a variable degree.

[0242] Gums and mucilage represent a wide range of different branched structures. Guar gum derived from the ground endosperm of guar seeds is a galactomannan. Guar gum is commercially available (e.g., Benefiber sold by Novartis AG). Other gums such as gum arabic and pectin also have different structures. Other gums also include xanthan gum, gellan gum, tara gum, psyllium seed husk gum and locust bean gum.

[0243] Wax is an ester of ethylene glycol and two fatty acids and usually exists as a hydrophobic liquid that is insoluble in water.

[0244] Inulin includes naturally occurring oligosaccharides belonging to a class of carbohydrates known as fructans. They are usually composed of fructose units linked by β-(2-1) glycosidic bonds with one terminal glucose unit. Oligosaccharides are sugar polymers that typically contain three to six component sugars. They are usually found O-linked or N-linked to compatible amino acid side chains in proteins or lipid molecules. Oligofructose is an oligosaccharide composed of short chains of fructose molecules.

[0245] Food sources of dietary fiber include, but are not limited to, cereals, beans, fruits, and vegetables. Cereals that provide dietary fiber include, but are not limited to, oats, rye, barley, wheat. Beans that provide fiber include, but are not limited to, peas and kidney beans such as soybeans. Fruits and vegetables that provide fiber sources include, but are not limited to, apples, oranges, pears, bananas, berries, tomatoes, green beans, broccoli, cauliflower, carrots, potatoes, celery. Plant foods such as bran, nuts and seeds (such as flaxseed) are also sources of dietary fiber. Plant parts that provide dietary fiber include, but are not limited to, stems, roots, leaves, seeds, pulp, and skin.

[0246] Although dietary fiber is usually derived from plant sources, indigestible animal products such as chitin are also classified as dietary fiber.Chitin is a polysaccharide composed of acetylglucosamine units linked by beta (1-4) bonds similar to the bonds of cellulose.

[0247] Dietary fiber sources are generally divided into soluble fiber and insoluble fiber categories based on their solubility in water. Both soluble and insoluble fibers are found in plant foods to varying degrees depending on the characteristics of the plant. Although insoluble in water, insoluble fiber has a passive hydrophilic characteristic that helps add bulk, soften stool, and shorten the transit time of stool solids through the intestines.

[0248] Unlike insoluble fiber, soluble fiber is easily dissolved in water.Soluble fiber undergoes active metabolic process by fermentation in the colon, thereby increasing colon flora and thereby increasing the quality of fecal solids.Fiber fermentation by colon bacteria also produces end products with significant health benefits.For example, the fermentation of food quality produces gas and short-chain fatty acids.The acid produced during the fermentation process includes butyric acid, acetic acid, propionic acid and valeric acid with different favorable characteristics, which are such as stabilizing blood sugar levels and providing liver control by glycogen degradation by acting on pancreatic insulin release.In addition, fiber fermentation can reduce atherosclerosis by reducing cholesterol synthesis in the liver and reducing blood LDL and triglyceride levels.The acid produced during the fermentation process reduces colon pH, thereby protecting colon mucosa from forming cancer polyps.The reduced colon pH also increases mineral absorption, improves the barrier properties of colon mucosal layer, and inhibits inflammation and adhesion stimulation.The fermentation of fiber can also be beneficial to the immune system by stimulating the production of T helper cells, antibodies, leukocytes, splenocytes, cytokines and lymphocytes.

[0249] fatty acid

[0250] In certain embodiments, the functional ingredient is at least one lipid acid. As used herein, this at least one lipid acid can be a single lipid acid or multiple lipid acids as a functional ingredient of the composition provided herein. Usually, according to a specific embodiment of the present invention, this at least one lipid acid is present in the composition with the amount that is enough to promote health and health care.

[0251] As used herein, "fatty acid" refers to any straight-chain monocarboxylic acid and includes saturated fatty acids, unsaturated fatty acids, long-chain fatty acids, medium-chain fatty acids, short-chain fatty acids, fatty acid precursors (including ω-9 fatty acid precursors), and esterified fatty acids. As used herein, "long-chain polyunsaturated fatty acids" refers to any polyunsaturated carboxylic acid or organic acid with a long aliphatic tail. As used herein, "ω-3 fatty acids" refers to any polyunsaturated fatty acid with a first double bond as the third carbon-carbon bond starting from the terminal methyl end of its carbon chain. In a specific embodiment, ω-3 fatty acids can include a long-chain ω-3 fatty acid. As used herein, "ω-6 fatty acids" refers to any polyunsaturated fatty acid with a first double bond as the sixth carbon-carbon bond starting from the terminal methyl end of its carbon chain.

[0252] The ω-3 fatty acids suitable for embodiments of the present invention can be derived from, for example, algae, fish, animals, plants or a combination thereof.The example of the ω-3 fatty acids suitable for includes, but is not limited to, linolenic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, octadecatetraenoic acid, eicosatetraenoic acid and a combination thereof.In some embodiments, the ω-3 fatty acids suitable for can be provided in fish oil (for example herring oil, tuna oil, salmon oil, bonito oil and cod oil), microalgae ω-3 oil or a combination thereof. In specific embodiments, suitable omega-3 fatty acids can be derived from commercially available omega-3 fatty acid oils, such as microalgae DHA oil (from Martek, Columbia, MD), OmegaPure (from Omega Protein, Houston, TX), Marinol C-38 (from Lipid Nutrition, Channahon, IL), Bonito oil and MEG-3 (from Ocean Nutrition, Dartmouth, NS), Evogel (from Symrise, Holzminden, Germany), marine oils from tuna or salmon (from Arista Wilden, CT). Wilton, CT), OmegaSource 2000, marine oil from menhaden, and marine oil from cod (from OmegaSource, RTP, NC).

[0253] Suitable omega-6 fatty acids include, but are not limited to, linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, and combinations thereof.

[0254] Suitable esterified fatty acids for use in embodiments of the present invention may include, but are not limited to, monoacylglycerols containing ω-3 and / or ω-6 fatty acids, diacylglycerols containing ω-3 and / or ω-6 fatty acids, or triacylglycerols containing ω-3 and / or ω-6 fatty acids, and combinations thereof.

[0255] Vitamins

[0256] In certain embodiments, the functional ingredient is at least one vitamin.

[0257] As used herein, the at least one vitamin can be a single vitamin or multiple vitamins as a functional ingredient of the composition provided herein. Generally, according to specific embodiments of the present invention, the at least one vitamin is present in the composition in an amount sufficient to promote health and wellness.

[0258] Vitamins are organic compounds that the human body needs in small amounts to function properly. The body uses vitamins without destroying them, unlike other nutrients such as carbohydrates and proteins. So far, 13 vitamins have been recognized, and one or more can be used in the composition herein. Suitable vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12 and vitamin C. Many vitamins also have alternative chemical names, and their non-limiting examples are provided below.

[0259]

[0260]

[0261] A variety of other compounds have been classified as vitamins by some authorities. These compounds may be referred to as pseudovitamins, and include, but are not limited to, compounds such as ubiquinone (coenzyme Q10), pancreatic acid, dimethylglycine, taestrile, amygdalin, flavonoids, p-aminobenzoic acid, adenine, adenylic acid, and s-methylmethionine. As used herein, the term vitamin includes pseudovitamins.

[0262] In some embodiments, the vitamin is a fat-soluble vitamin selected from vitamin A, vitamin D, vitamin E, vitamin K, and combinations thereof.

[0263] In other embodiments, the vitamin is a water-soluble vitamin selected from the following: vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, biotin, pantothenic acid, vitamin C and combinations thereof.

[0264] Glucosamine

[0265] In certain embodiments, the functional ingredient is glucosamine.

[0266] Generally, according to particular embodiments of this invention, glucosamine is present in these compositions in an amount sufficient to promote health and wellness.

[0267] Glucosamine (also known as chitosan) is an amino sugar that is considered to be an important precursor in the biochemical synthesis of glycosylated proteins and lipids. D-glucosamine is naturally present in cartilage in the form of glucosamine-6-phosphate, which is synthesized by fructose-6-phosphate and glutamine. However, glucosamine can also be obtained in other forms, and its non-limiting examples include glucosamine hydrochloride, glucosamine sulfate, N-acetyl-glucosamine or any other salt form or combination thereof. Glucosamine can be obtained by acid hydrolysis of lobster, crab, shrimp or prawn shells using methods well known to those of ordinary skill in the art. In a specific embodiment, glucosamine can be derived from fungal biomass containing chitin, as described in U.S. Patent Publication No. 2006 / 0172392.

[0268] These compositions may further comprise chondroitin sulfate.

[0269] Minerals

[0270] In certain embodiments, the functional ingredient is at least one mineral.

[0271] As used herein, the at least one mineral can be a single mineral or multiple minerals as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one mineral is present in the composition in an amount sufficient to promote health and wellness.

[0272] According to the teachings of the present invention, minerals include inorganic chemical elements required by organisms. Minerals are composed of a wide range of compositions (e.g., elements, simple salts, and complex silicates) and have widely varying crystalline structures. They can occur naturally in food and beverages, can be added as a supplement, or can be consumed or administered separately from food or beverages.

[0273] Minerals can be classified as bulk minerals, which are required in relatively large amounts, or trace minerals, which are required in relatively small amounts. Bulk minerals are generally required in amounts greater than or equal to about 100 mg per day and trace minerals are those minerals that are required in amounts less than about 100 mg per day.

[0274] In a specific embodiment of the present invention, the mineral substance is selected from a main mineral substance, a trace mineral substance or a combination thereof. The non-limiting examples of the main mineral substance include calcium, chlorine, magnesium, phosphorus, potassium, sodium and sulfur. The non-limiting examples of trace mineral substances include chromium, cobalt, copper, fluorine, iron, manganese, molybdenum, selenium, zinc and iodine. Although iodine is usually classified as a trace mineral substance, it requires a larger amount than other trace mineral substances and is often classified as a main mineral substance.

[0275] In other specific embodiments of the invention, the mineral is a trace mineral considered essential for human nutrition, non-limiting examples of which include bismuth, boron, lithium, nickel, rubidium, silicon, strontium, tellurium, tin, titanium, tungsten, and vanadium.

[0276] The minerals presented herein may be in any form known to those of ordinary skill in the art. For example, in one embodiment, the minerals may be in their ionic form having a positive or negative charge. In another embodiment, the minerals may be in their molecular form. For example, sulfur and phosphorus typically occur naturally as sulfates, sulfides, and phosphates.

[0277] preservative

[0278] In certain embodiments, the functional ingredient is at least one preservative.

[0279] As used herein, the at least one preservative can be a single preservative or multiple preservatives as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the invention, the at least one preservative is present in the composition in an amount sufficient to promote health and wellness.

[0280] In a specific embodiment of the present invention, the preservative is selected from an antimicrobial, an antioxidant, an anti-enzyme or a combination thereof. Non-limiting examples of antimicrobial include sulfites, propionates, benzoates, sorbates, nitrates, nitrites, bacteriocins, salts, sugars, acetic acid, dimethyl dicarbonate (DMDC), ethanol and ozone.

[0281] According to a specific embodiment, the preservative is a sulfite. Sulfites include, but are not limited to, sulfur dioxide, sodium bisulfite and potassium bisulfite.

[0282] According to another specific embodiment, the preservative is a propionate. Propionates include, but are not limited to, propionic acid, calcium propionate and sodium propionate.

[0283] According to yet another specific embodiment, the preservative is a benzoate. Benzoates include, but are not limited to, sodium benzoate and benzoic acid.

[0284] In another embodiment, the preservative is a sorbate. Sorbic acid salts include, but are not limited to, potassium sorbate, sodium sorbate, calcium sorbate, and sorbic acid.

[0285] In yet another specific embodiment, the preservative is a nitrate and / or a nitrite. Nitrates and nitrites include, but are not limited to, sodium nitrate and sodium nitrite.

[0286] In yet another particular embodiment, the at least one preservative is a bacteriocin, such as, for example, nisin.

[0287] In another specific embodiment, the preservative is ethanol.

[0288] In yet another particular embodiment, the preservative is ozone.

[0289] Non-limiting examples of anti-enzyme agents suitable for use as preservatives in particular embodiments of the present invention include ascorbic acid, citric acid, and metal chelators such as ethylenediaminetetraacetic acid (EDTA).

[0290] Hydration Agent

[0291] In certain embodiments, the functional ingredient is at least one hydrating agent.

[0292] As used herein, the at least one hydrating agent can be a single hydrating agent or multiple hydrating agents as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one hydrating agent is present in the composition in an amount sufficient to promote health and wellness.

[0293] Hydration products help the body to replace the body fluid lost by excretion. For example, body fluid is lost as sweat to regulate body temperature, as urine to excrete waste, and as water vapor to exchange the gas in the lung. Body fluid loss can also occur due to a wide range of external causes, and the non-limiting examples of these external causes include physical activity, exposure to dry air, diarrhea, vomiting, high fever, shock, blood loss and hypotension. Diseases causing body fluid loss include diabetes, cholera, gastroenteritis, shigellosis and yellow fever. Malnutrition forms causing body fluid loss include excessive consumption of alcohol, electrolyte imbalance, fasting and rapid weight loss.

[0294] In a specific embodiment, the hydration product is a composition that helps the body replace the body fluid lost during exercise. Therefore, in a specific embodiment, the hydration product is an electrolyte, and its non-limiting examples include sodium, potassium, calcium, magnesium, chloride, phosphate, bicarbonate and combinations thereof. Suitable electrolytes for use in specific embodiments of the present invention are also described in U.S. Patent No. 5,681,569, and the disclosure of the patent is clearly combined here by reference. In a specific embodiment, these electrolytes are obtained from their corresponding water-soluble salts. Non-limiting examples of salts used in specific embodiments include chlorides, carbonates, sulfates, acetates, bicarbonates, citrates, phosphates, hydrogen phosphates, tartrates, sorbates, citrates, benzoates or combinations thereof. In other embodiments, these electrolytes are provided by fruit juice, fruit extracts, vegetable extracts, tea or tea extracts.

[0295] In a specific embodiment of the present invention, hydration product is a kind of carbohydrate of the energy storage that supplements muscle burns.U.S. Patent number 4,312,856,4,853,237,5,681,569 and 6,989,171 describe the suitable carbohydrate for being used in a specific embodiment of the present invention, the disclosure content of these patents is clearly combined in this by reference.The limiting examples of being suitable for carbohydrate comprises monose, disaccharide, oligosaccharide, complex polysaccharide or its combination.The limiting examples of the monose of the suitable type for being used in a specific embodiment comprises triose, tetrose, pentose, hexose, heptose, octose and nonose. The limiting examples of suitable monosaccharides of a particular type include glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheltulose, octolose, and sialose. The limiting examples of suitable disaccharides include sucrose, lactose and maltose. The limiting examples of suitable oligosaccharides include sucrose, maltotriose and maltodextrin. In other specific embodiments, carbohydrate is provided by corn syrup, beet sugar, cane sugar, fruit juice or tea.

[0296] In another specific embodiment, the hydration is a kind of flavanol that provides cell rehydration.Flavanol is a class of natural substances present in plants, and generally includes the 2-phenylbenzopyrone molecular skeleton attached to one or more chemical parts.The limiting examples of suitable flavanols for specific embodiments of the present invention include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin 3 gallate, theaflavins, theaflavins 3-gallate, theaflavins 3'-gallate, theaflavins 3,3'-gallate, thearubigins or its combination.Several common sources of flavanols include tea tree, fruit, vegetables and flower.In a preferred embodiment, flavanols are extracted from green tea.

[0297] In a specific embodiment, the hydration product is a glycerol solution that enhances exercise endurance. Ingestion of a solution containing glycerol has been shown to provide a variety of beneficial physiological effects, such as expanded blood volume, reduced heart rate, and reduced rectal temperature.

[0298] Probiotics / Prebiotics

[0299] In certain embodiments, the functional ingredient is selected from at least one probiotic, prebiotic, and combinations thereof.

[0300] As used herein, the at least one probiotic or prebiotic can be a single probiotic or prebiotic or a plurality of probiotics or prebiotics as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the invention, the at least one probiotic, prebiotic or combination thereof is present in the composition in an amount sufficient to promote health and wellness.

[0301] According to the teachings of the present invention, probiotics include microorganisms that are beneficial to health when consumed in effective amounts. Ideally, probiotics favorably affect the human body's naturally occurring gastrointestinal microflora and impart health benefits beyond nutrition. Probiotics may include, but are not limited to, bacteria, yeast, and fungi.

[0302] According to the teachings of the present invention, prebiotics are compositions that promote the growth of beneficial bacteria in the intestine. Prebiotic substances can be consumed by a related probiotic, or in addition help to keep the related probiotic alive or stimulate its growth. When consumed in effective amounts, probiotics also favorably affect the naturally occurring gastrointestinal microflora of the human body, thereby conferring health benefits other than just nutrition. Prebiotic foods enter the colon and are used as substrates for endogenous bacteria, thereby indirectly providing energy, metabolic substrates and essential micronutrients to the host. The digestion and absorption of the prebiotic foods of the body depend on bacterial metabolic activity, which saves the host's energy from nutrients that escape digestion and absorption in the small intestine.

[0303] According to a specific embodiment, probiotics are beneficial microorganisms that favorably affect the gastrointestinal microflora naturally present in the human body and confer health benefits other than nutrition. Examples of probiotics include, but are not limited to, bacteria of the genus Lactobacilli, the genus Bifidobacteria, the genus Streptococci, or a combination thereof that confer beneficial effects on humans.

[0304] In a particular embodiment of the present invention, the at least one probiotic is selected from Lactobacillus. Lactobacillus (i.e., Lactobacillus bacteria, "L." thereafter) has been used as a food method for hundreds of years and is used to promote human health. The non-limiting examples of visible lactobacillus species in the human gastrointestinal tract include Lactobacillus acidophilus (L.acidophilus), Lactobacillus casei (L.casei), Lactobacillus fermentum (L.fermentum), Lactobacillus salivarius (L.saliva roes), Lactobacillus brevis (L.brevis), Lactobacillus leichmannii (L.leichmannii), Lactobacillus plantarum (L.plantarum), Lactobacillus cellobiosus (L.cellobiosus), Lactobacillus reuteri (L.reuteri), Lactobacillus rhamnosus (L.rhamnosus), Lactobacillus GG (L.GG), Lactobacillus bulgaricus (L.bulgaricus) and thermophilic lactic acid bacteria (L.thermophilus).

[0305] According to other specific embodiments of the present invention, the probiotic is selected from the genus Bifidobacterium. It is also known that bifidobacteria produce short-chain fatty acids (e.g., acetic acid, propionic acid and butyric acid), lactic acid and formic acid through carbohydrate metabolism to exert a beneficial effect on human health. Non-limiting types of bifidobacteria visible in the human gastrointestinal tract include infant bifidobacterium (B.angulatum), animal bifidobacterium (B.animalis), asteroid bifidobacterium (B.asteroides), bifidum bifidum (B.bifidum), boum bifidobacterium (B.boum), breve bifidobacterium (B.breve), catenulatum bifidum, choerinum bifidum (B.choerinum), coryneforme bifidobacterium (B.coryneforme), cuniculi bifidum (B.cuniculi), dentium bifidum (B.dentium), gallicum bifidum (B.gallicum), gallinarum bifidum (B.gallinarum), wild chrysanthemum bifidobacterium (B. indicum), B. longum, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum, B. pullorum, B. ruminantium, B. saeculare, B. scardovii, B. simiae, B. subtile, B. thermacidophilum, B. thermophilum, B. urinalis, and Bifidobacterium sp.

