Method for obtaining steviol glycosides

Abstract

The present disclosure provides a method of obtaining steviol glycosides from a stevia substance using an enzyme mixture, where the stevia substance may be an extract, by-product or waste product of a stevia extraction process involving the enzymes beta-1, 2-and beta-1, 3-glycosyltransferase as well as a sucrose synthase. Also provided are steviol glycoside compositions that can be prepared using such methods, as well as the use of the compositions in foods, beverages, and the like.

Description

Technical Field

[0001] This disclosure provides a method for obtaining steviol glycosides from stevia substances using an enzyme mixture, wherein the stevia substances may be extracts, byproducts, or waste products from the stevia extraction process. Also provided are steviol glycoside compositions that can be prepared using such methods, and the use of said compositions in food, beverages, etc. Background Technology

[0002] Steviol glycosides are plant secondary metabolites with desirable natural sweetness. Sweet-tasting steviol glycosides are extracted from stevia (stevia). These glycosides have commercial value and are used as calorie-free sweeteners. Stevia contains various steviol glycosides, but currently only rebaudioside A and steviol glycosides are widely available on the market. However, other glycosides extracted from stevia also have sweet characteristics and commercial value. The extraction of high-purity steviol glycosides is costly and has low yields.

[0003] Therefore, there is a need in the art for methods to obtain steviol glycosides, especially methods to obtain steviol glycosides at particularly low cost, which can lead to an increase in the yield of steviol glycosides.

[0004] Enzymatic conversion of steviol glycosides (such as rebaudioside A and steviol glycoside) to other steviol glycosides (especially rebaudioside M) is known in the art; however, these conversions are usually performed using high-purity starting materials for the purpose of studying specific conversions (see, for example, US2020 / 0032227A1, US2020 / 0157594 A1, WO 2022 / 115527 A1, WO 2023 / 121426A1 and WO 2018 / 144679 A3). Summary of the Invention

[0005] This invention provides a method for obtaining steviol glycosides from stevia substances using an enzyme mixture, wherein the stevia substances may be extracts, byproducts, or waste products from the stevia extraction process. The invention is based in part on the discovery that enzymatic conversion can be efficiently performed on low-quality and / or complex mixtures of starting materials, that enzymatic conversion can be implemented to improve the efficiency of the steviol glycoside extraction process, and that the enzymes can be used for desired and previously unconfirmed conversions of small amounts of steviol glycosides that may be present in unprocessed stevia extracts.

[0006] According to a first aspect of this disclosure, a method for obtaining one or more steviol glycosides is provided, the method comprising the steps of: adding an enzyme mixture to a stevia substance; incubating the enzyme mixture and the stevia substance; and obtaining the one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0007] In one embodiment of the first aspect of this disclosure, the total steviol glycoside content of the stevia substance is ≤90% on a dry weight basis.

[0008] In another embodiment of the first aspect of this disclosure, the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside D.

[0009] In another embodiment of the first aspect of this disclosure, the stevia substance comprises steviol glycosides and / or rhamnosyl-containing steviol glycosides and / or xylose-containing steviol glycosides.

[0010] Typically, the method is performed as part of an extraction process. Typically, the stevia substance is an extract, a byproduct, or a waste product. Therefore, a method for obtaining one or more steviol glycosides is provided, the method comprising the steps of: adding an enzyme mixture to a stevia substance during an extraction process, wherein the stevia substance is an extract, a byproduct, or a waste product; incubating the enzyme mixture and the stevia substance; and obtaining one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0011] In one embodiment, a mixture of one or more steviol glycosides is obtained. Therefore, a method for obtaining a mixture of one or more steviol glycosides is provided, the method comprising the steps of: adding an enzyme mixture to a stevia substance during an extraction process, wherein the stevia substance is an extract, byproduct, or waste product; incubating the enzyme mixture and the stevia substance; and obtaining a mixture of one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0012] In one embodiment, a purified mixture of one or more steviol glycosides is obtained. Therefore, a method for obtaining a purified mixture of one or more steviol glycosides is provided, the method comprising the steps of: adding an enzyme mixture to a stevia substance during an extraction process, wherein the stevia substance is an extract, byproduct, or waste product; incubating the enzyme mixture and the stevia substance; obtaining a mixture of one or more steviol glycosides from the enzyme mixture and the stevia substance; and purifying the mixture of one or more steviol glycosides.

[0013] According to a second aspect of this disclosure, a product comprising one or more steviol glycosides is provided, wherein the product is obtained by or can be obtained by the method of the first aspect of this disclosure. Therefore, a product comprising one or more steviol glycosides is provided, said one or more steviol glycosides being produced by: adding an enzyme mixture to a stevia substance during an extraction process, said stevia substance being an extract, byproduct, or waste product; incubating said enzyme mixture and stevia substance; and obtaining one or more steviol glycosides from said enzyme mixture and stevia substance.

[0014] These and other features, objects, and advantages of this disclosure will be better understood from the following description. In the description, reference is made to the accompanying drawings, which form part of the description, and embodiments of the disclosure are shown therein by way of illustration rather than limitation. The description of preferred embodiments is not intended to limit the disclosure to cover all modifications, equivalents, and alternatives. Therefore, the scope of this disclosure should be interpreted with reference to the claims set forth in this disclosure. Attached Figure Description

[0015] This disclosure will be better understood when considered in the following detailed description, and features, aspects, and advantages in addition to those set forth above will become apparent. This detailed description refers to the following figures.

[0016] Figure 1 A diagram showing the conversion of rebaudioside C to rebaudioside K or rebaudioside N2.

[0017] Figure 2 The LC-MS output of rebaudioside H4 produced from rebaudioside C using sucrase synthase and 1-3β-glycosylation enzyme is shown. The product was eluted between 9 and 10 minutes.

[0018] Figure 3 The LC-MS output shows the production of rebaudioside K from rebaudioside C using sucrase synthase and 1-2β-glycosylation enzyme. The product was eluted between 4 and 5 minutes.

[0019] Figure 4 The LC-MS outputs of rebaudioside N2 generated from rebaudioside C by treatment with sucrase synthase, ADP, sucrose, and 1-2 and 1-3 β-glycosylation enzymes are shown. As shown in the chromatograms at reaction time points 1, 3, and 24 hours, rebaudioside N2 is mainly generated via rebaudioside K. Rebaudioside N2 eluted at approximately 4 minutes, while rebaudioside K initially eluted between 4 and 5 minutes, and the consumption of rebaudioside K and rebaudioside C produced high-purity rebaudioside N2 eluted at 24 hours. The small peak eluted at approximately 4.8 minutes in the 24-hour reaction product is rebaudioside M and steviol glycoside generated by the glycosylation of rebaudioside A and D remaining in the starting material.

[0020] Figures 5A-5C Display using ADP ( Figure 5A ), sucrose synthase ( Figure 5B ) and one or both of sucrose and 1-2 and 1-3 glycosyltransferases ( Figure 5C LC-MS output of stevia extract processed by )

[0021] Figure 6 The starting material eluted at 22.086 minutes is shown.

[0022] Figure 7The LC-MS output of the reacted rebaudioside F is shown. The reacted rebaudioside F produces a peak at 16.785 min, which represents a glycoside with one xylose residue and five glycosyl bonds. The unlabeled peaks at 17.3 and 18.5 min represent minor isomers of this product, and the unlabeled peak at 15.4 min represents the product with one xylose residue and four glucose residues. Detailed Implementation

[0023] For the purpose of facilitating understanding of the principles of this disclosure, reference will now be made to embodiments, which will be described using specific language. However, it will be understood that this is not intended to limit the scope of this disclosure, and changes and further modifications to this disclosure, such as those shown herein, will commonly occur to those skilled in the art to which this disclosure pertains.

[0024] definition

[0025] As used in this specification, the article “a (kind)” means one or more (i.e., at least one) grammatical objects of the article. For example, “element” means at least one element and may include more than one element.

[0026] The term "approximately" is used to provide flexibility to the endpoints of a numerical range by specifying that a given value can be "slightly higher" or "slightly lower" than the endpoints without affecting the desired result. Typically, the term "approximately" in relation to a numerical value means that the value can vary by adding or subtracting 5% or less from that value.

[0027] Throughout this specification, unless the context otherwise requires, the words “comprise” and “include” and their variations (e.g., “comprises / comprising / includes / including”) shall be understood to mean that a specified component, feature, element, or step or a group of components, features, elements, or steps is included, but does not exclude any other whole or step or whole or group of steps.

[0028] As used herein, “and / or” means and covers any and all possible combinations of one or more related listed items, as well as the lack of combinations as explained in the alternative form (“or”).

[0029] Unless otherwise stated herein, the ranges of values ​​described herein are intended merely as a concise way of referring to each individual value falling within that range. Furthermore, each individual value is incorporated into the description as if it were described separately herein. For example, if the stated range is 1 to 50, this disclosure is intended to explicitly list values ​​such as 2 to 4, 10 to 30, or 1 to 3, etc. These are merely specific examples, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​are considered to be explicitly stated in this disclosure.

[0030] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0031] As used herein, the term "stevia" refers to a natural sweetener containing steviol glycosides. As used herein, the term "stevia plant" refers to the plant species *Stevia rebaudiana*. The stevia plant contains steviol glycosides, which are extracted to obtain sweetness.

[0032] As used herein, the term "stevioside" refers to naturally occurring sweet compounds derived from the stevia plant. Steviosides include, but are not limited to, steviol glycosides, rebaudioside, and rutin. Steviosides (STV) are the compounds primarily responsible for the sweetness in stevia. Rebaudiosides are steviol glycosides derived from the stevia plant. Rebaudiosides include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, and rebaudioside M. Rebaudiosides are often referred to as Reb (e.g., Reb A, Reb B, or Reb C). Rebaudioside A is the sweetest and second most abundant steviol glycoside in stevia. Rutin (RU) is a small amount of sweet steviol glycoside derived from the stevia plant. Rutin is typically derived from Rubus suavissimus and is used as a sugar substitute.

[0033] The method disclosed herein was developed using an enzyme capable of converting steviol glycosides to rebaudioside M. This method can be used with steviol glycosides containing sugars other than glucose alone, for example, rebaudioside K can be obtained from rebaudioside C, or rebaudioside N can be obtained from rebaudioside J. Additionally, other glycosides can be added.