[0306] According to other specific embodiments of the present invention, the probiotic is selected from the genus Streptococcus. Streptococcus thermophilus is a gram-positive facultative anaerobic bacterium. It is classified as a lactic acid bacterium and is commonly found in milk and dairy products, and is used to produce yogurt. Other non-limiting probiotic species of this bacterium include Streptococcus salivarus and Streptococcus cremoris.

[0307] Probiotics that can be used according to the present invention are well known to those skilled in the art. Non-limiting examples of foods containing probiotics include yogurt, sauerkraut, goat cheese, kimchi, fermented vegetables, and other foods containing microbial elements that favorably affect the host animal by improving the intestinal microbalance.

[0308] According to an embodiment of the present invention, prebiotics include, but are not limited to, mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins, and combinations thereof.

[0309] According to a specific embodiment of the present invention, the prebiotic is selected from dietary fiber, including but not limited to polysaccharides and oligosaccharides. These compounds have the ability to increase the number of probiotics, which produces the benefits given by these probiotics. The non-limiting examples of oligosaccharides classified as prebiotics according to a specific embodiment of the present invention include oligofructose, inulin, oligomalto-oligosaccharides, lactitol (Iactilol), oligolactose, lactulose, pyrodextrin, soybean oligosaccharides, oligomeric trans-galactose, and oligoxylose.

[0310] According to other specific embodiments of the present invention, the prebiotic is an amino acid. Although many known prebiotics break down to provide carbohydrates for probiotics, some probiotics also require amino acids for nutrition.

[0311] Prebiotics are found naturally in a variety of foods, including, but not limited to, bananas, berries, asparagus, garlic, wheat, oats, barley (and other whole grains), flaxseed, tomatoes, Jerusalem artichokes, onions and chicory, greens (e.g., dandelion greens, spinach, collard greens, beets, kale, mustard greens, turnip greens), and legumes (e.g., lentils, kidney beans, chickpeas, navy beans, white beans, black beans).

[0312] Weight Management Agents

[0313] In certain embodiments, the functional ingredient is at least one weight management agent.

[0314] As used herein, the at least one weight management agent can be a single weight management agent or multiple weight management agents as a functional ingredient of the composition provided herein. Generally, according to specific embodiments of the present invention, the at least one weight management agent is present in the composition in an amount sufficient to promote health and wellness.

[0315] As used herein, "weight management agent" includes an appetite suppressant and / or a thermogenic agent. As used herein, the phrases "appetite suppressant," "appetite satiety composition," "satiating agent," and "satiating ingredient" are synonymous. The phrase "appetite suppressant" describes a macronutrient, herbal extract, exogenous hormone, anorectic, anorectic drug, drug, and combinations thereof that, when delivered in an effective amount, suppress, inhibit, reduce, or otherwise shorten a person's appetite. The phrase "thermogenic agent" describes a macronutrient, herbal extract, exogenous hormone, anorectic, anorectic drug, drug, and combinations thereof that, when delivered in an effective amount, stimulate or otherwise enhance a person's thermogenesis or metabolism.

[0316] Suitable weight management agents include macronutrients selected from the group consisting of protein, carbohydrates, dietary fat, and combinations thereof. The consumption of protein, carbohydrates, and dietary fat stimulates the release of peptides that have an appetite suppressant effect. For example, the consumption of protein and dietary fat stimulates the release of the gastrointestinal hormone cholecystokinin (CCK), while the consumption of carbohydrates and dietary fat stimulates the release of glucagon-like peptide 1 (GLP-1).

[0317] Suitable macronutrient weight management agents also include carbohydrates. Carbohydrates generally include sugars, starches, cellulose and gums that the body converts into glucose for energy. Carbohydrates are generally divided into two categories, digestible carbohydrates (e.g., monosaccharides, disaccharides and starch) and indigestible carbohydrates (e.g., dietary fiber). Studies have shown that indigestible carbohydrates and complex polymer carbohydrates with reduced absorption and digestibility in the small intestine stimulate physiological responses that inhibit food intake. Therefore, the carbohydrates presented here ideally include indigestible carbohydrates or carbohydrates with reduced digestibility. Non-limiting examples of such carbohydrates include polydextrose; inulin; monosaccharide-derived polyols, such as erythritol, mannitol, xylitol and sorbitol; disaccharide-derived alcohols, such as isomalt, lactitol and maltitol; and hydrogenated starch hydrolysates. Carbohydrates are described in more detail below.

[0318] In another specific embodiment, the weight management agent is a dietary fat.Dietary fat is a lipid comprising a combination of saturated fatty acids and unsaturated fatty acids.Polyunsaturated fatty acids have been shown to have greater satiety than monounsaturated fatty acids.Therefore, the dietary fat presented here desirably includes polyunsaturated fatty acids, and its non-limiting examples include triglycerides.

[0319] In a specific embodiment, the weight management agent is a herbal extract. Extracts from various types of plants have been identified as having appetite suppressant properties. Non-limiting examples of plants whose extracts have appetite suppressant properties include plants of the genus Hoodia, the genus Trichocaulon, the genus Caralluma, the genus Stapelia, the genus Orbea, the genus Asclepias, and the genus Camelia. Other embodiments include extracts derived from Gymnema Sylvestre, Kola Nut, Citrus Aurantium, Yerba Mate, Griffonia Simplicifolia, Guarana, myrrh, guggul Lipid, and black currentseed oil.

[0320] Herbal extracts can be prepared from any type of plant material or plant biomass. Non-limiting examples of plant material and biomass include stems, roots, leaves, dry powders obtained from plant material, and sap or dried sap. Herbal extracts are typically prepared by extracting sap from the plant and then spray drying the sap. Alternatively, a solvent extraction procedure may be used. After the initial extraction, it may be desirable to further fractionate the initial extract (e.g., by column chromatography) to obtain an herbal extract with enhanced activity. Such techniques are well known to those of ordinary skill in the art.

[0321] In a specific embodiment, the herbal extract is derived from a plant of the genus H. alstonii, H. currorii, H. dregei, H. flava, H. gordonii, H. jutatae, H. mossamedensis, H. officinalis, H. parviflorai, H. pedicellata, H. pilifera, H. ruschii, and H. triebneri. H. plants are fleshy stem plants native to South Africa. A sterol glycoside of H. called P57 is believed to be responsible for the appetite suppressant effects of H. species.

[0322] In another embodiment, the herbal extract is derived from a plant of the genus Caralluma, species of which include C. indica, C. fimbriata, C. attenuate, C. tuberculata, C. edulis, C. adscendens, C. stalagmifera, C. umbellate, C. penicillata, C. russeliana, C. retrospicens, C. Arabica, and C. lasiantha. The Caralluma plant belongs to the same subtribe Asclepiadaceae as the genus Caralluma. Caratuberside A, Caratuberside B, Bouceroside I, Bouceroside II, Bouceroside III, Bouceroside IV, Bouceroside V, Bouceroside VI, Bouceroside VII, Bouceroside VIII, Bouceroside IX, and Bouceroside X are short, erect, and fleshy plants native to India that have medicinal properties such as appetite suppression, which are generally attributed to glycosides belonging to the group of glycoside pregnanes, non-limiting examples of which include caratuberside A, caratuberside B, bouceroside I, bouceroside II, bouceroside III, bouceroside IV, bouceroside V, bouceroside VI, bouceroside VII, bouceroside VIII, bouceroside IX, and bouceroside X.

[0323] In another embodiment, the at least one herbal extract is derived from a plant of the genus Thunbergia. Thunbergia plants are succulent plants typically native to South Africa, similar to the genus Thunbergia, and include T. piliferum and T. officinale.

[0324] In another embodiment, the herbal extract is derived from a plant of the genus Staphlomis or Obea, species of which include S. gigantean and O. variegate, respectively. Both Staphlomis and Obea belong to the same subfamily Asclepiadaceae as the genus Hoodia. Without wishing to be bound by any theory, it is believed that the compounds that exhibit appetite suppressant activity are saponins, such as pregnane glycosides, which include stavaroside A, B, C, D, E, F, G, H, I, J, and K.

[0325] In another specific embodiment, the herbal extract is derived from a plant of the genus Asclepiadaceae. Asclepiadaceae plants also belong to the family Asclepiadaceae. Non-limiting examples of Asclepiadaceae plants include A. incarnate, A. curassayica, A. syriaca, and A. tuberose. Without wishing to be bound by any theory, it is believed that these extracts contain steroidal compounds such as pregnane glycosides and pregnane aglycones that have an appetite suppressant effect.

[0326] In a specific embodiment, the weight management agent is an exogenous hormone with a weight management effect. Non-limiting examples of such hormones include CCK, peptide YY, ghrelin, bombesin and gastrin-releasing peptide (GRP), enterostatin, apolipoprotein A-IV, GLP-1, amylin, somastatin, and leptin.

[0327] In another embodiment, the weight management agent is a drug. Non-limiting examples include phentermine, diethylpropion, phendimetrazine, sibutramine, rimonabant, oxyntomodulin, fluoxetine hydrochloride, ephedrine, phenylethylamine, or other stimulants.

[0328] Osteoporosis Management Agents

[0329] In certain embodiments, the functional ingredient is at least one osteoporosis management agent.

[0330] As used herein, the at least one osteoporosis management agent can be a single osteoporosis management agent or multiple osteoporosis management agents as a functional ingredient of the composition provided herein. Generally, according to specific embodiments of the present invention, the at least one osteoporosis management agent is present in the composition in an amount sufficient to promote health and wellness.

[0331] Osteoporosis is a skeletal disorder of impaired bone strength, which leads to an increased risk of bone fracture. In general, osteoporosis is characterized by a decrease in bone mineral density (BMD), disruption of bone micro-architecture, and changes in the amount and type of non-collagenous proteins within the bone.

[0332] In certain embodiments, the osteoporosis management agent is at least one calcium source. According to a specific embodiment, the calcium source is any compound containing calcium, including salt complexes, dissolved substances, and other forms of calcium. Non-limiting examples of calcium sources include amino acid chelated calcium, calcium carbonate, calcium oxide, calcium hydroxide, calcium sulfate, calcium chloride, calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium citrate, calcium malate, calcium citrate malate, calcium gluconate, calcium tartrate, calcium lactate, dissolved substances thereof, and combinations thereof.

[0333] According to a specific embodiment, the osteoporosis management agent is a magnesium source. The magnesium source is any compound containing magnesium, including salt complexes, dissolved substances, and other forms of magnesium. Non-limiting examples of magnesium sources include magnesium chloride, magnesium citrate, magnesium glucoheptonate, magnesium gluconate, magnesium lactate, magnesium hydroxide, magnesium picolate, magnesium sulfate, dissolved substances thereof, and mixtures thereof. In another specific embodiment, the magnesium source includes an amino acid chelated magnesium or a creatine chelated magnesium.

[0334] In other embodiments, the osteoporosis agent is selected from vitamin D, C, K, precursors thereof and / or beta-carotene and combinations thereof.

[0335] Various plants and plant extracts have also been identified as being effective for preventing and treating osteoporosis. Without wishing to be bound by any theory, it is believed that these plants and plant extracts stimulate osteogenic proteins and / or inhibit bone resorption, thereby promoting bone regeneration and strength. Non-limiting examples of suitable plants and plant extracts for osteoporosis management agents include Taraxacum and Amelanchier species as disclosed in U.S. Patent Publication No. 2005 / 0106215, and Lindera, Artemisia, Acorus, Carthamus, Carum, Cnidium, Curcuma, Cyperus, Juniperus, Prunus, and Rhizoma Cnidium as disclosed in U.S. Patent Publication No. 2005 / 0079232. and species of the genera Prunus, Iris, Cichorium, Dodonaea, Epimedium, Erigonoum, Soya, Mentha, Ocimum, Thymus, Tanacetum, Plantago, Spearmint, Bixa, Vitis, Rosemarinus, Rhus, and Anethum.

[0336] Phytoestrogens

[0337] In certain embodiments, the functional ingredient is at least one phytoestrogen.

[0338] As used herein, the at least one phytoestrogen can be a single phytoestrogen or a plurality of phytoestrogens as a functional ingredient of the compositions provided herein. Generally, according to particular embodiments of the present invention, the at least one phytoestrogen is present in the composition in an amount sufficient to promote health and wellness.

[0339] Phytoestrogens are compounds found in plants that can typically be delivered to the human body by ingesting plants or plant parts that have these phytoestrogens. As used herein, "phytoestrogen" refers to any substance that causes any degree of estrogen-like effects when introduced into the body. For example, a phytoestrogen can bind to estrogen receptors in the body and have little estrogen-like effects.

[0340] Examples of suitable phytoestrogens for use in embodiments of the present invention include, but are not limited to, isoflavones, stilbenes, lignans, resorcyclic acid lactone, coumarins, coumestrol, coumestrol, equol, and combinations thereof. Suitable sources of phytoestrogens include, but are not limited to, whole grains, cereals, fibers, fruits, vegetables, black cohosh, agave root, black currant, cherry leaf viburnum, chasteberry, cramp bark, angelica root, devil's club root, false unicorn root, and thyme root. , walnuts ...

[0341] Isoflavones belong to a group of phytonutrients called polyphenols. In general, polyphenols (also called "polyphenols") are a group of chemicals found in plants that are characterized by the presence of more than one phenolic group per molecule.

[0342] Suitable phytoestrogen isoflavones according to embodiments of the present invention include genistein, daidzein, glycitein, biochanin A, formononetin, their respective naturally occurring glycosides and glycoside conjugates, matai resinol, secoisolariciresinol, enterodiol, enterodiol, plant tissue protein, and combinations thereof.

[0343] Suitable sources of isoflavones for use in embodiments of the present invention include, but are not limited to, soybeans, soybean products, legumes, alfalfa spouts, chickpeas, peanuts, and red clover.

[0344] Long chain primary aliphatic saturated alcohol

[0345] In certain embodiments, the functional ingredient is at least one long chain primary aliphatic saturated alcohol.

[0346] As used herein, the at least one long-chain aliphatic saturated primary alcohol can be a single long-chain aliphatic saturated primary alcohol or a plurality of long-chain aliphatic saturated primary alcohols as a functional ingredient of the composition provided herein. Typically, according to a specific embodiment of the present invention, the at least one long-chain aliphatic saturated primary alcohol is present in the composition in an amount sufficient to promote health and wellness.

[0347] Long chain aliphatic saturated primary alcohols are a different group of organic compounds. The term "alcohol" refers to the fact that the characteristic of these compounds is one hydroxyl group (-OH) bonded to one carbon atom. The term primary refers to the fact that in these compounds, the carbon atom to which the hydroxyl group is bonded is bonded to only one other carbon atom. The term saturated refers to the fact that the characteristic of these compounds is the absence of carbon-carbon pi bonds. The term aliphatic refers to the fact that the carbon atoms in these compounds are linked together in straight or branched chains rather than in rings. The term long chain refers to the fact that the number of carbon atoms in these compounds is at least 8 carbons.

[0348] Non-limiting examples of specific long chain aliphatic saturated primary alcohols for use in specific embodiments of the present invention include 8 carbon atom 1-octanol, 9 carbon atom 1-nonanol, 10 carbon atom 1-decanol, 12 carbon atom 1-dodecanol, 14 carbon atom 1-tetradecanol, 16 carbon atom 1-hexadecanol, 18 carbon atom 1-octadecanol, 20 carbon atom 1-eicosanol, 22 carbon atom 1-docosanol, 24 carbon atom 1-tetracosanol, 26 carbon atom 1-hexacosanol, 27 carbon atom 1-heptacosanol, 28 carbon atom 1-octanosol, 29 carbon atom 1-nonacosanol, 30 carbon atom 1-triacontanol, 32 carbon atom 1-dotriacontanol, and 34 carbon atom 1-tetracosanol.

[0349] In a particularly desirable embodiment of the present invention, the long chain primary aliphatic saturated alcohol is policosanol. Policosanol is a term for a mixture of long chain aliphatic saturated primary alcohols consisting primarily of 28 carbon 1-octacosanol and 30 carbon 1-triacontanol, and lower concentrations of other alcohols such as 22 carbon 1-docosanol, 24 carbon 1-tetracosanol, 26 carbon 1-hexacosanol, 27 carbon 1-heptacosanol, 29 carbon 1-nonacosanol, 32 carbon 1-dotriacontanol, and 34 carbon 1-tetracontanol.

[0350] Long chain primary aliphatic saturated alcohols are derived from natural fats and oils. They can be obtained from these sources by using extraction techniques well known to those of ordinary skill in the art. Policosanols can be isolated from a variety of plants and materials, including sugar cane (Saccharum officinarium), yam (e.g., Dioscorea opposite), rice bran (e.g., Oryza sativa), and beeswax. Policosanols can be obtained from these sources by using extraction techniques well known to those of ordinary skill in the art. Descriptions of such extraction techniques can be found in U.S. Patent Application No. 2005 / 0220868, the disclosure of which is expressly incorporated by reference.

[0351] Phytosterols

[0352] In certain embodiments, the functional ingredient is at least one phytosterol, phytostanol, or a combination thereof.

[0353] Generally, according to particular embodiments of this invention, the at least one phytosterol, phytostanol, or combination thereof is present in the composition in an amount sufficient to promote health and wellness.

[0354] As used herein, the phrases "stanols," "plant stanols," and "phytostanols" are synonyms.

[0355] Plant sterols and stanols are naturally present in small amounts in many fruits, vegetables, nuts, seeds, grains, beans, vegetable oils, barks and other plant sources. Although people normally consume plant sterols and stanols every day, the amount consumed is not enough to have a significant cholesterol-lowering effect or other health benefits. Therefore, it is desirable to supplement foods and beverages with plant sterols and stanols.

[0356] Sterols are a subgroup of steroids with a hydroxyl group at C-3. Typically, phytosterols have a double bond within the steroid nucleus, such as cholesterol; however, phytosterols may also contain a substituted side chain (R) at C-24, such as ethyl or methyl, or an additional double bond. The structure of phytosterols is well known to those skilled in the art.

[0357] At least 44 naturally occurring phytosterols have been found, and they are usually derived from plants, such as corn, soybeans, wheat and tung oil; however, they can also be produced synthetically to form compositions identical to those of nature or compositions having properties similar to those of naturally occurring phytosterols. According to a specific embodiment of the present invention, non-limiting examples of phytosterols well known to those of ordinary skill in the art include 4-demethyl sterols (e.g., β-sitosterol, campesterol, stigmasterol, rapeseed sterol, 22-dehydro-rapeseed sterol, and Δ5-avenasterol), 4-monomethyl sterols and 4,4-dimethyl sterols (triterpenoids) (e.g., cycloartenol, 24-methylenecycloartanol and cyclostanol (cyclobranol)).

[0358] As used herein, the phrases "stanols," "plant stanols," and "phytostanols" are synonyms. Phytostanols are saturated sterols found only in trace amounts in nature and may also be produced synthetically, for example, by hydrogenation of phytosterols. According to a particular embodiment of the invention, non-limiting examples of phytostanols include β-sitostanol, campestanol, cycloartanol, and saturated forms of other triterpene alcohols.

[0359] Phytosterols and phytostanols as used herein include various isomers such as alpha and beta isomers (eg, alpha-sitosterol and beta-sitostanol, which comprise one of the most effective phytosterols and phytostanols, respectively, for lowering serum cholesterol in mammals).

[0360] Phytosterol and phytostanol of the present invention can also be in its ester form.The suitable method for obtaining the ester of phytosterol and phytostanol is well known to those of ordinary skill in the art, and discloses in U.S. Patent number 6,589,588, 6,635,774, 6,800,317 and U.S. Patent Publication No. 2003 / 0045473, the disclosures of these patents are combined herein by reference in their entirety.The limiting examples of the ester of suitable phytosterol and phytostanol include acetic acid sitosterol ester, oleic acid sitosterol ester, oleic acid stigmasterol ester and its corresponding phytostanol ester.Phytosterol and phytostanol of the present invention can also include its derivative.

[0361] In general, the amount of the functional ingredient in the composition varies widely depending on the specific composition and the desired functional ingredient. One of ordinary skill in the art will readily determine the appropriate amount of the functional ingredient for each composition.

[0362] In one embodiment, a method for preparing a composition comprises combining rebaudioside I and at least one sweetener and / or additive and / or functional ingredient.

[0363] consumer goods

[0364] In one embodiment, the composition of the present invention is a consumable comprising rebaudioside I or a consumable containing a composition comprising rebaudioside I (eg, a sweetener composition).

[0365] Rebaudioside I or compositions containing it can be incorporated into any known edible or oral composition (referred to herein as a "consumer product"), such as, for example, pharmaceutical compositions, edible gel mixtures and compositions, dental compositions, foods (sweets, condiments, chewing gum, cereal compositions, baked goods, dairy products, and tabletop sweetener compositions), beverages and beverage products.

[0366] As used herein, consumables refer to substances that come into contact with the mouth of a person or animal, including substances that are ingested and subsequently excreted from the mouth and substances that are drunk, eaten, swallowed or otherwise ingested, and are healthy for human or animal consumption when used within generally acceptable ranges.

[0367] For example, a beverage is a consumable. Before adding rebaudioside I or a composition comprising rebaudioside I, the beverage can be sweetened or unsweetened. Rebaudioside I or a composition comprising rebaudioside I can be added to a beverage or a beverage base to sweeten the beverage or enhance its existing sweetness or flavor.

[0368] In one embodiment, the present invention is a consumable comprising rebaudioside I. The concentration of rebaudioside I in the consumable may be above, at or below its threshold sweetness concentration.

[0369] In a particular embodiment, the present invention is a beverage comprising rebaudioside I. The concentration of rebaudioside I in the beverage may be above, at or below its threshold sweetness concentration.

[0370] The consumable may optionally include additives, additional sweeteners, functional ingredients, and combinations thereof, as described herein.Any of the additives, additional sweeteners, and functional ingredients described above may be present in the consumable.

[0371] Pharmaceutical composition

[0372] In one embodiment, the present invention is a pharmaceutical composition comprising a pharmaceutically active substance and rebaudioside I.

[0373] In another embodiment, the present invention is a pharmaceutical composition comprising a pharmaceutically active substance and a composition comprising rebaudioside I.

[0374] Rebaudioside I or a composition comprising rebaudioside I may be present in the pharmaceutical composition as an excipient material that can mask the bitterness or other undesirable taste of a pharmaceutically active substance or another excipient material. The pharmaceutical composition may be in the form of a tablet, capsule, liquid, aerosol, powder, effervescent tablet or powder, syrup, emulsion, suspension, solution, or any other form to provide the pharmaceutical composition to the patient. In a specific embodiment, the pharmaceutical composition may be in a form for oral administration, buccal administration, sublingual administration, or any other administration route known in the art.

[0375] As referred to herein, "pharmaceutically active substance" means any drug, pharmaceutical preparation, medicament, prophylactic agent, therapeutic agent, or other substance having biological activity. As referred to herein, "excipient material" refers to any inactive substance used as a vehicle for an active ingredient, such as any material that aids in the handling, stability, dispersibility, wettability, and / or release kinetics of a pharmaceutically active substance.

[0376] Suitable pharmaceutical actives include, but are not limited to, agents for the gastrointestinal tract or digestive system, agents for the cardiovascular system, agents for the central nervous system, agents for pain or consciousness, agents for musculoskeletal disorders, agents for the eyes, agents for the ears, nose and oropharynx, agents for the respiratory system, agents for endocrine problems, agents for the reproductive system or urinary system, agents for contraception, agents for obstetrics and gynecology, agents for the skin, agents for infections and infections, agents for immunology, agents for allergic disorders, agents for nutrition, agents for hematological oncological diseases, agents for diagnosis, agents for euthanasia, or agents for other biological functions or disorders. Examples of suitable pharmaceutically active substances for embodiments of the present invention include, but are not limited to, antacids, reflux inhibitors, antiflatulents, antidopaminers, proton pump inhibitors, cytoprotectants, prostaglandin analogs, laxatives, antispasmodics, antidiarrheals, bile acid sequestrants, opioids, beta-blockers, calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, nitrates, antianginal drugs, vasoconstrictors, vasodilators, peripheral vascular activators, ACE inhibitors, angiotensin receptor blockers, , alpha blockers, anticoagulants, heparin, antiplatelet agents, fibrinolytics, antihemophilic factor, hemostatics, lipid-lowering agents, statins, hypnotics (hynoptics), anesthetics, antipsychotics, antidepressants, antiemetics, anticonvulsants, antiepileptics, anxiolytics, barbiturates, movement disorder drugs, stimulants, benzodiazepines, ciclopirox, dopamine antagonists, antihistamines, cholinergics, anticholinergics, emetics, cannabinoids, analgesics, muscle relaxants, antibiotics, aminoglycosides , antivirals, antifungals, anti-inflammatory drugs, antiglaucoma drugs, sympathomimetics, steroids, ceruminolytics, bronchodilators, NSAIDS, antitussives, mucolytics, decongestants, corticosteroids, androgens, antiandrogens, gonadotropins, growth hormones, insulin, antidiabetic drugs, thyroid hormones, calcitonin, bisphosphonates, antidiuretic hormone analogs, alkalinizing agents, quinolones, anticholinesterases, sildenafil, oral contraceptives, Hormone replacement therapy, bone regulator, follicle stimulating hormone, luteinizing hormone, gamolenic acid, progestin, dopamine agonist, estrogen, prostaglandin, gonadotropin releasing factor, clomiphene, tamoxifen, diethylstilbestrol, anti-leprosy drugs, anti-tuberculosis drugs, antimalarial drugs, anthelmintics, antiprotozoal drugs, antiserum, vaccine, interferon, nutritional supplements, vitamins, cytotoxic drugs, sex hormones, aromatase inhibitors, somatostatin inhibitors, or similar types of substances, or combinations thereof. Such components are generally regarded as safe (GRAS) and / or approved by the U.S. Food and Drug Administration (FDA).

[0377] The pharmaceutically active substance is present in the pharmaceutical composition in a wide range of amounts, depending on the specific pharmaceutically active agent used and its intended application. An effective dose of any pharmaceutically active substance described herein can be easily determined by using conventional techniques and by observing the results obtained under similar circumstances. In determining the effective dose, a variety of factors are considered, including, but not limited to, the type of patient; its size, age and general health; the specific disease involved; the degree or severity of the disease involved; the response of individual patients; the specific pharmaceutically active agent administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; and the use of combined medication. The pharmaceutically active substance is contained in a pharmaceutically acceptable carrier, diluent or excipient in an amount sufficient to be delivered to the patient, which is an effective amount of the pharmaceutically active substance in vivo, and there is no serious toxic effect when used in a generally acceptable amount. Therefore, a suitable amount can be easily judged by a person skilled in the art.

[0378] According to a specific embodiment of the present invention, the concentration of the pharmaceutical active substance in the pharmaceutical composition will depend on the absorption, inactivation and excretion rate of the drug and other factors known to those skilled in the art. It should be noted that the dosage value will change as the severity of the condition is alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the professional judgment of the person who needs and gives or monitors the giving of the pharmaceutical composition, and the dosage range described herein is only exemplary and is not intended to limit the scope and practice of the claimed composition. The pharmaceutical active substance can be given once, or can be divided into multiple smaller doses to be given at different time intervals.

[0379] Pharmaceutical composition can also comprise other pharmaceutically acceptable excipient materials.Examples of suitable excipient materials for embodiments of the present invention include but are not limited to, antiadhesive, adhesive (for example, microcrystalline cellulose, tragacanth or gelatin), coating, disintegrant, filler, diluent, softener, emulsifier, flavoring agent, coloring agent, adjuvant, lubricant, functional agent (for example, nutrition), viscosity modifier, expander, glidant (for example, colloidal silicon dioxide), surfactant, permeate, diluent or any other inactive ingredient or its combination.For example, pharmaceutical composition of the present invention can comprise the excipient material selected from lower group, and this group is made up of the following: calcium carbonate, coloring agent, brightener, preservative and flavoring, triacetin, magnesium stearate, hydrogenated vegetable oil (sterote), natural flavoring or artificial flavoring, essential oil, plant extract, fruit essence, gelatin or its combination.

[0380] The excipient material of pharmaceutical composition can optionally include other artificial sweeteners or natural sweeteners, filler sweeteners or their combination.Filler sweeteners include both caloric compounds and no caloric compounds.In a specific embodiment, additives are used as filler sweeteners.The limiting examples of filler sweeteners include sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (for example, sorbitol, mannitol, xylitol, lactitol, erythritol and maltitol), hydrogenated starch hydrolysate, isomalt, trehalose and mixtures thereof.In a specific embodiment, filler sweeteners are present in pharmaceutical composition with the amount of a wide range, and this amount depends on the desired sweetness.The applicable amount of two kinds of sweeteners is that those skilled in the art easily distinguish.

[0381] Edible gel mixture and edible gel composition

[0382] In one embodiment, the present invention is an edible gel or edible gel mix comprising rebaudioside I. In another embodiment, the present invention is an edible gel or edible gel mix comprising a composition comprising rebaudioside I.

[0383] Edible gels are gels that can be eaten. Gels are colloidal systems in which a network of particles spans the volume of a liquid medium. Although gels are primarily composed of liquids and therefore exhibit a density similar to that of liquids, gels have the structural coherence of solids due to the network of particles spanning the liquid medium. For this reason, gels typically appear as solid, jelly-like materials. Gels can be used in a variety of applications. For example, gels can be used in foods, pigments, and adhesives.

[0384] The non-limiting examples of the edible gel compositions of the specific embodiments include gel desserts, puddings, jellies, pastes, muffins, aspics, marshmallows, colloid milk candies, etc. Edible gel mixtures are typically powdered or granular solids, in which a fluid can be added to form an edible gel composition. The non-limiting examples of the fluids used in the specific embodiments include water, dairy fluids, dairy analog fluids, fruit juices, alcohol, alcoholic beverages, and combinations thereof. The non-limiting examples of dairy fluids that can be used in the specific embodiments include milk, yogurt, cream, fluid whey, and mixtures thereof. The non-limiting examples of dairy analog fluids that can be used in the specific embodiments include, for example, soy milk and non-dairy coffee whiteners. Because the edible gel products visible on the market are typically sweetened with sucrose, it is desirable to use an edible gel sweetened with an alternative sweetener so that a low-calorie or no-calorie substitute is provided.

[0385] As used herein, the term "gelling ingredient" refers to any material that can form a colloidal system in a liquid medium. Non-limiting examples of gelling ingredients used in specific embodiments include gelatin, alginate, carrageenan, gum, pectin, konjac, agar, edible acid, rennet, starch, starch derivatives, and combinations thereof. It is well known to those of ordinary skill in the art that the amount of gelling ingredient used in an edible gel mixture or edible gel composition is appropriately varied according to a variety of factors, such as the specific gelling ingredient used, the specific base fluid used, and the desired gel properties.

[0386] Edible gel mixtures and edible gels can be prepared using ingredients comprising the following items: edible acid, edible acid salt, buffer system, swelling agent, chelating agent, cross-linking agent, one or more flavorings, one or more pigments, and combinations thereof. Non-limiting examples of edible acid for specific embodiments include citric acid, adipic acid, fumaric acid, lactic acid, malic acid, and combinations thereof. Non-limiting examples of edible acid salts for specific embodiments include sodium salts of edible acid, potassium salts of edible acid, and combinations thereof. Non-limiting examples of swelling agents for specific embodiments include oligofructose (raftilose), isomalt, sorbitol, polydextrose, maltodextrin, and combinations thereof. Non-limiting examples of chelating agents for specific embodiments include disodium calcium ethylene tetraacetate, glucono delta-lactone, sodium gluconate, potassium gluconate, ethylenediaminetetraacetic acid (EDTA), and combinations thereof. Non-limiting examples of cross-linking agents for specific embodiments include calcium ions, magnesium ions, sodium ions, and combinations thereof.

[0387] Dental composition

[0388] In one embodiment, the present invention is a dental composition comprising rebaudioside I. In another embodiment, the present invention is a dental composition comprising a composition containing rebaudioside I. Dental compositions typically comprise an active tooth substance and a base material. Rebaudioside I or a composition comprising rebaudioside I can be used as a base material to sweeten the dental composition. The dental composition can be in the form of any oral composition for oral use, such as, for example, a mouth freshening preparation, a mouthwash, a mouth rinse, a dentifrice, a tooth polish, a dentifrice, a mouth spray, a tooth whitener, a dental floss, etc.

[0389] As referred to herein, "active dental material" means any composition that can be used to improve the aesthetic appearance and / or health of teeth or gums or to prevent dental caries. As referred to herein, "base material" means any inactive material used as a vehicle for an active dental material, such as any material that aids in the handling, stability, dispersibility, wettability, foaming, and / or release kinetics of an active dental material.

[0390] Suitable active tooth substances for embodiments of the present invention include, but are not limited to, substances that remove plaque, substances that remove food from teeth, substances that help eliminate and / or mask bad breath, substances that prevent caries, and substances that prevent gum disease (i.e., gums). Examples of suitable active tooth substances for embodiments of the present invention include, but are not limited to, anticaries, fluoride, sodium fluoride, sodium monofluorophosphate, stannous fluoride, hydrogen peroxide, urea peroxide (i.e., carbamide peroxide), antibacterial agents, plaque removers, detergents, anticalculus agents, abrasives, baking soda, percarbonate, perborates of alkali metals and alkaline earth metals, or substances of similar types, or combinations thereof. Such components are generally regarded as safe (GRAS) and / or are approved by the U.S. Food and Drug Administration (FDA).

[0391] According to specific embodiments of the invention, the active dental substance is present in the dental composition in an amount ranging from about 50 ppm to about 3000 ppm of the dental composition. Typically, the active dental composition is present in the dental composition in an amount effective to at least slightly improve the aesthetic appearance and / or health of teeth or gums or to prevent dental caries. For example, a dental composition comprising a dentifrice may comprise an active dental substance comprising fluorine in an amount of about 850 to 1,150 ppm.

[0392] The dental composition may also include a base material in addition to rebaudioside I or a composition comprising rebaudioside I. Examples of suitable base materials for embodiments of the present invention include, but are not limited to, water, sodium lauryl sulfate or other sulfates, humectants, enzymes, vitamins, herbs, calcium, flavorings (e.g., mint, bubble gum, cinnamon, lemon, or orange), surfactants, binders, preservatives, gelling agents, pH adjusters, peroxide activators, stabilizers, colorants, or similar types of materials, and combinations thereof.

[0393] The base material of dental composition can optionally include other artificial sweeteners or natural sweeteners, filler sweeteners or their combination. Filler sweeteners include both caloric compounds and non-caloric compounds. The non-limiting examples of filler sweeteners include sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (for example, sorbitol, mannitol, xylitol, lactitol, erythritol and maltitol), hydrogenated starch hydrolysate, isomalt, trehalose and mixtures thereof. Usually, the wide range of the amount of the filler sweetener present in dental composition depends on the specific embodiment and the desired sweetness of dental composition. Those of ordinary skill in the art will easily determine the appropriate amount of filler sweeteners. In a specific embodiment, filler sweeteners are present in dental composition in an amount of about 0.1 % by weight to about 5 % by weight of the scope of dental composition.

[0394] According to specific embodiments of the invention, the base material is present in the dental composition in an amount ranging from about 20% to about 99% by weight of the dental composition. Typically, the base material is present in an amount effective to provide a vehicle for an active dental substance.

[0395] In a specific embodiment, a dental composition comprises rebaudioside I and an active dental substance. In another specific embodiment, a dental composition comprises a composition comprising rebaudioside I and an active dental substance. Generally, the amount of the sweetener varies widely depending on the nature of the particular dental composition and the desired sweetness.

[0396] Food products include, but are not limited to, sweets, condiments, chewing gum, cereals, baked goods, and dairy products.

[0397] Sweets

[0398] In one embodiment, the present invention is a dessert comprising rebaudioside I. In another embodiment, the present invention is a dessert comprising a composition comprising rebaudioside I.

[0399] As mentioned herein, "sweets" may refer to candies, lollies, confectionery or similar terms. Sweets typically contain a base composition component and a sweetener component. Rebaudioside I or a composition comprising Rebaudioside I can be used as a sweetener component. The sweet can be in any food form, which is typically considered to be rich in sugar or typically candy. According to a specific embodiment of the present invention, the sweet food can be baked food, such as cakes; desserts, such as yogurt, jelly, drinkable jelly, pudding, Bavarian cream, milk jelly, cake, chocolate cake, mousse, etc., sweet food eaten at afternoon tea or after a meal; frozen food; cold desserts, for example, ice cream types, such as ice cream, ice milk, lacto-ice, etc. (food in which sweeteners and various other types of raw materials are added to dairy products and the resulting mixture is stirred and frozen), and frozen desserts, such as sherbet, dessert ice cream, etc. (food in which various other types of raw materials are added with a sugary liquid and the resulting mixture is stirred and frozen); general sweets, such as baked sweets or steamed sweets, such as crackers, biscuits, buns with bean jam fillings, sesame crisps , alfajor, etc.; rice cakes and snacks; tabletop products; general sugar sweets, such as chewing gum (for example, including compositions containing a substantially water-insoluble, chewable gum base, such as chicle or its substitutes, including jetulong, guttakay rubber or certain edible natural synthetic resins or waxes), hard candies, soft candies, mints, nougat, jelly beans, cream fudge, toffee, Swiss milk tablets, liquorice candies, chocolate candies, jelly candies, marzipan, divinity, cotton candy, etc.; sauces, including fruit flavored sauces, chocolate sauces, etc.; edible gels; creams, including butter cream, flour paste, whipped cream, etc.; jams, including strawberry jam, citrus jam, etc.; and breads, including sweet breads, etc. or other starch products, and combinations thereof.

[0400] As referred to herein, "base composition" means any composition that can be a food product and provides a matrix for carrying a sweetener component.

[0401] Suitable base compositions for embodiments of the present invention can include flour, yeast, water, salt, butter, egg, milk, milk powder, spirits, gelatin, nuts, chocolate, citric acid, tartaric acid, fumaric acid, natural flavor seasoning, artificial flavor seasoning, pigment, polyol, sorbitol, isomalt, maltitol, lactitol, malic acid, magnesium stearate, lecithin, hydrogenated glucose syrup, glycerine, natural gum or synthetic gum, starch etc., and combination thereof. Such components are generally considered as safe (GRAS) and / or are approved by the U.S. Food and Drug Administration (FDA). According to a specific embodiment of the present invention, this base composition is to be present in this sweet with the amount of the scope from about 0.1 % by weight to about 99 % by weight by the sweet weight. Generally, base composition is present in the sweet with the amount providing food.