[0034] As used herein, the term "enzyme mixture" refers to any mixture containing one or more glycosyltransferases. Typically, an "enzyme mixture" includes glycosyltransferases and sucrose synthases, as disclosed in U.S. Patent Application Publication No. US20200032227 A1.

[0035] As used herein, the term "stevia substance" refers to any substance containing one or more steviol glycosides. Typically, stevia substances are extracts, byproducts, or waste products derived from the stevia extraction process. Stevia extracts or extracts are products of the stevia plant extraction process containing 40-95% steviol glycosides by dry weight. Stevia extracts or extracts can be unpurified products. Stevia waste products or waste products are unpurified products generated from the first step of the stevia extraction process. Enrichment of steviol glycosides cannot be achieved by crystallization of stevia waste products.

[0036] As used herein, “mother liquor” refers to a byproduct stream obtained from the crystallization of a stevia stream. The mother liquor contains 40-95% steviol glycosides by dry weight.

[0037] As used herein, the term "minimal processed stevia extract" is understood to include extraction from stevia leaves with water and / or alcohol, flocculation with metal salts such as ferric sulfate and calcium hydroxide, adsorption of the aqueous solution onto a macroporous resin, and subsequent desorption using alcohol, ion exchange resin, activated carbon, or filtration (including perfiltration). Minimally processed stevia extract contains 50-95% steviol glycosides by dry weight.

[0038] As used in this article, “quality efficiency” refers to the increase in the percentage of steviol glycosides in stevia by dry weight.

[0039] Enzymatic conversion methods

[0040] As described above, a first aspect of this disclosure provides a method for obtaining one or more steviol glycosides, the method comprising the steps of: (a) adding an enzyme mixture to a stevia substance; (b) incubating the enzyme mixture and the stevia substance; and (c) obtaining the one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0041] Typically, stevia substances are extracts, byproducts, or waste products obtained from the stevia extraction process.

[0042] As stated above, this disclosure is based in part on the discovery that low-quality starting materials can be efficiently enzymatically converted. Therefore, in the first mode of the first aspect of this disclosure, the total steviol glycoside content of the stevia substance is ≤90% on a dry weight basis. In one embodiment of the first mode, the total steviol glycoside content of the stevia substance is ≤85% or ≤80% on a dry weight basis. Typically, the total steviol glycoside content of the stevia substance used in the first mode is ≥40% on a dry weight basis. More typically, the total steviol glycoside content of the stevia substance is ≥50% or ≥60% on a dry weight basis. Therefore, it is conceivable, for example, that the total steviol glycoside content of the stevia substance is 40% to 90%, 50% to 85%, 60% to 85%, or 60% to 80%, all percentages on a dry weight basis.

[0043] Typically, the stevia substance used in the first mode comprises a mixture of steviol glycosides. For example, the stevia substance used in the first mode may comprise rebaudioside A, steviol glycoside, optionally one or more additional steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M and steviol glycoside, and optionally one or more further steviol glycosides.

[0044] Typically, the total content of rebaudioside A and steviol glycosides in the stevia used in the first mode is 40 to 100% by weight of the total steviol glycoside content of the stevia. More typically, the total content of rebaudioside A and steviol glycosides is 50 to 95% by weight of the total steviol glycoside content of the stevia. Even more typically, the total content of rebaudioside A and steviol glycosides is 60 to 90% by weight.

[0045] It will be understood that, in the case where the stevia substance used in the first mode contains rebaudioside A and steviol glycoside, rebaudioside A and steviol glycoside can be present in any ratio, for example, 1:99 to 99:1 by weight.

[0046] Typically, in the first mode, when the total content of rebaudioside A and steviol glycosides in the stevia substance is 40 to 100% by weight of the total steviol glycoside content of the stevia substance, the content of rebaudioside A is 5 to 95% by weight of the total steviol glycoside content, and the content of steviol glycosides is 5 to 95% by weight. More typically, in such an embodiment, the content of rebaudioside A is 10 to 85% by weight, and the content of steviol glycosides is 10 to 85% by weight. Even more typically, the content of rebaudioside A is 20 to 80% by weight, and the content of steviol glycosides is 10 to 60% by weight. Still more typically, the content of rebaudioside A is 35 to 60% by weight, and the content of steviol glycosides is 15 to 50% by weight.

[0047] When the stevia used in the first mode contains rebaudioside D, the content of rebaudioside D in the stevia is typically 0.1 to 50% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside D is 0.5 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside D is 1 to 10% by weight of the total steviol glycoside content of the stevia. Still more typically, the content of rebaudioside D is 2 to 5% by weight of the total steviol glycoside content of the stevia.

[0048] Furthermore, as stated above, this disclosure is further based in part on the discovery that complex mixtures of starting materials can be efficiently enzymatically converted. Therefore, in the second mode of the first aspect of this disclosure, the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside D. In one embodiment of the second mode of the first aspect of this disclosure, the stevia substance further comprises one or more additional steviol glycosides selected from rebaudioside E, rebaudioside I, rebaudioside M, and stevia glycoside, and optionally one or more further steviol glycosides. For example, in one embodiment, the stevia substance comprises at least rebaudioside A, steviol glycoside, rebaudioside D, and rebaudioside M. In another embodiment, the stevia substance comprises at least rebaudioside A, steviol glycoside, rebaudioside D, and stevia glycoside.

[0049] What will be learned is that the stevia substances used in the second mode can contain rebaudioside A, steviol glycoside, and rebaudioside D in any ratio.

[0050] Typically, the total content of rebaudioside A and steviol glycosides in the stevia substance used in the second mode of the first aspect of this disclosure is 40 to 99.9% by weight of the total steviol glycoside content of the stevia substance. More typically, the total content of rebaudioside A and steviol glycosides is 50 to 95% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the total content of rebaudioside A and steviol glycosides is 60 to 90% by weight.

[0051] Typically, in the second mode, when the total content of rebaudioside A and steviol glycosides in the stevia substance is 40 to 99.9% by weight of the total steviol glycoside content of the stevia substance, the content of rebaudioside A is 5 to 95% by weight of the total steviol glycoside content, and the content of steviol glycosides is 5 to 95% by weight. More typically, in such an embodiment, the content of rebaudioside A is 10 to 85% by weight, and the content of steviol glycosides is 10 to 85% by weight. Even more typically, the content of rebaudioside A is 20 to 80% by weight, and the content of steviol glycosides is 10 to 60% by weight. Still more typically, the content of rebaudioside A is 35 to 60% by weight, and the content of steviol glycosides is 15 to 50% by weight.

[0052] Typically, the rebaudioside D content of the stevia substance used in the second mode of the first aspect of this disclosure is 0.1 to 50% by weight of the total steviol glycoside content of the stevia substance. More typically, the rebaudioside D content is 0.5 to 15% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the rebaudioside D content is 1 to 10% by weight of the total steviol glycoside content of the stevia substance. Still more typically, the rebaudioside D content is 2 to 5% by weight of the total steviol glycoside content of the stevia substance.

[0053] In cases where the stevia substance used in the first or second mode of this disclosure comprises rebaudioside E, typically, the content of rebaudioside E is 0.01 to 10% by weight of the total steviol glycoside content of the stevia substance. More typically, the content of rebaudioside E is 0.1 to 5% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the content of rebaudioside E is 0.5 to 1.5% by weight of the total steviol glycoside content of the stevia substance.

[0054] In cases where the stevia substance used in the first or second mode of this disclosure comprises rebaudioside I, typically, the content of rebaudioside I is 0.01 to 10% by weight of the total steviol glycoside content of the stevia substance. More typically, the content of rebaudioside I is 0.05 to 2% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the content of rebaudioside I is 0.1 to 0.5% by weight of the total steviol glycoside content of the stevia substance.

[0055] In cases where the stevia substance used in the first or second mode of this disclosure comprises rebaudioside M, typically, the content of rebaudioside M is 0.01 to 20% by weight of the total steviol glycoside content of the stevia substance. More typically, the content of rebaudioside M is 0.1 to 15% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the content of rebaudioside M is 0.2 to 10% by weight of the total steviol glycoside content of the stevia substance.

[0056] In cases where the stevia substance used in the first or second mode of this disclosure comprises stevia glycosides, typically, the stevia glycoside content is 0.01 to 10% by weight of the total steviol glycoside content of the stevia substance. More typically, the stevia glycoside content is 0.1 to 5% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the stevia glycoside content is 0.2 to 2% by weight of the total steviol glycoside content of the stevia substance.

[0057] Typically, according to the second mode of the first aspect of this disclosure, the total steviol glycoside content of the stevia substance is ≤95% on a dry weight basis. Typically, the total steviol glycoside content of the stevia substance used in the second mode is ≥40% on a dry weight basis. Thus, for example, the stevia substance may have a total steviol glycoside content of 40% to 95% on a dry weight basis.

[0058] In one embodiment, the method is according to a first mode and a second mode of the first aspect of this disclosure. In other words, a method for obtaining one or more steviol glycosides is provided, the method comprising the steps of: adding an enzyme mixture to a stevia substance; incubating the enzyme mixture and the stevia substance; and obtaining the one or more steviol glycosides from the enzyme mixture and the stevia substance, wherein the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside D, and wherein the total steviol glycoside content of the stevia substance is ≤90% on a dry weight basis.

[0059] Furthermore, as discussed above, it has been found that enzymes can be used to perform ideal and previously unconfirmed conversions of small amounts of steviol glycosides that may be present within unprocessed stevia extracts. For example, using enzymes developed for converting steviol glycosides to rebaudioside M, it has been demonstrated that these enzymes readily glycosylate steviol glycosides at glucose sites directly linked to the steviol core, selectively forming β1-2 and β1-3 bonds. For example, steviol glycosides contain two glucosyl residues linked to the C-13 and C-19 positions of the steviol core, which can be fully glycosylated to rebaudioside M, yielding a total of four glucosyl residues with these enzymes. When the enzyme mixture described herein is applied to minimally processed stevia extracts, even without process optimization, the recovery rate of steviol glycosides is expected to increase from approximately 80% to approximately 90% due to the increased yield efficiency. When applied to the mother liquor from the extraction process, 104 kg of steviol glycosides can be recovered after the reaction for every 100 kg of steviol glycosides in the mother liquor.