[0402] The base material of sweet food can optionally include other artificial sweeteners or natural sweeteners, filler sweeteners or their combination. Filler sweeteners include both caloric compounds and no caloric compounds. The limiting examples of filler sweeteners include sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (for example, sorbitol, mannitol, xylitol, lactitol, erythritol and maltitol), hydrogenated starch hydrolysate, isomalt, trehalose and mixtures thereof. Usually, the wide range of the amount of the filler sweetener present in the sweet food depends on the specific embodiment and the desired sweetness of the sweet food. Those of ordinary skill in the art will easily determine the appropriate amount of filler sweeteners.

[0403] In a specific embodiment, the sweet comprises rebaudioside I or a composition comprising rebaudioside I and a base composition. Generally, the amount of rebaudioside I in the sweet varies widely depending on the specific embodiment of the sweet and the desired sweetness. One of ordinary skill in the art will readily determine the appropriate amount. In a specific embodiment, rebaudioside I is present in the sweet in an amount in the range of about 30 ppm to about 6000 ppm of the sweet. In another embodiment, rebaudioside I is present in the sweet in an amount in the range of about 1 ppm to about 10,000 ppm of the sweet. In embodiments where the sweet comprises hard candy, rebaudioside I is present in an amount in the range of about 150 ppm to about 2250 ppm of the hard candy.

[0404] Seasoning composition

[0405] In one embodiment, the present invention is a condiment comprising rebaudioside I. In another embodiment, the present invention is a condiment comprising a composition containing rebaudioside I. As used herein, a condiment is a composition for enhancing or improving the flavor of a food or beverage. Non-limiting examples of condiments include tomato sauce (ketchup); mustard greens; barbecue sauce; butter; Worcestershire sauce; chutney; appetizer sauce; curry powder; dips; fish sauce; horseradish sauce; hot sauce; jelly, jam, marmalade, or preserves; mayonnaise; peanut butter; relish; mayonnaise; salad dressings (e.g., oil and vinegar, Caesar, French, ranch, blue cheese, Russian, Thousand Island, Italian, and vinaigrette), salsa; sauerkraut; soy sauce; steak sauce; syrup; tartar sauce; and Worcestershire sauce.

[0406] The seasoning base generally comprises a mixture of different ingredients, which include, without limitation, vehicles (e.g., water and vinegar); spices or seasonings (e.g., salt, pepper, garlic, mustard seeds, onions, chili peppers, turmeric, and combinations thereof); fruits, vegetables, or products thereof (e.g., tomatoes or tomato-based products (paste, puree), juice, fruit juice peel, and combinations thereof); oils or oil emulsions, particularly vegetable oils; thickeners (e.g., xanthan gum, food starch, other hydrocolloids, and combinations thereof); and emulsifiers (e.g., egg yolk solids, proteins, gum arabic, carob gum, guar gum, karaya gum, tragacanth gum, carrageenan, pectin, propylene glycol esters of alginic acid, sodium carboxymethylcellulose, polysorbates, and combinations thereof). Recipes for seasoning bases and methods of preparing seasoning bases are well known to those of ordinary skill in the art.

[0407] Typically, condiments also include caloric sweeteners, such as sucrose, high fructose corn syrup, molasses, honey, or brown sugar. In exemplary embodiments of condiments provided herein, rebaudioside I or a sweetener composition comprising rebaudioside I is used, rather than a traditional caloric sweetener. Therefore, a condiment composition ideally includes rebaudioside I or a composition comprising rebaudioside I and a condiment base.

[0408] The condiment composition optionally may include other natural and / or synthetic high-potency sweeteners, bulk sweeteners, pH modifiers (e.g., lactic acid, citric acid, phosphoric acid, hydrochloric acid, acetic acid, and combinations thereof), fillers, functional agents (e.g., pharmaceutical agents, nutrients, or components of foods or plants), flavoring agents, coloring agents, or combinations thereof.

[0409] Chewing gum composition

[0410] In one embodiment, the present invention is a chewing gum composition comprising rebaudioside I. In another embodiment, the present invention is a chewing gum composition comprising a composition containing rebaudioside I. Chewing gum compositions typically comprise a water-soluble portion and a water-insoluble chewable gum base portion. The water-soluble portion, typically comprising the composition of the present invention, dissipates over a period of time during chewing along with a portion of the flavoring, while the insoluble gum base portion remains in the mouth. The insoluble gum base typically determines whether a gum is considered a chewing gum, bubble gum, or functional gum.

[0411] Insoluble gum base is usually present in the chewing gum composition in an amount ranging from about 15% to about 35% by weight of the chewing gum composition, and it generally comprises a combination of elastomers, softeners (plasticizers), emulsifiers, resins, and fillers. Such components are generally considered food grade, regarded as safe (GRA) and / or approved by the U.S. Food and Drug Administration (FDA).

[0412] Elastomers are the main component of gum base, which provide the gum with rubbery, adhesive properties, and may include one or more natural rubbers (e.g., smoked latex, liquid latex, or guayule); natural gums (e.g., jelutong, perillo, tonka bean gum, bidentate gum, chocolate gum, nispero, rosindinha, chicle, and gutta hang kang); or synthetic elastomers (e.g., butadiene-styrene copolymers, isobutylene-isoprene copolymers, polybutadiene, polyisobutylene, and vinyl polymerized elastomers). In a particular embodiment, the elastomer is present in the gum base in an amount ranging from about 3% to about 50% by weight of the gum base.

[0413] Use resin to change the firmness of gum base, and help to soften the elastomer component of gum base.The limiting examples of applicable resin comprises rosin ester, terpene resin (for example, from the terpene resin of α-pinene, β-pinene and / or d-limonene), polyvinyl acetate, polyvinyl alcohol, ethylene vinyl acetate and vinyl acetate-vinyl laurate copolymer.The limiting examples of rosin ester comprises the glyceride of glyceride of partially hydrogenated rosin, the glyceride of polymerized rosin, the glyceride of part dimerized rosin, the methyl ester of pentaerythritol ester of partially hydrogenated rosin, rosin methyl ester or partially hydrogenated rosin.In a specific embodiment, this resin is present in the gum base with the amount of the scope of about 5 weight % to about 75 weight % of gum base.

[0414] Softener is also referred to as plasticizer, and they are used to change the mouthfeel of chewing ease and / or chewing gum composition.Usually, softener comprises oil, fat, wax and emulsifier.The limiting examples of oil and fat comprises tallow, hydrogenated tallow, large hydrogenation or partially hydrogenated vegetable oil (such as soybean oil, rapeseed oil, cottonseed oil, sunflower oil, palm oil, coconut oil, corn oil, safflower oil or palm kernel oil)), cocoa butter, glyceryl monostearate, triacetin, rosin glyceride, lecithin, monoglyceride, diglyceride, triglyceride acetylated monoglyceride and free fatty acid.The limiting examples of wax comprises polypropylene / polyethylene / Fisher-Tropsch wax, paraffin and microcrystalline wax and natural wax (for example, candelilla wax, beeswax and palm wax).Microcrystalline wax, particularly those microcrystalline waxes with high crystallinity and high melting point can also be regarded as thickener or structural modifier. In a particular embodiment, the softeners are present in the gum base in an amount ranging from about 0.5% to about 25% by weight of the gum base.

[0415] Emulsifiers are used to form a uniform dispersion of the insoluble phase and soluble phase of the chewing gum composition and also have plasticizing properties. Suitable emulsifiers include glyceryl monostearate (GMS), lecithin (phosphatidylcholine), polyglycerol polyricinoleic acid (PPGR), mono- and diglycerides of fatty acids, glyceryl distearate, tristriacetin, acetylated monoglycerides, triacetin, and magnesium stearate. In a specific embodiment, these emulsifiers are present in the gum base in an amount ranging from about 2% by weight to about 30% by weight of the gum base.

[0416] The chewing gum composition can also include adjuvants or fillers in the gum base and / or the soluble part of the chewing gum composition. Suitable adjuvants and fillers include lecithin, inulin, polydextrin, calcium carbonate, magnesium carbonate, magnesium silicate, limestone powder, aluminum hydroxide, aluminum silicate, talcum, clay, aluminum oxide, titanium dioxide and calcium phosphate. In a specific embodiment, lecithin can be used as a kind of inert filler to reduce the viscosity of the chewing gum composition. In other specific embodiments, lactic acid copolymer, protein (for example, gluten and / or zein) and / or guar gum can be used to form a chewing gum that is more biodegradable. These adjuvants or fillers are normally present in the gum base with the amount of up to about 20% by weight of the gum base. Other optional ingredients include coloring agent, whitening agent, preservative and flavoring.

[0417] In specific embodiments of chewing gum compositions, the gum base constitutes from about 5 weight % to about 95 weight % of the chewing gum composition, more desirably from about 15 weight % to about 50 weight % of the chewing gum composition and even more desirably from about 20 weight % to about 30 weight % of the chewing gum composition.

[0418] The soluble part of the chewing gum composition can optionally include other artificial sweeteners or natural sweeteners, bulk sweeteners, softeners, emulsifiers, flavorings, coloring agents, adjuvants, fillers, functional agents (e.g., drugs or nutrients), or combinations thereof. Examples of suitable softeners and emulsifiers are described above.

[0419] Filling sweeteners include both caloric compounds and no caloric compounds. The limiting examples of filling sweeteners include sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, tagatose, polyols (for example, sorbitol, mannitol, xylitol, lactitol, erythritol and maltitol), hydrogenated starch hydrolysate, isomalt, trehalose and mixtures thereof. In a particular embodiment, filling sweeteners are present in the chewing gum composition in an amount ranging from about 1 % by weight to about 75 % by weight of the chewing gum composition.

[0420] Flavoring agent can be used in the insoluble gum base or the soluble part of chewing gum composition.This type of flavoring agent can be natural flavor seasoning or artificial flavor seasoning.In a specific embodiment, this flavoring agent comprises essential oil, as the oil that comes from plant or fruit, peppermint oil, spearmint oil, other peppermint oils, clove oil, cinnamon oil, wintergreen oil, bay oil, thyme oil, cedar leaf oil, nutmeg oil, allspice oil, sage oil, mace oil and almond oil.In another specific embodiment, this flavoring agent comprises plant extract or fruit essence, as apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot and its mixture.In another specific embodiment, this flavoring agent comprises citrus flavoring agent, as the oil of extract, essence or lemon, sour orange, orange, tangerine, grapefruit, citron or kumquat.

[0421] In a particular embodiment, a chewing gum composition comprises rebaudioside I or a composition comprising rebaudioside I and a gum base. In a particular embodiment, rebaudioside I is present in the chewing gum composition in an amount ranging from about 1 ppm to about 10,000 ppm of the chewing gum composition.

[0422] Cereal composition

[0423] In one embodiment, the present invention is a cereal composition comprising rebaudioside I. In another embodiment, the present invention is a cereal composition comprising a composition containing rebaudioside I. Cereal compositions are typically consumed as a staple food or as a snack. Non-limiting examples of cereal compositions used in specific embodiments include ready-to-eat cereals and hot cereals. Ready-to-eat cereals are cereals that can be eaten without further processing (i.e., cooking) by the consumer. Examples of ready-to-eat cereals include breakfast cereals and snack bars. Breakfast cereals are typically processed to produce a shredded, flaked, expanded or extruded form. Breakfast cereals are usually consumed cold and are usually mixed with milk and / or fruit. Snack bars include, for example, energy bars, rice cakes, granola bars, and nutritional bars. Hot cereals are usually cooked with milk or water before eating. Non-limiting examples of hot cereals include grits, porridge, corn porridge, rice, and oatmeal.

[0424] Cereal compositions typically include at least one cereal component. As used herein, the term "cereal component" refers to materials such as all or part of grain, all or part of seed, and all or part of grass. Non-limiting examples of cereal components used in specific embodiments include corn, wheat, rice, barley, bran, bran endosperm, bulgur, sorghum, millet, oats, rye, triticale, buckwheat, fonio, quinoa, kidney beans, soybeans, amaranth, teff, spelt, and kaniwa.

[0425] In a specific embodiment, the cereal composition comprises rebaudioside I or a composition comprising rebaudioside I and at least one cereal ingredient. Rebaudioside I or a composition comprising rebaudioside I can be added to the cereal composition in a variety of ways, for example, as a coating, as a frosting, as a glaze, or as a matrix blend (i.e., added as an ingredient to the cereal formulation before preparing the final cereal product).

[0426] Thus, in a specific embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the cereal composition as a base blend. In one embodiment, rebaudioside I or a composition comprising rebaudioside I is blended with a hot cereal prior to cooking to provide a sweetened hot cereal product. In another embodiment, rebaudioside I or a composition comprising rebaudioside I is blended with a cereal base prior to extruding the cereal.

[0427] In another specific embodiment, rebaudioside I or a composition comprising rebaudioside I is used as a coating, such as by combining rebaudioside I or a composition comprising rebaudioside I with an edible grade oil and applying the mixture to cereals to add to the cereal composition. In a different embodiment, rebaudioside I or a composition comprising rebaudioside I and an edible grade oil can be applied to cereals separately by first applying oil or a sweetener. Non-limiting examples of edible grade oils for specific embodiments include vegetable oils, such as corn oil, soybean oil, cottonseed oil, peanut oil, coconut oil, rapeseed oil, olive oil, sesame seed oil, palm oil, palm kernel oil, and mixtures thereof. In another embodiment, edible grade fats can be used to replace these oils, as long as the fat is melted before the fat is applied to the cereals.

[0428] In another embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the cereal composition as a glaze. Non-limiting examples of glazing agents used in specific embodiments include corn syrup, honey, syrup and honey syrup solids, maple syrup and maple syrup solids, sucrose, isomalt, polydextrose, polyols, hydrogenated starch hydrolysate, its aqueous solution and mixtures thereof. In another such embodiment, rebaudioside I or a composition comprising rebaudioside I is added as a glaze by combining with a glazing agent and edible grade oil or fat and applying the mixture to cereals. In another embodiment, a gum system, such as, gum arabic, carboxymethyl cellulose or algin, can be added to the glaze to provide structural support. In addition, the glaze can also include a coloring agent, and can also include a spice.

[0429] In another embodiment, rebaudioside I or a composition comprising rebaudioside I is added to the cereal composition as a frosting. In one such embodiment, rebaudioside I or a composition comprising rebaudioside I is combined with water and a frosting agent and then applied to cereals. Non-limiting examples of frosting agents used in specific embodiments include maltodextrin, sucrose, starch, polyols, and mixtures thereof. The frosting can also include a food grade oil, food grade fat, coloring agent, and / or spices.

[0430] Typically, the amount of rebaudioside I in a cereal composition varies widely depending on the specific type of the cereal composition and its desired sweetness. One of ordinary skill in the art can easily determine the appropriate amount of the sweetener added to the cereal composition. In a specific embodiment, rebaudioside I is present in the cereal composition in an amount ranging from about 0.02% by weight to about 1.5% by weight of the cereal composition, and the at least one additive is present in the cereal composition in an amount ranging from about 1% by weight to about 5% by weight of the cereal composition.

[0431] Bakery

[0432] In one embodiment, the present invention is a baked good comprising rebaudioside I. In another embodiment, the present invention is a baked good comprising a composition comprising rebaudioside I. As used herein, baked goods include ready-to-eat and all ready-to-bake products, flours, and mixes that require preparation prior to serving. Non-limiting examples of baked goods include cakes, crackers, biscuits, brownies, muffins, rolls, bagels, donuts, strudel, pastries, croissants, small pastries, bread, bread products, and buns.

[0433] Preferred baked goods according to embodiments of the present invention can be classified into three groups: bread-type doughs (e.g., white bread, variety bread, soft bread, hard rolls, bagels, pizza dough, and tortillas), sweet doughs (e.g., Danish pastry, croissants, crackers, puff pastry, pie crust, small pastries, and sweet cookies), and batters (e.g., cakes such as sponge cakes, pound cakes, devil's food cakes, cheesecakes, and layer cakes, donuts or other yeast-raised cakes, brownies, and muffins). Doughs are generally characterized as flour-based, while batters are more water-based.

[0434] Baked food according to a specific embodiment of the present invention generally comprises a combination of sweetener, water and fat. Baked food prepared according to many embodiments of the present invention also contains flour, so as to prepare a dough or paste. The term "dough" as used herein is a mixture of flour and other ingredients, and its hardness is enough to knead or roll. The term "paste" as used herein is composed of flour, liquid such as milk or water, and other ingredients and is thin enough to pour or drip from a spoon. Ideally, according to a specific embodiment of the present invention, the flour is present in the baked food in an amount of about 15% to about 60% on a dry weight basis, more preferably about 23% to about 48% on a dry weight basis.

[0435] The type of flour can be selected based on the desired product. Typically, flour comprises edible non-toxic flour commonly used in baked goods. According to a specific embodiment, flour can be a bleached baking flour, all-purpose flour, or unbleached flour. In other specific embodiments, flour processed in other ways can also be used. For example, in a specific embodiment, flour can be enriched with additional vitamins, minerals or proteins. Non-limiting examples of flour suitable for specific embodiments of the present invention include wheat flour, corn flour, whole grains, parts of whole grains (wheat, bran and oatmeal), and combinations thereof. In a specific embodiment, starch or starch-containing materials can also be used as flour. Common food starch is usually derived from potatoes, corn, wheat, barley, oats, cassava, arrowroot and sago. In a specific embodiment of the present invention, modified starch and pregelatinized starch can be used.

[0436] The type of fat or oil used in a specific embodiment of the present invention can include any edible fat, oil or combination thereof that is applicable to baking. The non-limiting examples of fats that are applicable to a specific embodiment of the present invention include vegetable oils, tallow, lard, marine animal oils and combinations thereof. According to a specific embodiment, these fats can be fractionated, partially hydrogenated and / or strengthened. In another specific embodiment, the fat desirably includes reduced, low-calorie or indigestible fats, fat substitutes or synthetic fats. In another specific embodiment, a mixture of shortening, fat or hard fat and soft fat can also be used. In a specific embodiment, shortening can be mostly derived from triglycerides, and the triglycerides are derived from plant sources (for example, cottonseed oil, soybean oil, peanut oil, linseed oil, sesame oil, palm oil, palm kernel oil, rapeseed oil, safflower oil, coconut oil, corn oil, sunflower seed oil and mixtures thereof). In a specific embodiment, synthetic triglycerides or natural triglycerides with fatty acids of a chain length from 8 to 24 carbon atoms can also be used. Desirably, according to particular embodiments of this invention, the fat is present in the baked good in an amount ranging from about 2% to about 35% by weight on a dry basis, more desirably from about 3% to about 29% by weight on a dry basis.

[0437] Baked goods according to particular embodiments of the present invention also contain water in an amount sufficient to provide the desired consistency so that the baked goods can be properly shaped, machined, and cut before or after cooking. The total moisture content of the baked goods includes any water added directly to the baked goods as well as water present in separately added ingredients (e.g., flour, which typically contains about 12% to about 14% moisture by weight). Ideally, according to particular embodiments of the present invention, water is present in the baked goods in an amount up to about 25% by weight of the baked goods.

[0438] Baked food according to a specific embodiment of the present invention may also include a variety of additional common ingredients, such as leavening agents, flavorings, pigments, milk, milk byproducts, eggs, egg byproducts, cocoa, vanilla or other flavoring agents, and inclusions, such as nuts, raisins, cherries, apples, apricots, peaches, other fruits, citrus peels, preservatives, coconut, flavored flakes (such as chocolate flakes, butterscotch flakes and caramel flakes) and combinations thereof. In a specific embodiment, the baked food may also include an emulsifier, such as lecithin and monoglyceride.