[0060] Furthermore, steviol glycosides can be linked to sugars other than glucose, such as carbohydrates containing rhamnose or xylose. These were first discovered in rebaudioside C and rebaudioside F, respectively. The enzyme mixture described herein can also add β1-2 and β1-3 glucosyl bonds to steviol glycosides containing rhamnose and xylose by implementing the disclosed method. Based on liquid chromatography-mass spectrometry data, it can be determined that at least three steviol glycosides having five glucosyl residues and one xylose or rhamnose residue were generated by the method disclosed herein. The method, combined with the enzyme disclosed in U.S. Patent Application Publication No. US20200032227 A1, results in the complete glycosylation of at least two isomers of rebaudioside C and F.

[0061] These methods were applied to minimally processed stevia extracts or mother liquors, enabling multiple glycosylation of various steviol glycosides, such as rebaudioside C, rebaudioside F, and stevia glycosides, using enzymes originally designed for the conversion of steviol glycosides to rebaudioside M. Bioconversion was carried out at optimal enzyme, sucrose, and ADP concentrations to achieve increased yield efficiency. As shown in Example 5, glucose was added to steviol glycosides having rhamnose and xylose residues. This resulted in an increased yield of steviol glycosides from the stevia processing equipment.

[0062] Therefore, in the third mode of the first aspect of this disclosure, the stevia substance comprises steviol glycosides and / or rhamnosyl-containing steviol glycosides and / or xylose-containing steviol glycosides.

[0063] In one implementation of the third mode, the stevia substance comprises stevia glycosides. Stevia glycosides can be extracted from the stevia plant or from Rubus tectorius.

[0064] In one embodiment, stevia glycosides are the major component of stevia. For example, stevia may contain ≥50%, ≥75%, ≥85%, or ≥95% stevia glycosides by dry weight.

[0065] Alternatively, stevia glycosides may be a minor component of stevia. For example, the stevia glycoside content of stevia may be ≤10% by weight, ≤5% by weight, or ≤2% by weight of the total steviol glycoside content of stevia. Typically, when stevia glycosides are a minor component of stevia, the stevia glycoside content is ≥0.01% by weight, ≥0.1% by weight, or ≥0.5% by weight of the steviol glycoside content of stevia. Therefore, it is conceivable, for example, that the stevia glycoside content of stevia may be from 0.01% by weight to 10% by weight, from 0.1% by weight to 5% by weight, or from 0.5% by weight to 2% by weight of the total steviol glycoside content of stevia.

[0066] In one embodiment where stevia glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≤95% on a dry weight basis. Typically, when stevia glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≥40% on a dry weight basis. Thus, for example, the stevia may have a total steviol glycoside content of 40% to 95% on a dry weight basis. Optionally, when stevia glycosides are a minor component of stevia, the method is also according to a first mode of the first aspect of this disclosure. In other words, the total steviol glycoside content of the stevia is ≤90% on a dry weight basis.

[0067] Typically, where stevioside is a minor component of stevioside, the stevioside further comprises rebaudioside A, steviol glycoside, one or more additional steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I and rebaudioside M, and one or more further steviol glycosides.

[0068] Typically, when stevia glycosides are a minor component of stevia, the total content of rebaudioside A and stevia glycosides in the stevia used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia. More typically, the total content of rebaudioside A and stevia glycosides is 50 to 95% by weight of the total steviol glycoside content of the stevia. Even more typically, the total content of rebaudioside A and stevia glycosides is 60 to 90% by weight. It will be understood that when the stevia used contains rebaudioside A and stevia glycosides, rebaudioside A and stevia glycosides can be present in any ratio, for example, 1:99 to 99:1 by weight.

[0069] Typically, when stevia glycosides are a minor component of the stevia compound and the total content of rebaudioside A and steviol glycosides in the stevia compound used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia compound, the content of rebaudioside A is 5 to 95% by weight of the total steviol glycoside content, and the content of steviol glycosides is 5 to 95% by weight. More typically, in such embodiments, the content of rebaudioside A is 10 to 85% by weight, and the content of steviol glycosides is 10 to 85% by weight. Even more typically, the content of rebaudioside A is 20 to 80% by weight, and the content of steviol glycosides is 10 to 60% by weight. Still more typically, the content of rebaudioside A is 35 to 60% by weight, and the content of steviol glycosides is 15 to 50% by weight.

[0070] When stevia glycosides are a minor component of stevia and the stevia used contains rebaudioside D, the content of rebaudioside D in the stevia is typically 0.1 to 50% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside D is 0.5 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside D is 1 to 10% by weight of the total steviol glycoside content of the stevia. Still more typically, the content of rebaudioside D is 2 to 5% by weight of the total steviol glycoside content of the stevia.

[0071] When stevia glycosides are a minor component of stevia and the stevia used contains rebaudioside E, typically, the content of rebaudioside E is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside E is 0.1 to 5% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside E is 0.5 to 1.5% by weight of the total steviol glycoside content of the stevia.

[0072] When stevia glycosides are a minor component of stevia and the stevia used contains rebaudioside I, typically, the content of rebaudioside I is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside I is 0.05 to 2% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside I is 0.1 to 0.5% by weight of the total steviol glycoside content of the stevia.

[0073] When stevia glycosides are a minor component of stevia and the stevia used contains rebaudioside M, typically, the content of rebaudioside M is 0.01 to 20% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside M is 0.1 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside M is 0.2 to 10% by weight of the total steviol glycoside content of the stevia.

[0074] In one embodiment where stevia glycoside is a minor component of stevia, the method is also according to a second mode of the first aspect of this disclosure. In other words, the stevia contains rebaudioside A, steviol glycoside, rebaudioside D, and stevia glycoside as a minor component.

[0075] In another embodiment of the third mode, the stevia substance comprises rhamnosyl-containing steviol glycosides, such as rebaudioside C, dulcioside A, and / or dulcioside B. Typically, in such embodiments, the stevia substance comprises rebaudioside C.

[0076] In one embodiment, rhamnosyl-containing steviol glycosides are the major components of the stevia substance. For example, the stevia substance may contain ≥50%, ≥75%, ≥85%, or ≥95% of rhamnosyl-containing steviol glycosides by dry weight. For example, the stevia substance may contain ≥75%, ≥85%, or ≥95% of rebaudioside C by dry weight.

[0077] Alternatively, rhamnosyl-containing steviol glycosides may be a minor component of the stevia substance. Typically, in such embodiments, the content of rhamnosyl-containing steviol glycosides (e.g., rebaudine C content) in the stevia substance is <50% by weight of the total steviol glycoside content of the stevia substance. For example, the content of rhamnosyl-containing steviol glycosides (e.g., rebaudine C content) in the stevia substance may be ≤25% by weight, ≤10% by weight, or ≤5% by weight of the total steviol glycoside content of the stevia substance. Typically, when rhamnosyl-containing steviol glycosides are a minor component of the stevia substance, the content of rhamnosyl-containing steviol glycosides (e.g., rebaudine C content) may be ≥0.1% by weight, ≥0.5% by weight, or ≥1% by weight of the total steviol glycoside content of the stevia substance. Therefore, it can be envisioned that the content of rhamnosyl-containing steviol glycosides (e.g., rebaudine C content) of stevia substances is 0.1% to 25% by weight, or 0.5% to 10% by weight, or 1% to 5% by weight of the total steviol glycoside content of stevia substances.

[0078] In one embodiment where rhamnosyl-containing steviol glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≤95% on a dry weight basis. Typically, when rhamnosyl-containing steviol glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≥40% on a dry weight basis. Thus, for example, the stevia may have a total steviol glycoside content of 40% to 95% on a dry weight basis. Optionally, when rhamnosyl-containing steviol glycosides are a minor component of stevia, the method is also according to a first mode of the first aspect of this disclosure. In other words, the total steviol glycoside content of the stevia is ≤90% on a dry weight basis.

[0079] Typically, where rhamnosyl-containing steviol glycosides are minor components of stevia, the stevia further comprises rebaudioside A, steviol glycoside, one or more additional steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M and steviol glycoside, and one or more further steviol glycosides.

[0080] Typically, when rhamnosyl-containing steviol glycosides are a minor component of the stevia substance, the total content of rebaudioside A and steviol glycosides in the stevia substance used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia substance. More typically, the total content of rebaudioside A and steviol glycosides is 50 to 95% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the total content of rebaudioside A and steviol glycosides is 60 to 90% by weight. It will be understood that when the stevia substance used contains rebaudioside A and steviol glycosides, rebaudioside A and steviol glycosides can be present in any ratio, for example, 1:99 to 99:1 by weight.

[0081] Typically, when rhamnosyl-containing steviol glycosides are a minor component of the stevia substance and the total content of rebaudioside A and steviol glycosides in the stevia substance used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia substance, the content of rebaudioside A is 5 to 95% by weight of the total steviol glycoside content, and the content of steviol glycosides is 5 to 95% by weight. More typically, in such embodiments, the content of rebaudioside A is 10 to 85% by weight, and the content of steviol glycosides is 10 to 85% by weight. Even more typically, the content of rebaudioside A is 20 to 80% by weight, and the content of steviol glycosides is 10 to 60% by weight. Still more typically, the content of rebaudioside A is 35 to 60% by weight, and the content of steviol glycosides is 15 to 50% by weight.

[0082] When rhamnosyl steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside D, the content of rebaudioside D in the stevia is typically 0.1 to 50% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside D is 0.5 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside D is 1 to 10% by weight of the total steviol glycoside content of the stevia. Still more typically, the content of rebaudioside D is 2 to 5% by weight of the total steviol glycoside content of the stevia.

[0083] When rhamnosyl steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside E, typically, the content of rebaudioside E is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside E is 0.1 to 5% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside E is 0.5 to 1.5% by weight of the total steviol glycoside content of the stevia.

[0084] When rhamnosyl steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside I, typically, the content of rebaudioside I is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside I is 0.05 to 2% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside I is 0.1 to 0.5% by weight of the total steviol glycoside content of the stevia.

[0085] When rhamnosyl steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside M, typically, the content of rebaudioside M is 0.01 to 20% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside M is 0.1 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside M is 0.2 to 10% by weight of the total steviol glycoside content of the stevia.

[0086] When rhamnosyl steviol glycosides are a minor component of stevia and the stevia used contains steviosides, typically, the stevioside content is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the stevioside content is 0.1 to 5% by weight of the total steviol glycoside content of the stevia. Even more typically, the stevioside content is 0.2 to 2% by weight of the total steviol glycoside content of the stevia.