[0439] According to a specific embodiment of the present invention, the leavening agent may include a chemical leavening agent or a yeast leavening agent. Non-limiting examples of chemical leavening agents suitable for use in specific embodiments of the present invention include baking soda (e.g., sodium bicarbonate, potassium bicarbonate, or aluminum bicarbonate), fermentation acid (e.g., sodium aluminum phosphate, monocalcium phosphate, or dicalcium phosphate), and combinations thereof.

[0440] According to another embodiment of the present invention, the cocoa may comprise natural chocolate or "alkali-treated" chocolate, the majority of which fat or cocoa butter has been expressed or removed by solvent extraction, extrusion or other means. In one embodiment, it may be desirable to reduce the amount of fat in a baked good containing chocolate due to the presence of additional fat in cocoa butter. In one embodiment, it may be desirable to add a greater amount of chocolate than cocoa in order to provide an equivalent amount of flavor and color.

[0441] Baked goods also typically include caloric sweeteners, such as sucrose, high fructose corn syrup, erythritol, molasses, honey, or brown sugar. In the exemplary embodiment of the baked goods provided herein, the caloric sweeteners are partially or completely replaced by Rebaudioside I or a composition comprising Rebaudioside I. Therefore, in one embodiment, a baked food comprises Rebaudioside I or a composition comprising Rebaudioside I and fat, water, and optionally flour combination. In a specific embodiment, the baked food may optionally include other natural and / or synthetic high-efficiency sweeteners and / or bulking sweeteners.

[0442] Dairy

[0443] In one embodiment, the consumable of the present invention is a dairy product comprising rebaudioside I. In another embodiment, the consumable of the present invention is a dairy product comprising a composition comprising rebaudioside I. Dairy products suitable for use in the present invention and methods for preparing dairy products are well known to those of ordinary skill in the art. As used herein, dairy products include milk or foods produced from milk. Non-limiting examples of dairy products suitable for embodiments of the present invention include milk, cream, sour cream, crème brûlée, buttermilk, fermented buttermilk, milk powder, condensed milk, evaporated milk, butter, cheese, cottage cheese, cream cheese, yogurt, ice cream, soft jelly, frozen yogurt, gelato, mayonnaise (vla), piima, yogurt Kajmak, kephir, viili, kumiss, airag, ice milk, casein, ayran, lassi, khoa, or a combination thereof.

[0444] Milk is a fluid secreted by the mammary glands of female mammals for the nourishment of their young. The female ability to produce milk is one of the defining mammalian characteristics and provides a primary source of nutrients for newborns before they are able to digest more diverse foods. In a particular embodiment of the invention, the dairy products are raw milk derived from cattle, goats, sheep, horses, donkeys, camels, buffaloes, yaks, reindeers, moose, or humans.

[0445] In a specific embodiment of the present invention, processing dairy products from raw milk generally includes the steps of pasteurization, creaming and homogenization. Although raw milk can be consumed without pasteurization, it is generally pasteurized to destroy harmful microorganisms, such as bacteria, viruses, protozoa, molds, and yeasts. Pasteurization generally involves heating the milk to a high temperature for a short period of time to substantially reduce the number of microorganisms, thereby reducing the risk of disease.

[0446] Creaming is traditionally after the pasteurization step and involves separating the milk into a higher fat cream layer and a lower fat milk layer. The milk will separate into the milk layer and the cream layer after standing for twelve to twenty-four hours. The cream rises to the top of the milk layer and can be skimmed off and used as a separate dairy product. As an alternative, centrifugation can be used to separate the cream from the milk. The remaining milk is classified according to its fat content, non-limiting examples of which include whole milk, 2% fat milk, 1% fat milk, and skim milk.

[0447] Milk is often homogenized after removing the fat of the desired amount from the milk by emulsification. Homogenization prevents cream from separating from milk and generally involves pumping milk through a small diameter tube under high pressure to destroy the fat globules in the milk. Pasteurization, emulsification and homogenization of milk are common, but are not necessary for producing consumable dairy products. Therefore, the dairy products suitable for use in embodiments of the present invention may be without processing steps, through a single processing step or through a combination of processing steps described herein. The dairy products suitable for use in embodiments of the present invention may be through processing steps except processing steps described herein.

[0448] Specific embodiments of the present invention include dairy products produced from milk by additional processing steps. As described above, cream can be skimmed from the top of the milk or separated from the milk using a machine centrifuge. In one specific embodiment, the dairy product includes sour cream, which is a fat-rich dairy product obtained by fermenting cream using a bacterial culture. The bacteria produce lactic acid during the fermentation process, which makes the cream sour and thicker. In another specific embodiment, the dairy product includes crème fraîche, which is a heavy cream slightly acidified with a bacterial culture in a manner similar to sour cream. French cream is generally not as thick or as sour as sour cream. In another specific embodiment, the dairy product includes fermented buttermilk. Fermented buttermilk is obtained by adding bacteria to milk. The resulting fermentation in which the bacterial culture converts lactose into lactic acid produces a sour-tasting fermented buttermilk. Although it is produced in a different way, fermented buttermilk is generally similar to traditional buttermilk, which is a byproduct of butter manufacturing.

[0449] According to other specific embodiments of the present invention, the dairy product comprises milk powder, condensed milk, evaporated milk or a combination thereof. Milk powder, condensed milk and evaporated milk are usually produced by removing water from milk. In a specific embodiment, the dairy product comprises a milk powder containing dry milk solids, and the dry milk solids have a low moisture content. In another specific embodiment, the dairy product comprises condensed milk. Condensed milk usually comprises milk with reduced moisture content and an added sweetener, thereby producing a thick sweet product with a long shelf life. In another specific embodiment, the dairy product comprises evaporated milk. Evaporated milk usually comprises fresh, homogenized milk from which about 60% of the water has been removed, which has been cooled, fortified with additives such as vitamins and stabilizers, packaged, and finally sterilized. According to another specific embodiment of the present invention, the dairy product comprises dry creamer and rebaudioside I or a composition comprising rebaudioside I.

[0450] In another specific embodiment, the dairy products provided herein include butter. Butter is typically prepared by churning fresh or fermented cream or milk. Butter typically contains milk fat around small droplets that contain mostly water and milk proteins. The churning process damages the membranes around the milk fat globules, allowing the milk fat to bind and separate from the rest of the cream. In another specific embodiment, the dairy product includes buttermilk, which is the sour liquid that remains after butter is produced from whole milk by the churning process.

[0451] In another specific embodiment, dairy products include cheese, which is a solid food produced by coagulating milk using a combination of rennet or rennet substitute and acidification. Rennet is a natural enzyme complex produced in the stomach of mammals that digest milk, and it is used to coagulate milk in cheese preparation, so that it is separated into a solid called curd and a liquid called whey. Usually, rennet is obtained from the stomach of young ruminants such as calves; However, alternative sources of rennet include some plants, microbial organisms, and genetically modified bacteria, fungi or yeast. In addition, milk can be coagulated by adding acid such as citric acid. Usually, a combination of rennet and / or acidification is used to coagulate milk. After milk is separated into curd and whey, some cheeses are made by simple drainage, salting and packaging these curds. However, for most cheeses, more processing is required. Many different methods can be used to produce hundreds of available cheese varieties. Processing methods include heating the cheese, cutting it into small pieces to drain, salting, stretching, forming cheddar cheese, washing, making it moldy, ripening and maturing. Some cheeses such as blue cheese have additional bacteria or molds introduced before or during ripening, thereby giving flavor and fragrance to the final product. Cottage cheese is a kind of cheese curd product with suitable flavor, which is discharged but not squeezed, so that some whey is retained. Usually the curd is washed to remove acidity. Cream cheese is a kind of soft, moderate-taste cottage cheese with high fat content, which is produced by adding cream to milk and then coagulating it to form a kind of abundant curd. Or, cream cheese can be made from skim milk, in which cream is added to the curd. It should be understood that cheese as used herein includes all solid foods produced by coagulating milk.

[0452] In another specific embodiment of the present invention, the dairy product includes yogurt. Yogurt is usually produced by bacterial fermentation of milk. The fermentation of lactose produces lactic acid, which acts on the protein in the milk to produce a gel-like texture and sour taste. In a particularly desirable embodiment, the yogurt can be sweetened and / or flavored with a sweetener. Non-limiting examples of flavoring agents include, but are not limited to, fruit (e.g., peach, strawberry, banana), vanilla, and chocolate. As used herein, yogurt also includes yogurt varieties with different consistencies and viscosities, such as dahi, dadih or dadih, 1abneh or labaneh, bulgarian, kefir, and matsoni. In another specific embodiment, the dairy product includes a beverage based on yogurt, which is also referred to as drinkable yogurt or a yogurt mousse (yogurt smoothie). In a particularly desirable embodiment, a beverage based on yogurt can include sweeteners, flavoring agents, other ingredients, or a combination thereof.

[0453] In particular embodiments of the present invention, other dairy products other than those described herein may be used. Such dairy products are well known to those of ordinary skill in the art, and non-limiting examples thereof include milk, milk and juice, coffee, tea, vla, healthy yogurt, filmjolk, kajmak, kephir, viili, kumiss, airag, ice milk, casein, ayran, lassi, and khoa.

[0454] According to a specific embodiment of the present invention, dairy composition can also include other additives.The limiting examples of applicable additives include sweeteners and flavorings, such as chocolate, strawberry and banana.The specific embodiment of the dairy composition provided here can also include additional nutritional supplements such as vitamins (e.g., vitamin D) and minerals (e.g., calcium) to improve the nutritional composition of milk.

[0455] In a particularly desirable embodiment, the dairy composition comprises rebaudioside I or a composition comprising rebaudioside I is combined with a dairy product. In a specific embodiment, rebaudioside I is present in the dairy composition in an amount ranging from about 200% to about 20,000% by weight of the dairy composition.

[0456] Rebaudioside I or a composition comprising rebaudioside I is also suitable for processed agricultural products, livestock products or seafood; processed meat products such as sausages, etc.; retorted foods, pickles, jams boiled in soy sauce, delicacies, side dishes; soups; snacks such as potato chips, sweet biscuits, etc.; chopped stuffings, leaves, stems, stalks, homogenized roasted leaves and animal feed.

[0457] Tabletop Sweetener Composition

[0458] In one embodiment, the present invention is a tabletop sweetener comprising rebaudioside I. The tabletop composition may further comprise at least one bulking agent, additive, anti-caking agent, functional ingredient, or a combination thereof.

[0459] Suitable "bulking agents" include, but are not limited to, maltodextrin (10DE, 18DE, or 5DE), corn syrup solids (20DE or 36DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribulose, mannose, xylitol, mannitol, galactitol, erythritol, maltitol, lactitol, isomalt, maltose, tagatose, lactose, inulin, glycerol, propylene glycol, polyols, polydextrose, oligofructose, cellulose and cellulose derivatives, etc., and mixtures thereof. In addition, according to other embodiments of the present invention, granulated sugar (sucrose) or other caloric sweeteners such as crystalline fructose, other carbohydrates or sugar alcohols can be used as a bulking agent because they provide good content uniformity without adding a large amount of calories.

[0460] As used herein, the phrases "anti-caking agent" and "glidant" refer to any composition that contributes to content uniformity and uniform dissolution. According to specific embodiments, non-limiting examples of anti-caking agents include tartar, calcium silicate, silicon dioxide, microcrystalline cellulose (Avicel, FMC BioPolymer, Philadelphia, Pennsylvania), and tricalcium phosphate. In one embodiment, the anti-caking agent is present in the tabletop sweetener composition in an amount from about 0.001% to about 3% by weight of the tabletop sweetener composition.

[0461] These tabletop sweetener compositions can be packaged in any form known in the art. Non-limiting forms include, but are not limited to, powder form, granular form, packet, tablet, capsule, pellet, cube, solid, and liquid.

[0462] In one embodiment, the tabletop sweetener composition is a disposable (portion control) package containing a dry blend. The dry blend formulation can generally include powders or granules. Although the tabletop sweetener composition can be in a packet of any size, a non-limiting embodiment of a conventional portion control tabletop sweetener packet is about 2.5×1.5 inches and maintains about 1 gram of sweetener composition with a sweetness equivalent to 2 teaspoons of granulated sugar (about 8g). The amount of rebaudioside I in a tabletop sweetener formulation of a dry blend can be changed. In a specific embodiment, a tabletop sweetener formulation of a dry blend can contain rebaudioside I in an amount from about 1% (w / w) to about 10% (w / w) of the tabletop sweetener composition.

[0463] Solid tabletop sweetener embodiments include cubes and tablets. A non-limiting example of a conventional cube is equivalent in size to a standard cube of granulated sugar, which is approximately 2.2 x 2.2 x 2.2 cm. 3 and weighs about 8 g. In one embodiment, the solid tabletop sweetener is in tablet form or any other form known to those skilled in the art.

[0464] Tabletop sweetener compositions can also be embodied as in liquid form, wherein rebaudioside I is combined with a liquid carrier. Suitable non-limiting examples of carrier agents of liquid tabletop sweeteners include water, alcohol, polyols, glyceryl or citric acid groups dissolved in water, and mixtures thereof. The sweetness equivalent of any form of tabletop sweetener compositions described herein or known in the art can be changed to obtain a desired sweetness profile. For example, the tabletop sweetener compositions can include a sweetness comparable to a considerable amount of standard sugar. In another embodiment, the tabletop sweetener compositions can include a sweetness up to 100 times of a considerable amount of sugar. In another embodiment, the tabletop sweetener compositions can include a sweetness up to 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, and 2 times of a considerable amount of sugar.

[0465] Beverages and beverage products

[0466] In one embodiment, the present invention is a beverage or beverage product comprising rebaudioside I. In another embodiment, the present invention is a beverage or beverage comprising a composition (eg, a sweetener composition) comprising rebaudioside I.

[0467] As used herein, a kind of "beverage product" is a kind of drinkable beverage, a kind of beverage concentrate, a kind of beverage syrup or a kind of beverage powder brewed beverage.Suitable drinkable beverages include carbonated beverages and non-carbonated beverages.Carbonated beverages include, but are not limited to, enhanced sparkling beverages, cola, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger wine, soda and root beer.Non-carbonated beverages include, but are not limited to, fruit juice, fruit flavored juice, fruit juice drinks, nectar, vegetable juice, vegetable flavored juice, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks (for example, water with natural or synthetic flavoring agents), coconut juice, tea type drinks (for example, black tea, green tea, black tea, oolong tea), coffee, cocoa drinks, beverages containing milk components (for example, milk drinks, coffee containing milk components, cafe au lait, milk tea, fruit milk drinks), beverages containing cereal extracts, ice smoothies and combinations thereof.

[0468] Beverage concentrates and beverage syrups are prepared with an initial volume of a liquid base (e.g., water) and the desired beverage ingredients. Full strength beverages are then prepared by adding an additional volume of water. Powdered beverages are prepared by dry mixing all beverage ingredients in the absence of a liquid base. Full strength beverages are then prepared by adding the full volume of water.

[0469] The beverage contains a liquid matrix, i.e., a base component in which the ingredients (including the composition of the present invention) are dissolved. In one embodiment, the beverage contains beverage-quality water as the liquid matrix, such as, for example, deionized water, distilled water, reverse osmosis water, carbon-treated water, pure water, softened water, and combinations thereof. Additional suitable liquid matrices include, but are not limited to, phosphoric acid, phosphate buffer, citric acid, citrate buffer, and carbon-treated water.

[0470] In one embodiment, the consumable product of the present invention is a beverage comprising rebaudioside I.

[0471] In another embodiment, a beverage contains a composition comprising rebaudioside I.

[0472] In yet another embodiment, the present invention is a beverage product comprising rebaudioside I.

[0473] In another embodiment, the present invention is a beverage product containing a composition comprising rebaudioside I.

[0474] The concentration of rebaudioside I in a beverage or beverage product may be above, at, or below its threshold sweetness or recognition concentration.

[0475] In a specific embodiment, the concentration of rebaudioside I in the beverage or beverage product is above its threshold sweetness or flavor recognition concentration. In one embodiment, the concentration of rebaudioside I is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or more above its threshold sweetness or flavor recognition concentration.

[0476] In another specific embodiment, the concentration of rebaudioside I in the beverage or beverage product is at or about the threshold sweetness or flavor recognition concentration of rebaudioside I.

[0477] In yet another specific embodiment, the concentration of rebaudioside I in the beverage or beverage product is below the threshold sweetness or flavor recognition concentration of rebaudioside I. In one embodiment, the concentration of rebaudioside I is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or more below its threshold sweetness or flavor recognition concentration.

[0478] In one embodiment, the present invention is a beverage or beverage product, the amount of rebaudioside I contained in it is in the range of from about 1ppm to about 10,000ppm, for example, from about 5ppm to about 10,000ppm, from about 10ppm to about 10,000ppm, from about 15ppm to about 10,000ppm, from about 20ppm to about 10,000ppm or from about 25ppm to about 10,000ppm. In another embodiment, rebaudioside I is present in the beverage in an amount ranging from about 100ppm to about 600ppm. In other embodiments, rebaudioside I is present in the beverage in an amount ranging from about 100 to about 200ppm, from about 100ppm to about 300ppm, from about 100ppm to about 400ppm or from about 100ppm to about 500ppm. In another embodiment, rebaudioside I is present in the beverage or beverage product in an amount ranging from about 300ppm to about 700ppm, such as, for example, from about 400ppm to about 600ppm. In another embodiment, rebaudioside I is present in a beverage or beverage product in an amount ranging from about 200 ppm to about 400 ppm, such as about 200 to about 250 ppm, about 250 ppm to about 300 ppm, about 300 ppm to about 350 ppm or about 350 ppm. In another embodiment, rebaudioside I is present in a beverage or beverage product in an amount ranging from about 450 ppm to about 650 ppm, such as about 450 ppm to about 500 ppm, about 500 ppm to about 550 ppm, about 550 ppm to about 600 ppm or about 600 to about 650 ppm.

[0479] In an exemplary embodiment, the beverage of the present invention comprises rebaudioside I in an amount from about 200 to about 300 ppm, more particularly about 200 ppm, about 225, about 250 ppm, about 275 ppm or about 300 ppm.

[0480] In an exemplary embodiment, the beverage of the present invention comprises rebaudioside I in an amount from about 500 to about 600 ppm, more specifically about 500 ppm, about 525 ppm, about 550 ppm, about 575 ppm or about 600 ppm.

[0481] The beverage may further comprise at least one additional sweetener. Any sweetener described in detail herein may be used, including natural sweeteners, non-natural sweeteners or synthetic sweeteners. These may be added to the beverage before, simultaneously or after the rebaudioside I.

[0482] In one embodiment, the beverage comprises a carbohydrate sweetener at a concentration of from about 100 ppm to about 140,000 ppm. Synthetic sweeteners can be present in the beverage at a concentration of from about 0.3 ppm to about 3,500 ppm. Natural high potency sweeteners can be present in the beverage at a concentration of from about 0.1 ppm to about 3,000 ppm.

[0483] The beverage may further comprise additives, including but not limited to carbohydrates, 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, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighting agents, juices, dairy products, cereals and other plant extracts, flavonoids, alcohols, polymers and combinations thereof. Any suitable additives described herein may be used.

[0484] In one embodiment, the polyol can be present in the beverage in a concentration from about 100 ppm to about 250,000 ppm, such as, for example, from about 5,000 ppm to about 40,000 ppm.

[0485] In another embodiment, the amino acid can be present in the beverage at a concentration from about 10 ppm to about 50,000 ppm, such as, for example, from about 1,000 ppm to about 10,000 ppm, from about 2,500 ppm to about 5,000 ppm, or from about 250 ppm to about 7,500 ppm.