[0087] In one embodiment where rhamnosyl-containing steviol glycosides are minor components of stevia, the method is also according to a second mode of the first aspect of this disclosure. In other words, the stevia substance comprises rebaudioside A, steviol glycoside, rebaudioside D, and rhamnosyl-containing steviol glycosides, such as rebaudioside C, as minor components.

[0088] In another implementation of the third mode, the stevia substance comprises xylose-containing steviol glycosides, such as rebaudioside F.

[0089] In one embodiment, xylose-containing steviol glycosides are the major components of the stevia substance. For example, the stevia substance may contain ≥50%, ≥75%, ≥85%, or ≥95% xylose-containing steviol glycosides by dry weight. For example, the stevia substance may contain ≥75%, ≥85%, or ≥95% rebaudioside F by dry weight.

[0090] Alternatively, xylose-containing steviol glycosides may be a minor component of the stevia substance. Typically, in such embodiments, the content of xylose-containing steviol glycosides in the stevia substance (e.g., rebaudine C content) is < 50% by weight of the total steviol glycoside content of the stevia substance. For example, the content of xylose-containing steviol glycosides in the stevia substance (e.g., rebaudine F content) may be ≤15% by weight, ≤10% by weight, or ≤5% by weight of the total steviol glycoside content of the stevia substance. Typically, when xylose-containing steviol glycosides are a minor component of the stevia substance, the content of xylose-containing steviol glycosides (e.g., rebaudine F content) may be ≥0.1% by weight, ≥0.5% by weight, or ≥1% by weight of the total steviol glycoside content of the stevia substance. Therefore, it can be envisioned that the content of xylose-containing steviol glycosides (e.g., rebaudin F content) of stevia substances is 0.1% to 15% by weight, or 0.5% to 10% by weight, or 1% to 5% by weight of the total steviol glycoside content of stevia substances.

[0091] In one embodiment where xylose-containing steviol glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≤95% on a dry weight basis. Typically, when xylose-containing steviol glycosides are a minor component of stevia, the total steviol glycoside content of the stevia is ≥40% on a dry weight basis. Thus, for example, the stevia may have a total steviol glycoside content of 40% to 95% on a dry weight basis. Optionally, when xylose-containing steviol glycosides are a minor component of stevia, the method is also according to a first mode of the first aspect of this disclosure. In other words, the total steviol glycoside content of the stevia is ≤90% on a dry weight basis.

[0092] Typically, where xylose-containing steviol glycosides are minor components of stevia, the stevia further comprises rebaudioside A, steviol glycoside, one or more additional steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M and steviol glycoside, and one or more further steviol glycosides.

[0093] Typically, when xylose-containing steviol glycosides are a minor component of stevia, the total content of rebaudioside A and steviol glycosides in the stevia used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia. More typically, the total content of rebaudioside A and steviol glycosides is 50 to 95% by weight of the total steviol glycoside content of the stevia. Even more typically, the total content of rebaudioside A and steviol glycosides is 60 to 90% by weight. It will be understood that when the stevia used contains rebaudioside A and steviol glycosides, rebaudioside A and steviol glycosides can be present in any ratio, for example, 1:99 to 99:1 by weight.

[0094] Typically, when xylose-containing steviol glycosides are a minor component of the stevia substance and the total content of rebaudioside A and steviol glycosides in the stevia substance used is 40 to 99.9% by weight of the total steviol glycoside content of the stevia substance, the content of rebaudioside A is 5 to 95% by weight of the total steviol glycoside content, and the content of steviol glycosides is 5 to 95% by weight. More typically, in such embodiments, the content of rebaudioside A is 10 to 85% by weight, and the content of steviol glycosides is 10 to 85% by weight. Even more typically, the content of rebaudioside A is 20 to 80% by weight, and the content of steviol glycosides is 10 to 60% by weight. Still more typically, the content of rebaudioside A is 35 to 60% by weight, and the content of steviol glycosides is 15 to 50% by weight.

[0095] When xylose-containing steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside D, the content of rebaudioside D in the stevia is typically 0.1 to 50% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside D is 0.5 to 15% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside D is 1 to 10% by weight of the total steviol glycoside content of the stevia. Still more typically, the content of rebaudioside D is 2 to 5% by weight of the total steviol glycoside content of the stevia.

[0096] When xylose-containing steviol glycosides are a minor component of stevia and the stevia used contains rebaudioside E, typically, the content of rebaudioside E is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the content of rebaudioside E is 0.1 to 5% by weight of the total steviol glycoside content of the stevia. Even more typically, the content of rebaudioside E is 0.5 to 1.5% by weight of the total steviol glycoside content of the stevia.

[0097] When xylose-containing steviol glycosides are a minor component of the stevia substance and the stevia substance used contains rebaudioside I, typically, the content of rebaudioside I is 0.01 to 10% by weight of the total steviol glycoside content of the stevia substance. More typically, the content of rebaudioside I is 0.05 to 2% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the content of rebaudioside I is 0.1 to 0.5% by weight of the total steviol glycoside content of the stevia substance.