[0486] In yet another embodiment, the nucleotide may be present in the beverage at a concentration from about 5 ppm to about 1,000 ppm.

[0487] In another embodiment, the organic acid additive may be present in the beverage at a concentration from about 10 ppm to about 5,000 ppm.

[0488] In yet another embodiment, the inorganic acid additive may be present in the beverage at a concentration from about 25 ppm to about 25,000 ppm.

[0489] In another embodiment, the bitter compound may be present in the beverage at a concentration from about 25 ppm to about 25,000 ppm.

[0490] In another embodiment, the flavorant may be present in the beverage in a concentration from about 0.1 ppm to about 4,000 ppm.

[0491] In yet another embodiment, the polymer may be present in the beverage at a concentration from about 30 ppm to about 2,000 ppm.

[0492] In another embodiment, the protein hydrolysate may be present in the beverage at a concentration from about 200 ppm to about 50,000 ppm.

[0493] In another embodiment, the surfactant additive may be present in the beverage at a concentration from about 30 ppm to about 2,000 ppm.

[0494] In yet another embodiment, the flavonoid additive may be present in the beverage at a concentration from about 0.1 ppm to about 1,000 ppm.

[0495] In another embodiment, the alcohol additive may be present in the beverage at a concentration from about 625 ppm to about 10,000 ppm.

[0496] In yet another embodiment, the astringent additive may be present in the beverage at a concentration from about 10 ppm to about 5,000 ppm.

[0497] The beverage may further contain one or more functional ingredients described in detail above. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols and combinations thereof. Any suitable functional ingredient described herein may be used.

[0498] It is contemplated that the pH of a consumer's product (e.g., beverage) will not substantially or adversely affect the taste of the sweetener. A non-limiting example of the pH range of the beverage can be from about 1.8 to about 10. Another example includes a pH range from about 2 to about 5. In a specific embodiment, the pH of the beverage can be from about 2.5 to about 4.2. It will be appreciated by those skilled in the art that the pH of the beverage can change based on the type of beverage. For example, a dairy beverage can have a pH greater than 4.2.

[0499] The titratable acidity of a beverage comprising rebaudioside I can range, for example, from about 0.01% to about 1.0% by weight of the beverage.

[0500] In one embodiment, the sparkling beverage product has an acidity of from about 0.01% to about 1.0% by weight of the beverage, such as, for example, from about 0.05% to about 0.25% by weight of the beverage.

[0501] A carbonated beverage product has carbonation of 0 to about 2% (w / w) carbon dioxide or its equivalent, such as from about 0.1% to about 1.0% (w / w).

[0502] The temperature of the beverage can range, for example, from about 4°C to about 100°C, such as, for example, from about 4°C to about 25°C.

[0503] The beverage may be a calorie-rich beverage having up to about 120 calories per 8 ounce serving.

[0504] The beverage can be a mid-calorie beverage having up to about 60 calories per 8 ounce serving.

[0505] The beverage can be a low calorie beverage having up to about 40 calories per 8 ounce serving.

[0506] The beverage can be a zero calorie beverage having less than about 5 calories per 8 ounce serving.

[0507] How to use

[0508] The compounds and compositions of the invention can be used to impart sweetness or enhance the flavor or sweetness of consumables or other compositions.

[0509] In another aspect, the present invention is a method for preparing a consumable product, the method comprising (i) providing a consumable product base and (ii) adding rebaudioside I to the consumable product base to provide a consumable product.

[0510] In a specific embodiment, the present invention is a method for preparing a beverage, the method comprising (i) providing a beverage base and (ii) adding rebaudioside I to the consumable base to provide a beverage.

[0511] In another aspect, the present invention is a method of preparing a sweetened consumable product, the method comprising (i) providing a sweetenable consumable product and (ii) adding rebaudioside I to the sweetenable consumable product to provide a sweetened consumable product.

[0512] In a specific embodiment, the present invention is a method for preparing a sweetened beverage, the method comprising (i) providing a sweetenable beverage and (ii) adding rebaudioside I to the sweetenable beverage to provide a sweetened beverage.

[0513] In the above method, rebaudioside I can be provided as is, or in the form of a composition. When rebaudioside I is provided as a composition, when the composition is added to a consumable (e.g., a beverage), the amount of the composition is effective to provide a concentration of rebaudioside I above, at, or below its threshold flavor or sweetness recognition concentration. When rebaudioside I is not provided as a composition, it can be added to the consumable at a concentration above, at, or below its threshold flavor or sweetness recognition concentration.

[0514] In one embodiment, the present invention is a method for enhancing the sweetness of a consumable, the method comprising (i) providing a consumable comprising one or more sweet ingredients, and (ii) adding rebaudioside I (1) to the consumable to provide a consumable with enhanced sweetness, wherein rebaudioside I is added to the consumable at a concentration at or below its threshold sweetness recognition concentration. In a specific embodiment, rebaudioside I is added to the consumable at a concentration below its threshold sweetness recognition concentration.

[0515] In another embodiment, the present invention is a method of enhancing the sweetness of a consumable, the method comprising (i) providing a consumable comprising one or more sweet ingredients, and (ii) adding a composition comprising rebaudioside I to the consumable to provide a consumable having enhanced sweetness, wherein when the composition is added to the consumable, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold sweetness recognition concentration. In a specific embodiment, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I below its threshold sweetness recognition concentration.

[0516] In a specific embodiment, the present invention is a method for enhancing the sweetness of a beverage, the method comprising (i) providing a beverage comprising at least one sweet ingredient, and (ii) adding rebaudioside I to the beverage to provide a beverage with enhanced sweetness, wherein rebaudioside I is added to the beverage in an amount effective to provide a concentration at or below its threshold sweetness recognition concentration. In a specific embodiment, rebaudioside I is added to a consumable in an amount effective to provide a concentration below its threshold sweetness recognition concentration.

[0517] In another specific embodiment, the present invention is a method of enhancing the sweetness of a beverage, the method comprising (i) providing a beverage comprising one or more sweet ingredients, and (ii) adding a composition comprising rebaudioside I to the beverage to provide a beverage with enhanced sweetness, wherein when the composition is added to the beverage, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold sweetness recognition concentration. In a specific embodiment, when the composition is added to the beverage, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I below its threshold sweetness recognition concentration.

[0518] In another embodiment, the present invention is a method for enhancing the flavor of a consumable, the method comprising (i) providing a consumable comprising at least one flavor component, and (ii) adding rebaudioside I to the consumable to provide a consumable with enhanced flavor, wherein rebaudioside I is added to the consumable at a concentration at or below its threshold flavor recognition concentration. In a specific embodiment, rebaudioside I is added to the consumable at a concentration below the threshold flavor recognition concentration.

[0519] In another embodiment, the present invention is a method for enhancing the flavor of a consumable product, the method comprising (i) providing a consumable product comprising at least one flavor ingredient, and (ii) adding a composition of rebaudioside I to the consumable product to provide a consumable product having enhanced flavor, wherein when the composition is added to the consumable product, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold flavor recognition concentration. In a specific embodiment, when the composition is added to the consumable product, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I below its threshold flavor recognition concentration.

[0520] In a specific embodiment, the present invention is a method for enhancing the flavor of a beverage, the method comprising (i) providing a beverage comprising at least one flavor component, and (ii) adding rebaudioside I to the beverage to provide a beverage with enhanced flavor, wherein rebaudioside I is added to the beverage at a concentration at or below its threshold flavor recognition concentration. In a specific embodiment, rebaudioside I is added to a consumable at a concentration below the threshold flavor recognition concentration.

[0521] In a specific embodiment, the present invention is a method for enhancing the flavor of a beverage, the method comprising (i) providing a beverage comprising at least one flavor component, and (ii) adding a composition comprising rebaudioside I to the beverage to provide a beverage having enhanced flavor, wherein when the composition is added to the beverage, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I at or below its threshold flavor recognition concentration. In a specific embodiment, when the composition is added to the consumable, rebaudioside I is present in the composition in an amount effective to provide a concentration of rebaudioside I below its threshold flavor recognition concentration.

[0522] The present invention also includes methods of preparing sweetening compositions (e.g., sweetened consumables) and flavor-enhanced compositions (e.g., flavor-enhanced consumables) by adding rebaudioside I or compositions comprising rebaudioside I to such compositions / consumables.

[0523] The following examples illustrate preferred embodiments of the present invention. It should be understood that the present invention is not limited to the materials, proportions, conditions and procedures in these examples, which are merely illustrative.

[0524] Example 1

[0525] In vivo production of UGT76G1

[0526] NcoI and NdeI restriction sites were added to the original nucleic acid sequence described in Genbank Accession No. AAR06912.1. After codon optimization, the following nucleic acid sequence was obtained:

[0527]

[0528] After synthesizing gene and this gene is subcloned into pET30A+ carrier utilizing NdeI and XhoI cloning site, UGT76G1_pET30a+ plasmid is introduced into Escherichia coli R121 (DE3) and Escherichia coli EC100 by electroporation.The cell that obtains is cultivated in culture dish under the situation that kanamycin exists, and selects appropriate bacterium colony, and allows it to grow in liquid LB substratum (Erenmayer flask).Glycerol is added in suspension as cryoprotectant and aliquots of 400 μ L are stored at-20 ℃ and at-80 ℃.

[0529] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGT76G1 plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was continued at 30° C. for 8 h with shaking at 135 rpm.

[0530] The production medium contained 60 g / L overnight rapid expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The medium was stirred at 20°C while taking samples to measure OD and pH. The culture produced significant growth and achieved good OD. After 40 hours, the cells were harvested by centrifugation and frozen to produce a wet cell weight of 12.7 g.

[0531] Lysis was performed by adding Bugbuster master mix (Novagen), and the lysate was recovered by centrifugation and kept frozen. Activity test was performed on the thawed lysate.

[0532] Example 2

[0533] In vitro production of UGT76G1

[0534] Use the S30 T7 high yield protein expression system kit from Promega. 4 μg of UGT76G1_pET30a+ plasmid from Escherichia coli EC100 was mixed with 80 μL of S30 premix plus, and 72 μL of S30T7 extract was added. Nuclease-free water was added to obtain a total volume of 200 μL, and the resulting solution was incubated at 30°C for 2h. 180 μL was used for catalytic test reactions.

[0535] Example 3

[0536] In vitro production of UGT91D2

[0537] NcoI and NdeI restriction sites were added to the original nucleic acid sequence described in Genbank Accession No. ACE87855.1. After codon optimization, the following nucleic acid sequence was obtained:

[0538]

[0539] After synthesizing gene and this gene is subcloned into pET30A+ carrier utilizing NcoI and XhoI cloning site, UGT91D2_pET30a+ plasmid is introduced into Escherichia coli EC100 by electroporation.The cell that obtains is cultivated in the presence of kanamycin, and appropriate bacterium colony is selected and allowed to grow in liquid LB substratum (Erenmayer flask).Glycerol is added in suspension as cryoprotectant and aliquots of 400 μ L are stored at-20 ℃ and at-80 ℃.

[0540] The S30T7 High Yield Protein Expression System Kit from Promega was used for in vitro synthesis of proteins.

[0541] 4 μg of UGT91D2_pET30a+ plasmid was mixed with 80 μL of S30 premix plus, and 72 μL of S30T7 extract was added. Nuclease-free water was added to obtain a total volume of 200 μL, and the resulting solution was incubated at 30°C for 2 h. 5 μL was used for SDS-page analysis, and the remaining 45 μL was used in the catalytic test reaction.

[0542] Example 4

[0543] In vivo production of UGTSL

[0544] The pET30A+ carrier containing the gene corresponding to this enzyme (GI 460409128, Version XP_004249992.1) is introduced into Escherichia coli BL21 (DE3) by heat shock. The cells obtained are cultured in a culture dish in the presence of kanamycin, and appropriate bacterium colonies are selected and grown in liquid LB medium (Erlenmayer flask). Glycerol is added to the suspension as a cryoprotectant and aliquots of 400 μ L are stored at -20 ℃ and -80 ℃.

[0545] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGT plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was performed at 30° C. with shaking at 135 rpm for 8 hours.

[0546] The production medium contains 60g / L overnight fast expression TB medium (Novagen company), 10g / L glycerol and 50mg / L kanamycin. The pre-culture is added to 400mL of this medium and the solution is stirred at 20 ℃, and the sample is taken to measure OD and pH simultaneously. The culture produces significant growth and obtains good OD. After 40 hours, by centrifugal harvesting cells, and it is frozen. Cell wet weight (CWW) is 6.8g.

[0547] Lysis was performed by adding Bugbuster Master Mix (Novagen), and the lysate was recovered by centrifugation and used fresh.

[0548] Example 5

[0549] In vivo production of UGTSL2

[0550] UGTSL2 (GI_460410132 / XP_004250485.1) amino acid sequence:

[0551]

[0552] The pET30A+ vector containing the UGTSL2 gene was introduced into Escherichia coli B121 (DE3) by heat shock. The cells obtained were cultured in a culture dish in the presence of kanamycin, and appropriate bacterium colonies were selected and grown in liquid LB medium (Erlenmayer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μL aliquots were stored at -20 °C and -80 °C.

[0553] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGTSL2 plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was continued at 30° C. for 8 h with shaking at 135 rpm.

[0554] The production medium contains 60 g / L overnight fast expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The pre-culture is added to 200 mL of this medium and the solution is stirred at 20 ° C while taking samples to measure OD and pH. The culture produces significant growth and obtains a good OD. After 40 hours, the cells are harvested by centrifugation, frozen, and a cell wet weight of 6.22 g is obtained.

[0555] Lysis was performed by adding Bugbuster Master Mix (Novagen) to 1.4 g of cells, and the lysate was recovered by centrifugation and used freshly.

[0556] Example 6

[0557] Catalytic reactions with in vivo generated UGT76G1

[0558] The total reaction volume was 5.0 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM stevioside and 500 μL of UGT76G1 thawed lysate. The reaction was run at 135 rpm on an orbital shaker at 30 ° C. For each sample, 460 μL of the reaction mixture was quenched with 40 μL of 2N H2S04 and 420 μL of methanol / water (6 / 4). The sample was immediately centrifuged and maintained at 10 ° C and then analyzed by HPLC (CAD). HPLC indicated that stevioside was almost completely converted to rebaudioside A ( Figure 4 ).

[0559] Example 7

[0560] Preparation and activity of UGT76G1 produced by pET30a+ plasmid and BL21(DE3) expression strain

[0561] The pET30a+_UGT76G1 plasmid was transformed into the BL21DE3) expression strain (Lucigen E. EXPRESS electrocompetent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0562] The storage aliquot is thawed and added to the LBGKP substratum of 30mL.Under 30 ℃ of shaking, this cultivation is carried out for 8h, and it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the kanamycin of 50mg / L of 400mL subsequently.This substratum is stirred at 20 ℃, and sample is taken to measure OD (600nm) and pH simultaneously.After 40 hours, by centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 10.58g.

[0563] 3.24 g of the obtained precipitate were lysed by adding 8.1 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.5 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0564] Example 8

[0565] Preparation and activity of UGT76G1 produced by pET30a+ plasmid and Tuner (DE3) expression strain

[0566] The pET30a+_UGT76G1 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner tm The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin). Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0567] The storage aliquot was thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. This culture was continued for 15 h at 30 ° C with shaking. 4.4 mL of this culture was used to inoculate 200 mL of production medium containing LB. The medium was stirred at 37 ° C until an OD (600 nm) of 0.9 was obtained, followed by the addition of 400 μ L of 100 mM IPTG solution, and the medium was stirred at 30 ° C for 4 hours. The cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 1.38 g.

[0568] The obtained pellet was lysed by adding 4.9 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 2.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0569] Example 9

[0570] Preparation and activity of UGT76G1 produced by pMAL plasmid and BL21 expression strain

[0571] After the synthetic UGT76G1 gene was subcloned into the pMAL plasmid using the Nde1 and Sal1 cloning sites, the pMAL_UGT76G1 plasmid was transformed into a BL21 expression strain (New England Biolabs BL21 competent E. coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a culture dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0572] The storage aliquot was thawed and added to 30 mL of LBGKP medium. The culture was carried out for 8 h at 30 ° C with shaking, and then the culture was used to inoculate 400 mL of production medium containing 60 g / L of "Overnight Rapid Expression TB Medium" (Novagen, reference 71491-5), 10 g / L of glycerol and 50 mg / L of ampicillin. The medium was stirred at 20 ° C and samples were taken to measure OD and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 5.86 g.

[0573] 2.74 g of the obtained precipitate were lysed by adding 9.6 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0574] Example 10

[0575] Preparation and activity of UGT76G1 produced by pMAL plasmid and ArcticExpress expression strain

[0576] By heat shock treatment, pMAL_UGT76G1 plasmid is transformed into ArticExpress expression strain (Agilent (Agilent) ArcticExpress competent cells). The cells obtained are grown on LB agar medium in a culture dish in the presence of ampicillin and geneticin. Select suitable bacterium colonies, and grow them in the liquid LBGKP medium containing ampicillin and geneticin. Add glycerol and store 400 μ L of aliquots at -20 ℃ and at -80 ℃.

[0577] The storage aliquot is thawed and added to 30mL of LBGKP culture medium (containing ampicillin and geneticin). Under 30 ℃ of shaking, the culture is carried out for 8h, and then used to inoculate 400mL of the production culture medium containing "overnight rapid expression TB culture medium" (Novagen company, reference 71491-5) of 60g / L, 10g / L of glycerol and 50mg / L of ampicillin. The culture medium is stirred at 12 ℃, and the sample is taken to measure OD (600nm) and pH simultaneously. After 68 hours, the cells are harvested by centrifugation, and they are frozen. The wet weight of the cells obtained is 8.96g.

[0578] 2.47 g of the obtained precipitate were lysed by adding 8.73 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.79 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0579] Example 11

[0580] Preparation and activity of UGT76G1 produced by pCOLDIII plasmid and ArcticExpress expression strain

[0581] After using Nde1 and Xhol cloning sites to subclone the synthetic UGT76G1 gene into pCOLDIII plasmid, pCOLDIII_UGT76G1 plasmid was transformed into ArcticExpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The cells obtained were grown on LB agar medium in a culture dish under the presence of ampicillin and geneticin. Select suitable bacterium colonies, and grow them in the liquid LBGKP medium containing ampicillin and geneticin. Add glycerol and store 400 μ L of aliquots at -20 ℃ and at -80 ℃.

[0582] The storage aliquot is thawed and added in the LBGKP substratum (containing ampicillin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out for 8h, and it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the kanamycin of 50mg / L of 400mL subsequently.This substratum is stirred at 12 ℃, and sample is taken and measured OD (600nm) and pH simultaneously.After 63 hours, by centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 6.54g.

[0583] 2.81 g of the obtained precipitate were lysed by adding 9.8 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.2 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0584] Example 12

[0585] Preparation and activity of UGT76G1 produced by pCOLDIII plasmid and Origami2(DE3) expression strain

[0586] The pCOLDIII_UGT76G1 plasmid was transformed into the Origami2 (DE3) expression strain (Novagen Origami IM 2 (DE3) competent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0587] The storage aliquot was thawed and added to 30mL of LBGKP culture medium (containing ampicillin). The culture was carried out for 8h at 30°C of shaking, and then used to inoculate 400mL of the production medium of "overnight rapid expression TB culture medium" (Novagen, reference 71491-5) containing 60g / L, 10g / L of glycerol and 50mg / L of kanamycin. The culture medium was stirred at 12°C, and samples were taken to measure OD (600nm) and pH simultaneously. After 68 hours, cells were harvested by centrifugation, and were frozen. The wet weight of the cells obtained was 2.53g.

[0588] 1.71 g of the obtained precipitate were lysed by adding 6.0 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 1.9 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0589] Example 13

[0590] Preparation of UGT91D2 using pMAL plasmid and BL21 expression strain

[0591] After the synthetic UGT91D2 gene was subcloned into the pMAL plasmid using the Nde1 and Sa]1 cloning sites, the pMAL_UGT91D2 plasmid was transformed into a BL21 expression strain (New England Biolabs BL21 competent Escherichia coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a culture dish in the presence of ampicillin. Suitable bacterium colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and aliquots of 400 μL were stored at 20°C and 80°C.

[0592] The storage aliquot was thawed and added to 30 mL of LBGKP culture medium. The culture was carried out for 8 h at 30 ° C with shaking, and then used to inoculate 400 mL of a production medium containing 60 g / L of "Overnight Rapid Expression TB Culture Medium" (Novagen, reference 71491-5), 10 g / L of glycerol and 50 mg / L of ampicillin. The culture medium was stirred at 20 ° C, and samples were taken to measure OD and pH at the same time. After 40 hours, the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 12.32 g.

[0593] 2.18 g of the obtained precipitate were lysed by adding 7.7 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.2 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0594] Example 14

[0595] Preparation of UGT91D2 using pMAL plasmid and ArcticExpress expression strain

[0596] By heat shock treatment, pMAL_UGT91D2 plasmid is transformed into ArcticExpress expression strain (Agilent ArcticExpress competent cells).The cells obtained are grown on LB agar medium in a culture dish in the presence of ampicillin and geneticin.Select suitable bacterium colonies, and grow them in the liquid LBGKP medium containing ampicillin and geneticin.Add glycerol and store 400 μ L of aliquots at -20 ℃ and at -80 ℃.

[0597] The storage aliquot is thawed and added in the LBGKP substratum (containing ampicillin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out 8h, subsequently it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the ampicillin of 50mg / L of 400mL.This substratum was stirred 16 hours at 20 ℃, then stirred 50 hours in addition at 12 ℃, sampled and measured OD (600nm) and pH simultaneously.By centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 15.77g.

[0598] 2.57 g of the obtained precipitate were lysed by adding 9.0 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.8 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0599] Example 15

[0600] Preparation of UGT91D2 using pET30a+ plasmid and Tuner (DE3) expression strain

[0601] The pET30a+_UGT91D2 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner tm The cells were grown on LB agar medium in a culture dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium (containing kanamycin). Glycerol was added and aliquots of 400 μL were stored at -20°C and -80°C.

[0602] The storage aliquot was thawed and added to 100 mL of LB culture medium containing 50 mg / L kanamycin. This culture was continued for 15 h at 30 ° C with shaking. 6.2 mL of this culture was used to inoculate 500 mL of production culture medium containing LB. The culture medium was stirred at 37 ° C until an OD (600 nm) of 0.9 was obtained, followed by the addition of 500 μ L of 100 mM IPTG solution (the IPTG concentration in the culture medium was 100 μ M), and the culture medium was stirred at 30 ° C for 4 hours, harvested by centrifugation, and frozen. The wet weight of the cells obtained was 4.02 g.

[0603] 1.92 g of the obtained precipitate were lysed by adding 6.8 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 2.8 mL of water. The lysate was recovered by centrifugation and directly subjected to the activity test.

[0604] Example 16

[0605] Preparation of UGT91D2 using pET30a+ plasmid and ArcticExpress expression strain

[0606] By heat shock treatment, pET30a+_UGT91D2 plasmid is transformed into ArcticExpress (DE3) expression strain (Agilent ArcticExpress competent cells).The cell obtained is grown on LB agar medium in culture dish in the presence of kanamycin and geneticin.Select suitable bacterium colony, and make it grow in the liquid LBGKP medium containing kanamycin and geneticin.Add glycerol and the aliquots of 400 μ L are stored at 20 ℃ and at 80 ℃.

[0607] The storage aliquot is thawed and added in the LBGKP substratum (containing kanamycin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out for 8h, subsequently it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the ampicillin of 50mg / L of 400mL.This substratum is stirred 16 hours at 20 ℃.Then at 12 ℃ again 50 hours.Simultaneously sample is taken to measure OD (600nm) and pH.After 60 hours, by centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 16.07g.

[0608] 3.24 g of the precipitate obtained were lysed by adding 11.4 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.8 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0609] Example 17

[0610] Activity assay of in vivo preparations of UGT91D2

[0611] Activity testing for the conversion of rubusoside to stevioside was performed on a 5 mL scale using 1000 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl in 50 mM sodium phosphate buffer at pH 7.2. Samples were obtained and analyzed by HPLC. The results of different preparations of UGT91D2 are summarized in the table below.

[0612]

[0613] *Note: Activity is quoted per mL of lysate. 1 U is equivalent to 1 μmol of substrate converted in 1 hour at 30°C and pH 7.2.

[0614] Example 18

[0615] Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M

[0616] Starting from the amino acid sequence of UGT76G1 as described in Genbank (AAR06912.1), different mutations at various amino acid positions were identified that could alter the activity of the enzyme for the conversion of rebaudioside D (Reb D) to rebaudioside M (Reb M). TM The list of the sudden change of strategic design is used for synthesizing 96 variant genes subsequently, and these variant genes contain 3, 4 or 5 in these sudden changes of codon optimization for expression in intestinal bacteria.These genes are subcloned in pET30a+ plasmid and are used for transforming intestinal bacteria BL21 (DE3) chemical competent cells.The cell that obtains is grown on solid LB agar medium in culture dish under the existence of kanamycin.Select suitable bacterium colony, and make it grow in the liquid LB medium in test tube.Glycerol is added in suspension as cryoprotectant and the aliquot of 400 μ L is stored at-20 ℃ and at-80 ℃.

[0617] These stock aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76G1var plasmid were thawed and added to LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out in a 96 microtiter plate at 135 rpm at 30° C. with shaking for 8 h.

[0618] 50 μL of the above culture was used to inoculate 60 g / L overnight rapid expression TB medium (Overnight Express TM Instant TB medium) , 10g / L glycerol and 50mg / L kanamycin 3.95mL production medium. Stir the resulting culture in a 48-well plate at 20°C. The culture produced significant growth and obtained a good OD (600nm; 1cm). After 44 hours, the cells were harvested by centrifugation and frozen.

[0619] By adding Master Mix Lysis was performed and the lysate was recovered by centrifugation. Activity was tested with 100 μL of fresh lysate added to a solution of rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-glucose (final concentration 2.5 mM) in 50 mM phosphate buffer (pH 7.2).

[0620] The reaction was allowed to proceed at 30°C and samples were taken after 2, 4, 7 and 24 hours to determine the conversion and initial rate by HPLC (CAD detection) using the analytical method described above for the conversion of rebaudioside D to rebaudioside M. The results are depicted in the table below.

[0621]

[0622]

[0623]

[0624]

[0625] *Mutations are annotated as follows: original amino acid-position-new amino acid: for example, alanine to glycine mutation at position 33 is annotated as A33G.

[0626] Example 19

[0627] In vivo production of UGT76G1 in Saccharomyces cerevisiae

[0628] UGT76G1[Stevia](gi_37993653 / gb_AAR06912.1)

[0629]

[0630] The above amino acid sequence was codon optimized for expression in Saccharomyces cerevisiae. In addition, the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned into the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_UGT76G1_Sc vector was used to transform chemically competent Saccharomyces cerevisiae INVScl cells (Invitrogen).

[0631] Cells were grown on solid synthetic minimal medium containing 2% glucose lacking uracil, and single colonies were picked and grown on liquid synthetic minimal medium lacking uracil (SC-U containing 2% glucose). After centrifugation, cells were suspended with SC-U (containing 2% glucose) and 60% glycerol / water. Aliquots were stored at -80°C, and one aliquot was used to start culturing in SC-U (containing 2% glucose) for 43 hours at 30°C. A portion of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19 hours at 30°C.

[0632] Cells were harvested by centrifugation and washed with five volumes of CelLytic TM Y cell lysis reagent (Sigma) was used for lysis. The lysate was directly used for activity test (UGT76G1_Sc).

[0633] Example 20

[0634] Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M (round 2)

[0635] The most active clone from the first round of directed evolution of UGT76G1 (see Example 18, UGT76G1var94 containing mutations Q266E_P272A_R334K_G348P_L379G) was selected as the baseline clone for the second round. A list of 53 mutations was established, which contained different positive mutations from the first round of identification and new mutations obtained by the DNA2.0ProteinGPStm strategy. This mutation list was subsequently used to design 92 variant genes, each of which contained 3 different mutations. After codon optimization for expression in Escherichia coli, these genes were synthesized, subcloned into pET30a+ plasmids and used to transform Escherichia coli BL21 (DE3) chemically competent cells. The cells obtained were grown on solid LB agar medium in a culture dish in the presence of kanamycin. Suitable bacterium colonies were selected and grown in liquid LB medium in a test tube. Glycerol was added to the suspension as a cryoprotectant and aliquots of 400 μL were stored at -20°C and at -80°C.

[0636] These stock aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76G1var plasmid were thawed and added to LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out in a microtiter plate at 30°C with shaking for 8 h.

[0637] 50 μL of the above culture was used to inoculate 60 g / L overnight rapid expression TB medium (Overnight Express TM Instant TB medium) 10g / L glycerol and 3.95mL of the production medium of 50mg / L kanamycin. Stir the obtained culture in 48 deep well plates at 20°C. The culture produces significant growth and obtains good OD (600nm). After 44 hours, the cells are harvested by centrifugation and frozen.

[0638] By adding Master Mix Lysis was performed and the lysate was recovered by centrifugation. Activity was tested with 100 μL of fresh lysate added to a solution of rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-glucose (final concentration 2.5 mM) in 50 mM phosphate buffer (pH 7.2).

[0639] The reaction was carried out at 30°C and samples were taken after 2, 4, 7 and 24 hours to determine the conversion and initial rate using the analytical method for the conversion of rebaudioside D to rebaudioside M described above by HPLC (CAD detection). The experiment was carried out in parallel with the baseline clone round 1-Var94. The conversion rate after 22h and the initial rate for this baseline clone were defined as 100%, and the normalized conversion rate and initial rate for the second round of clones are depicted in the following table:

[0640]

[0641]

[0642]

[0643] *Mutations are annotated as follows: Reference gene - original amino acid - position - new amino acid: For example, for variant 94 from the first round of directed evolution of UGT76G1, the mutation of alanine to glycine at position 33 is annotated as Round 1-Var94(A33G)

[0644] Modeling of these results allowed a ranking of the effect of each mutation to be obtained. The following mutations were determined to be beneficial for activity: S42A, F46I, 1190L, S274G, I295M, K303G, F31S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, S456L.

[0645] Example 21

[0646] Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside X (round 3)

[0647] The most active clone from the second round of directed evolution of UGT76G1 (see Example 20, the second round of mutations S42A_F46I_I407V_UGT76G1var66) was selected as the baseline clone for the third round of directed evolution. A list of 56 mutations was established, which contained different positive mutations from the second round of identification and 30 new mutations obtained by the DNA2.0ProteinGPStm strategy. This mutation list was subsequently used to design 92 variant genes, each of which contained 3 or 4 different mutations. After codon optimization for expression in Escherichia coli, these genes were synthesized, subcloned into pET30a+ plasmids and used to transform Escherichia coli BL21 (DE3) chemically competent cells. The cells obtained were grown on solid LB agar medium in a culture dish in the presence of kanamycin. Suitable bacterium colonies were selected and grown in liquid LB medium in a test tube. Glycerol was added to the suspension as a cryoprotectant and aliquots of 400 μL were stored at -20°C and at -80°C.

[0648] These stock aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76G1var plasmid were thawed and added to LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out in a 96 microtiter plate at 30°C with shaking for 8 h.

[0649] 50 μL of the above culture was used to inoculate 60 g / L overnight rapid expression TB medium (Overnight Express TM Instant TB medium) 10g / L glycerol and 3.95mL of the production medium of 50mg / L kanamycin. Stir the obtained culture in 48 deep well plates at 20°C. The culture produces significant growth and obtains good OD (600nm). After 44 hours, the cells are harvested by centrifugation and frozen.

[0650] By adding Master Mix Lysis was performed and the lysate was recovered by centrifugation. Activity was tested with 100 μL of fresh lysate added to a solution of rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-glucose (final concentration 2.5 mM) in 50 mM phosphate buffer (pH 7.2).

[0651] The reaction was carried out at 30°C and samples were taken after 1, 2, 4, 6 and 22 hours to determine the conversion and initial rate using the analytical method for the conversion of rebaudioside D to rebaudioside M described above by HPLC (CAD detection). The experiment was carried out in parallel with the baseline clone, round 2 - Var66. The conversion rate after 22h and the initial rate for this baseline clone were defined as 100%, and the normalized conversion rate and initial rate for the 3rd round clone are depicted in the table below:

[0652]

[0653]

[0654]

[0655] *Mutations are annotated as follows: Reference gene - original amino acid - position - new amino acid: For example, for variant 66 from the second round of directed evolution of UGT76G1, the mutation of isoleucine to leucine at position 190 is annotated as Round 2-Var66(I190L)

[0656] Modeling of these results allowed a ranking of the effect of each mutation to be obtained. The following mutations were identified as beneficial for activity:

[0657] I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R, K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P, V264C, E449D, A352G.

[0658] Example 22

[0659] Conversion of rebaudioside A to rebaudioside I using UGT76G1

[0660] Reactions using UGT76G1-R1-F12 (also known as UGT76G1var94)

[0661] The total reaction volume was 40 mL with the following composition: 50 mM potassium phosphate buffer pH 7.5, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM rebaudioside A and 4 mL UGT76G1-R1-F12 lysate (2.5 U / mL). The reaction was run at 135 rpm on an orbital shaker at 30 °C. For sampling, 125 μL of the reaction mixture was quenched with 10 μL of 2N H2SO4 and 115 μL of methanol / water (7 / 3). The sample was centrifuged immediately and maintained at 10 °C before analysis by LC-MS. An Agilent 1200 series HPLC system equipped with a binary pump (G1312B), autosampler (G1367D), thermostatted column compartment (G1316B), DAD detector (G1315C) connected to an Agilent 6110A MSD and interfaced with "LC / MSD Chemstation" software was used.

[0662] Instrument conditions

[0663]

[0664] Mobile phase gradient program

[0665] Time (min) A(%): Formic acid 0.1% B(%): acetonitrile 0 76 24 8.5 76 24 10.0 71 29

[0666] 16.5 70 30

[0667] The response characteristic curve is shown in Figure 5 middle.

[0668] After 42 h of reaction, 20 mL of the reaction mixture was quenched with 20 mL of ethanol and used for structure elucidation.

[0669] In a similar manner, the best clones from UGT76G1 directed evolution rounds 1 (UGT76G1-R1-F12), 2 (UGT76G1-R2-B9 identified above as "Round 2-Var66"), and 3 (UGT76G1-R3-G3 identified above as "Round 3-Var21") and native UGT76G1 were tested for conversion of rebaudioside A to rebaudioside I. Results Figure 5 Shown in.

[0670] Example 23

[0671] Isolation and characterization of rebaudioside I

[0672] Crude reaction sample. A sample of batch number crude CB-2977-198 was prepared for isolation using UGT76G1 according to Example 22.

[0673] HPLC analysis. Preliminary HPLC analysis of the samples was performed using a Waters 2695 Alliance system according to the following method: Phenomenex Synergi Hydro-RP, 4.6×250 mm, 4 μm (p / n 00G-4375-E0); column temperature: 55° C.; mobile phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; mobile phase B: acetonitrile (MeCN); flow rate: 1.0 mL / min; injection volume: 10 μL. Detection was performed by UV (210 nm) and CAD.

[0674] gradient:

[0675] Time (min) ％A ％B 0.0-8.5 75 25 10.0 71 29 16.5 70 30 18.5-24.5 66 34 26.529.0 48 52 31-37 30 70 38 75 25

[0676] Separated by HPLC. Purification was performed using a Waters Atlantis dC18 (30×100 mm, 5 μm, p / n 186001375) column with an isocratic mobile phase of 80:20 water / MeCN. The flow rate was maintained at 45 mL / min and the injection load was 180 mg. The detector wavelength was set to 210 nm.

[0677] Fraction analysis was performed using: Waters Atlantis dC18 (4.6 x 150 mm, 5 μm, p / n 186001342) column; mobile phase A: water; mobile phase B: MeCN; flow rate: 1 mL / min; isocratic mobile phase conditions: 75:25 A / B for 30 minutes.

[0678] MS and MS / MS. MS and MS / MS data were generated using a Waters QT Micro mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. The samples were diluted to a concentration of 0.25 mg / mL with H2O:MeCN (1:1) and introduced by flow injection for MS data acquisition. The samples were further diluted to 0.01 mg / mL to produce a good s / n to tune for MS / MS and acquired by direct infusion. The collision energy was set to 60 V in order to obtain MS / MS data with increased fragment ion peaks due to the nature of the molecule.

[0679] NMR.Samples were prepared by dissolving approximately 1.0 mg in 180 μL of pyridine-d5 + TMS and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm trans probe or a 5 mm broadband probe. 13C and HMBC NMR data were acquired at Rensselaer Polytechnic Institute using their Bruker Avance 600 MHz and 800 MHz instruments, respectively, with a 5 mm cryo-probe. 1 H and 13 C NMR spectra referenced to TMS resonance (δ H 0.00ppm and δ C 0.0ppm).

[0680] Isolation of Reb I was performed using a semi-synthetic steviol glycoside mixture (lot number CB-2977-198). The material was analyzed by HPLC as described above. Figure 6 As shown, the Reb I peak was observed at a retention time (tR) of about 17 minutes.

[0681] Results and discussion

[0682] The reb I peak was isolated from the reaction crude as described above. The isolated fractions were combined and lyophilized. The purity of the final product was 91%, as confirmed by LC-CAD using the above method. Approximately 1 mg of reb I was provided for spectrophotometric and spectrometric analysis.

[0683] Mass spectrometry. The ESI-TOF mass spectrum obtained by perfusing a sample of reb I showed an [MH] ion at m / z 1127.4741. The mass of the [MH] ion was consistent with the expected molecular formula C for reb I. 50 H 79 O 28 (For C 50 H 79 O 28 Calculated: 1127.4758, error: -1.5 ppm) is in good agreement. MS data confirmed that reb I has a nominal mass of 1128 Daltons consistent with the molecular formula C 50 H 80 O 28 .

[0684] The MS / MS spectrum of reb I ([MH] ion at m / z 1127.4 was selected for fragmentation) indicated the loss of two sugar units at m / z 803.5301, but showed no additional fragmentation using a collision energy of 30 V. When a higher collision energy (60 V) was applied, no parent ion was observed, but the sequential loss of three sugar units at m / z 641.4488, 479.3897, and 317.3023 was observed from m / z 803.5301.

[0685] NMR spectroscopy. A series of NMR experiments were performed, including 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HSQC-DEPT, HMBC NOESY, and 1D TOCSY were performed to allow the assignment of reb I.

[0686] The reb I obtained at 300K 1 In the H NMR spectrum, one of the anomeric protons is completely obscured by the water resonance. Therefore, the H NMR spectrum of the sample was obtained at a lower temperature (292 K). 1 The H NMR spectrum was obtained at 292 K to remove the water resonance and to fully resolve the anomeric protons at this temperature. Therefore, all other NMR data for reb I were obtained at 292 K.

[0687] 1D and 2D NMR data indicate that the central core of the glycoside is a diterpene. H The methyl proton at 1.22 to the C The HMBC correlation of the carbonyl at 176.9 allowed the assignment of the tertiary methyl group (C-18) as well as one of the C-19s and provided a starting point for the assignment of the remaining aglycones. C Additional HMBC correlations of carbons at 38.5, 44.0, and 57.2 allowed assignment of C-3, C-4, and C-5. 1 H- 13 Analysis of C HSQC-DEPT data shows that C The carbon at 38.5 is a methylene group and at δ C The carbons at 57.2 are methines, which are designated C-3 and C-5, respectively. This leaves C The carbon at 44.0, which showed no correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. The HSQC-DEPT data were used to assign H 1.02 and 2.35) and C-5 (δ H 1.03) 1H chemical shift. One of the H-3 protons (δ H 1.02) and in δ H The COSY correlation between the protons at 1.44 allowed the assignment of one of the H-2 protons, which in turn showed a similarity to that assigned to H-1 at δ H Then, based on additional COSY and HSQC-DEPT correlations, the remaining 1 H and 13 C chemical shifts are assigned and summarized in the table below.

[0688] 1 H and 13 C NMR (500 and 150 MHz, pyridine-d5), assignment of rebaudioside I aglycone

[0689]

[0690]

[0691] In δ H The other tertiary methyl singlet observed at 1.26 showed HMBC correlations with C-1 and C-5 and was assigned to H-20. The methyl proton showed correlations with the quaternary carbons (δ C 39.8) and methine carbon (δ C 54.1) of the other HMBC association. Then, at H-5 (δ H 1.03) and in δ H COSY correlations between protons at 1.90 and 2.33 allowed assignment of the H-6 proton, which in turn showed a strong affinity to the proton at δ H The correlation of the protons at 1.29 and 1.31 was then determined from the HSQC-DEPT data for C-6 (δ C 22.2) and C-7 (δ C 41.7) 13 C chemical shift. H 0.88) and in δ H The COSY correlation between the protons at 1.67 and 1.70 allowed the assignment of the H-11 proton, which in turn showed a correlation with the proton assigned to H-12 at δ H COSY correlations for protons at 1.98 and 2.28. HSQC-DEPT data were then used to assign C-11 (δ C 20.5) and C-12 (δ C 37.3). HThe olefinic protons observed at 5.02 and 5.67 show similarities to C-13 (6 C 86.7) and was therefore assigned as H-17 (δ C 104.8). The methine proton H-9 shows delta with the protons assigned to C-8, C-14 and C-15, respectively. C HMBC correlations for carbons at 42.3, 44.3, and 47.6. Using HSQC-DEPT data, the carbons at C-14 (δ H 1.78 and 2.59) and C-15 (δ H 2.04) 1 H chemical shift. Additional HMBC correlations from H-9 to C-11 and H-12 to C-9 further confirmed the assignments made above. C HMBC correlation of the quaternary carbon at 154.0 allowed the assignment of C-16 to complete the assignment of the central core.

[0692] The relative stereochemistry of central diterpene core is specified using the association observed in the NOESY spectrum. In this NOESY spectrum, the NOE association observed between H-14 and H-20 shows that H-14 and H-20 are on the same face of ring. Similarly, NOE association is observed between H-9 and H-5 and H-5 and H-18. NOE association is not observed between H-9 and H-14. Therefore, NOESY data show that, as shown below, compared with H-14 and H-20, H-5, H-9 and H-18 are on the opposite face of ring. Therefore these data show that the relative stereochemistry in the central core is retained during the glycosylation step.

[0693] For reb I, 1 H- 13 Analysis of C HSQC-DEPT data confirmed the presence of five anomeric protons. H 6.14(δ C 95.3), 5.57(δ C 104.6), 5.38(δ C 104.7), 5.29(δ C 105.0), and 5.06(δ C All five anomeric protons were resolved in the spectrum obtained at 98.0). In addition, all five anomeric protons had large couplings (7.7 Hz-8.2 Hz), indicating that they have β-configuration. H The anomeric proton observed at 6.14 showed an HMBC correlation with C-19, indicating that it corresponds to Glc ISimilarly, in δ H The anomeric proton observed at 5.06 showed an HMBC correlation with C-13, allowing it to be assigned as Glc II terminal protons.

[0694] Glc I The terminal proton (δ H 6.14) shows that H The one at 4.18 is designated as Glc I COSY correlation of the protons of H-2. The COSY spectrum did not allow assignment of H-3 or H-4 due to data overlap. Therefore, Glc I A series of 1D TOCSY experiments were performed with several different mixing times using selective irradiation of the anomeric protons. I In addition to the assignment of H-2, TOCSY data showed that H The protons at 4.27, 4.25, and 3.93 were assigned to H-3, H-4, and H-5, respectively. H The proton observed at 4.37 was assigned to Glc I One of the H-6 protons. δ H The other H-6 methylene proton at 4.27 is based on the protons from H-5 to δ H The COSY correlation at 4.27 was assigned. Using HSQC-DEPT data, the Glc I C-2(δ C 72.5), C-3(δ C 89.4), C-4(δ C 69.2), C-5(δ C 78.2-78.8), and C-6 (δ C 61.7) 13 C chemical shift. HMBC correlations at H-1 to C-3 and H-4 to C-6 further confirmed the assignments made above, completing the Glc I Designation.

[0695] Of the four remaining unassigned glucose moieties, one was assigned as Glc based on HMBC correlation I The substituent at C-3 of H The anomeric proton observed at 5.29 shows similarity to Glc I C-3 was HMBC correlated and assigned as the anomeric proton of Glcv. I Reverse HMBC association of H-3 to the anomeric carbon of Glcv.

[0696] Regarding the glycoside at C-19 1 H and 13 A summary of the C chemical shifts is shown in the following table:

[0697] 1 H and 13 C NMR (500 and 150 MHz, pyridine-d5), assignment of rebaudioside I C-19 glycoside.

[0698]

[0699] Five carbon resonances in the range of 78.2-78.8 (78.16, 78.47, 78.50, 78.55 and 78.77) therefore could not be assigned unambiguously to chemical shifts.

[0700] A summary of the key HMBC and COSY correlations used to assign the C-19 glycosidic region is provided below:

[0701]

[0702] 1 H- 13 C HMBC association

[0703] 1 H- 1 H COSY association

[0704] Glcv(δ H 5.29) shows the same anomeric proton as in δ H The one at 4.04 is designated as Glc V COSY correlation of the H-2 protons. Then HSQC-DEPT data were used to assign Glc V C-2(δ C 75.3 or 75.5). Due to data overlap, the COSY spectrum does not allow assignment of the remaining protons. Therefore, a series of 1D TOCSY experiments were performed with several different mixing times using selective irradiation of the Glcv terminal protons. In addition to confirming the assignment of Glcv H-2, the TOCSY data allowed assignment of Glc V H-3(δ H 4.27), H-4(δ H 4.12), and H-5 (δ H 4.05). In the TOCSY spectrum, H The proton observed at 4.56 was assigned as one of the Glcv H-6 protons. HThe other H-6 methylene proton at 4.26 is based on the H-5 to δ H The HSQC-DEPT data were used to assign the Glcv C-3 (δ C 78.2-78.6), C-4(δ C 71.5 or 71.6), C-5(δ C 78.5 or 78.6) and C-6 (δ C 62.3 or 62.4) 13 C chemical shift, thus completing the assignment of G1cv.

[0705] In a similar manner, Glc II Designation of Glc II Anomeric proton (δ H 5.06) shows and is identified as Glc II H-2 in δ H The COSY correlation of the proton at 4.34 and furthermore the COSY correlation of the proton at δ H 4.20(Glc II COSY correlation of the protons at H-3) shows the H 3.97(Glc II H-4), which also shows an additional correlation with the proton at δ H 3.80(Glc II H-5) shows COSY correlation with the proton at δ designated as H-6. H Additional COSY correlations for protons at 4.18 and 4.49. Therefore, Glc II Selective irradiation of the anomeric protons A series of 1D TOCSY experiments were performed with several different mixing times. The TOCSY data confirmed the above proton assignments. II C-2(δ C 80.2), C-3(δ C 87.5), C-4(δ C 70.1), C-5(δ C 77.6), and C-6 (δ C 62.5) 13 The assignment of C chemical shifts is based on HSQC-DEPT data. II H-3 to C-2 and C-4 and also from Glc II HMBC correlations of H-4 to C-3, C-5, and C-6 confirmed the assignments made above, completing the Glc II Designation.

[0706] Based on HMBC correlation, the two remaining unassigned glucose moieties were assigned to be in the Glc II Substituents at C-2 and C-3. H The anomeric proton observed at 5.57 shows similarity to Glc II C-2 was HMBC associated and was designated as Glc III The terminal proton of H The anomeric proton observed at 5.38 shows similarity to Glc II C-3 was HMBC associated and designated as Glc IV It was also observed that the anomeric proton from Glc II H-2 to Glc III The terminal carbon and the II H-3 to Glc IV The mutual HMBC correlation of the terminal carbons.

[0707] Glc III (δ H 5.57) shows the same anomeric proton as in δ H The one at 4.21 is designated as Glc III COSY correlation of the H-2 protons. Then HSQC-DEPT data were used to assign Glc III C-2(δ C 76.3). Due to data overlap, the COSY spectrum did not allow assignment of the remaining protons. Therefore, Glc III Selective irradiation of the anomeric protons was performed in a series of 1D TOCSY experiments with several different mixing times. In addition to confirming the assignment of GlcIII H-2, the TOCSY data allowed the assignment of Glc III H-3(δ H 4.27), H-4(δ H 4.25) and H-5(δ H 3.94). In the TOCSY spectrum, H 4.41 and δ H The proton observed at 4.53 was assigned to Glc III The H-6 proton was assigned using HSQC-DEPT data about C-3 (δ C 78.2-78.6), C-4(δ C 72.1), C-5(δ C 78.2-78.8) and C-6 (δ C 63.1) 13 C chemical shift. From H-5 to δ C HMBC correlation of carbon at 63.1 further confirmed that GlcIII The designation of C-6 completes the Glc III Designation.

[0708] Glc IV (δ H 5.38) shows the same anomeric proton as in δ H The one at 4.01 is designated as Glc IV COSY correlation of the H-2 protons. Then HSQC-DEPT data were used to assign Glc IV C-2(δ C 75.3 or 75.5). Due to data overlap, the COSY spectrum did not allow assignment of the remaining protons. Therefore, Glc IV Selective irradiation of the anomeric protons was performed in a series of 1D TOCSY experiments with several different mixing times. IV In addition to the assignment of H-2, 1D TOCSY data allow the assignment of H-3 (δ H 4.28), H-4(δ H 4.11), H-5(δ H 4.13) and H-6(δ H 4.25 and 4.58). H The proton at 4.25 also shows a relationship with δ H The COSY correlation at 4.58 further confirmed that these protons belonged to H-6. Using HSQC-DEPT data, the protons on C-3 (δ C 78.2-78.6), C-4(δ C 72.1), C-5(δ C 78.2-78.6) and C-6 (δ C 62.3 or 62.4) 13 C chemical shift. HMBC correlations from H-4 to C-6 and H-5 to C-1 further confirmed that Glc IV The designation of C-6 completes the Glc IV Designation.

[0709] Regarding the glycoside at C-13 1 H and 13 A summary of the C chemical shifts is shown below:

[0710] 1 H and 13 C NMR (500 and 150 MHz, pyridine-d5), assignment of rebaudioside I C-13 glycoside.

[0711]

[0712] Five carbon resonances in the range of 78.2-78.8 (78.16, 78.47, 78.50, 78.55 and 78.77) therefore could not be assigned unambiguously to chemical shifts.

[0713] A summary of the key HMBC and COSY correlations used to assign the C-13 glycosidic region is provided below:

[0714]

[0715] 1 H- 13 C HMBC association

[0716] 1 H- 1 H COSY association

[0717] NMR and MS analysis of rebaudioside I allowed the structure to be fully assigned as (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl)oxy]ent-kaur-16-ene-19-oic acid (3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl) ester].

[0718] Example 24

[0719] Sensory characteristics of rebaudioside I

[0720] Rebaudioside I was evaluated to determine its sensory characteristics, including comparison with Rebaudioside M.

[0721] Sample preparation The samples were prepared at a concentration of 400 ppm in a water matrix. Analysis was performed to determine the actual concentration of the samples.

[0722] method

[0723] Seven panelists participated in the testing of Rebaudioside I and Rebaudioside M. Samples were provided at approximately 4°C. Panelists were instructed to drink 1 sip of sample (10 mL), hold it in the mouth for 5 seconds, spit it out and evaluate the taste attributes as described below. A 5-minute break was set between each sample, and panelists were instructed to rinse their palates with at least 1 sip of unsalted crackers and 2 sips of filtered water. Samples were randomized and each sample was present in duplicate during this period.

[0724] The attributes evaluated included: sweetness intensity (maximum sweetness level in the mouth over the course of 5 seconds); bitterness intensity (maximum bitterness level in the mouth over the course of 5 seconds); overall maximum sweetness intensity (maximum sweetness intensity experienced from a sip to 1 minute); overall maximum bitterness intensity (maximum bitterness intensity experienced from a sip to 1 minute); other intensity (intensity of any taste, aroma, or mouthfeel other than sweetness and bitter (e.g., metallic, plastic, licorice, etc.)); sweetness lingering intensity (sweetness intensity 1 minute after spitting out the sample); and bitter aftertaste intensity (bitterness intensity 1 minute after spitting out the sample).

[0725] Sweeteners were compared for each attribute using 3-way ANOVA analysis and significance was determined at 95% CL. Fishers LSD was used to compare significant differences between mean scores.

[0726] result

[0727] The sweetness intensity, overall maximum sweetness intensity and sweetness lingering intensity of rebaudioside I were lower.

[0728] Table 1: Sensory attributes of Rebaudioside I compared to Rebaudioside M at 400 ppm

[0729]

[0730] Example 25: Beverage Preparation

[0731] Flavored black tea : The taste profile of a flavored zero calorie black tea beverage containing Reb A at a concentration of 275 ppm was compared to a comparable flavored zero calorie black tea beverage having a concentration of 275 ppm Reb I. The beverage containing Reb I was determined to have a fresher aftertaste, with less sweetness lingering and a fuller overall sweetness profile.

[0732] Enhanced Water : The taste profile of a zero calorie enhanced water beverage containing Reb A at a concentration of 200 ppm was compared to a comparable zero calorie enhanced water beverage containing Reb I at a concentration of 200 ppm. The beverage containing Reb I had a fresher aftertaste and had a reduced sweetness linger and a fuller overall sweet taste quality.

[0733] Lemon-flavored sparkling drink : In zero calorie Sour Lemon Reb I levels were evaluated in flavored sparkling beverage bases to determine the effect of increasing sweetness. Sour LemonSamples of flavored sparkling beverages with Reb I levels between 400 and 750 ppm (in 50 ppm increments) and psicose at 3.5%. All samples tasted significantly better than comparable formulations containing Reb A, resulting in a fresher profile with increased sweetness intensity and no negative aftertaste characteristics. Samples with 550 ppm and 600 ppm Reb I were found to be comparable in sweetness level to a 10.0 Brix HFCS sweetened ... Sour Lemon Flavored sparkling beverage preparations come closest.

Claims

1. A method for enhancing the flavor of a consumable product, wherein the method comprises: 1) Prepare the rebaudioside I composition by the following steps: contacting a starting composition comprising rebaudioside A with a biocatalyst capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I; wherein the biocatalyst is UGT, wherein UGT is an abbreviation for UDP-glycosyltransferase; wherein the UGT is a UGT76G1 variant; and wherein the UGT76G1 variant is selected from: UGT76G1-R1-F12, where the mutations in UGT76G1-R1-F12 are: Q266E, P272A, R334K, G348P, and L379G; UGT76G1-R2-B9, which is UGT76G1-R1-F12 with additional mutations S42A, F46I, and I407V; and UGT76G1-R3-G3, which is UGT76G1-R2-B9 with additional mutation points I46L, K303G, and K393R; 2) Adding the Rebaudioside I composition to the consumer product.

2. A method for enhancing the sweetness of a consumable product, wherein the method comprises: 1) Prepare the rebaudioside I composition by the following steps: contacting a starting composition comprising rebaudioside A with a biocatalyst capable of converting rebaudioside A to rebaudioside I to provide a composition comprising rebaudioside I; wherein the biocatalyst is UGT, wherein UGT is an abbreviation for UDP-glycosyltransferase; wherein the UGT is a UGT76G1 variant; and wherein the UGT76G1 variant is selected from: UGT76G1-R1-F12, where the mutations in UGT76G1-R1-F12 are: Q266E, P272A, R334K, G348P, and L379G; UGT76G1-R2-B9, which is UGT76G1-R1-F12 with additional mutations S42A, F46I, and I407V; and UGT76G1-R3-G3, which is UGT76G1-R2-B9 with additional mutation points I46L, K303G, and K393R; 2) Adding the Rebaudioside I composition to the consumer product.

3. The method of claim 1 or 2, wherein the UGT is provided in a form selected from a pure form, a crude lysate or a whole cell suspension.

4. The method of claim 1 or 2, wherein the UGT is provided by a microorganism.

5. The method of claim 1 or 2, wherein rebaudioside A is provided in pure form, or in the form of a steviol glycoside mixture or a stevia extract containing at least 50% rebaudioside A by weight.

6. The method of claim 1 or 2, wherein the rebaudioside I composition comprises greater than 1% rebaudioside I by weight.

7. The method of claim 1 or 2, further comprising isolating rebaudioside I to provide an isolated rebaudioside I composition.

8. The method of claim 7, further comprising purifying the isolated rebaudioside I composition to provide highly purified rebaudioside I, comprising greater than 80% rebaudioside I by weight.

9. The method of claim 1, wherein the Rebaudioside I composition comprises Rebaudioside I in an amount effective to provide a concentration below a threshold flavor recognition concentration of Rebaudioside I when the Rebaudioside I composition is added to the consumable.

10. The method of claim 2, wherein the Rebaudioside I composition comprises Rebaudioside I in an amount effective to provide a concentration below a threshold sweetness recognition concentration of Rebaudioside I when the Rebaudioside I composition is added to the consumable.

11. The method of claim 9 or 10, wherein the Rebaudioside I composition further comprises one or more additives selected from the group consisting of: Carbohydrates, polyols, amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, weighting agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers and combinations thereof.

12. The method of claim 9 or 10, wherein the Rebaudioside I composition further comprises a polyamino acid or a corresponding salt thereof.

13. The method of claim 9 or 10, wherein the Rebaudioside I composition further comprises an emulsifier.

14. The method of claim 9 or 10, wherein the Rebaudioside I composition further comprises one or more functional ingredients selected from the group consisting of: Saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, plant sterols, and combinations thereof.

15. The method of claim 1 or 2, wherein the consumer product is selected from the group consisting of: A pharmaceutical composition, an edible gel mixture or composition, a dental composition, a confectionery, a savory composition, a cereal composition, a baked product, a dairy product, a beverage or a beverage product.

16. The method of claim 1 or 2, wherein the consumer product is a chewing gum composition.

17. The method of claim 1 or 2, wherein the consumable is a tabletop sweetener composition.

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