[0098] When xylose-containing steviol glycosides are minor components of the stevia substance and the stevia substance used contains rebaudioside M, typically, the content of rebaudioside M is 0.01 to 20% by weight of the total steviol glycoside content of the stevia substance. More typically, the content of rebaudioside M is 0.1 to 15% by weight of the total steviol glycoside content of the stevia substance. Even more typically, the content of rebaudioside M is 0.2 to 10% by weight of the total steviol glycoside content of the stevia substance.

[0099] When xylose-containing steviol glycosides are a minor component of stevia and the stevia used includes stevia glycosides, typically, the stevia glycoside content is 0.01 to 10% by weight of the total steviol glycoside content of the stevia. More typically, the stevia glycoside content is 0.1 to 5% by weight of the total steviol glycoside content of the stevia. Even more typically, the stevia glycoside content is 0.2 to 2% by weight of the total steviol glycoside content of the stevia.

[0100] In one embodiment where xylose-containing steviol glycosides are minor components of stevia, the method is also according to a second mode of the first aspect of this disclosure. In other words, the stevia substance comprises rebaudioside A, steviol glycoside, rebaudioside D, and xylose-containing steviol glycosides, such as rebaudioside F, as minor components.

[0101] In some embodiments applicable to the first aspect and any mode outlined above, a method for obtaining one or more steviol glycosides includes adding an enzyme mixture to a stevia substance during an extraction process, wherein the stevia substance is an extract, byproduct, or waste product; incubating the enzyme mixture and the stevia substance; and obtaining steviol glycosides from the enzyme mixture and the stevia substance.

[0102] In some embodiments, the stevia substance comprises an extract, such as minimally processed stevia extract. In some embodiments, the minimally processed stevia extract contains 60 to 95% total steviol glycosides on a dry weight basis.

[0103] In some implementations, stevia is a byproduct. In certain implementations, the byproduct is the mother liquor.

[0104] In some embodiments, the mother liquor contains about 40% to about 95% of steviol glycosides on a dry weight basis.

[0105] When the total steviol glycoside content of stevia is <100% on a dry weight basis, the remainder of the dry weight of the stevia typically contains one or more components selected from polysaccharides, carbohydrates, ash, polyphenols, flavonoids, proteins, amino acids, vitamins, fats, and plant metabolites. For example, the remainder of the dry weight of stevia may contain polysaccharides and / or carbohydrates, as well as at least one or more components selected from ash, polyphenols, flavonoids, proteins, amino acids, vitamins, fats, and plant metabolites.

[0106] More typically, the remainder of the dry weight of stevia consists essentially of one or more components selected from polysaccharides, carbohydrates, ash, polyphenols, flavonoids, proteins, amino acids, vitamins, fats, and plant metabolites. For example, the remainder of the dry weight of stevia may consist essentially of polysaccharides and / or carbohydrates, along with at least one or more components selected from ash, polyphenols, flavonoids, proteins, amino acids, vitamins, fats, and plant metabolites.

[0107] It will be understood that the step of incubating the enzyme mixture and the stevia substance results in the glycosylation of one or more steviol glycosides within the stevia substance. Typically, the step of incubating the enzyme mixture and the stevia substance results in the glycosylation of one or more steviol glycosides within the stevia substance. More typically, the step of incubating the enzyme mixture and the stevia substance results in the glycosylation of one or more steviol glycosides within the stevia substance via β1-2 and / or β1-3 bonds.

[0108] For example, in cases where the stevia compound contains one or more of stevioside, steviol, rebaudioside A, rebaudioside D, rebaudioside E, or rebaudioside I; stevioside can be converted to steviol; steviol can be converted to rebaudioside A and / or rebaudioside E; rebaudioside A can be converted to rebaudioside D and / or rebaudioside I; rebaudioside E can be converted to rebaudioside D; rebaudioside D can be converted to rebaudioside M; and rebaudioside I can be converted to rebaudioside M. Furthermore, chain reactions may occur, resulting in the formation of rebaudioside M, for example, from rebaudioside A (via rebaudioside D and / or rebaudioside I), from rebaudioside E (via rebaudioside D), from steviol (via rebaudioside A, D, E, and / or I), and / or from stevioside (via any one of steviol and / or rebaudioside A, D, E, and / or I).

[0109] Similarly, when the stevia contains rebaudioside C, rebaudioside C can be converted into β1-2 and / or β1-3 glucosylated derivatives of rebaudioside C, such as rebaudioside K, rebaudioside N2, and / or rebaudioside H4. And when the stevia contains rebaudioside F, rebaudioside F can be converted into β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F.

[0110] Typically, the obtained one or more steviol glycosides comprise a mixture of steviol glycosides. For example, the obtained one or more steviol glycosides may comprise (i) one or more steviol glycosides that have been glycosylated by an incubation step and (ii) a mixture of one or more residual steviol glycosides from stevia.

[0111] In some embodiments, the steviol glycosides obtained by the methods of this disclosure comprise rebaudin A, rebaudin D, rebaudin M, rebaudin E, rebaudin I, steviol glycosides, and / or steviosides. Embodiments according to the first or second mode of this disclosure, or the third mode where the stevioside contains steviosides, or the third mode where rhamnosyl-containing steviol glycosides or xylose-containing steviol glycosides are minor components of the stevioside, are typical.

[0112] In a further embodiment, the obtained steviol glycoside comprises rebaudioside A, rebaudioside D, rebaudioside I, and / or rebaudioside M. Again, embodiments according to the first or second mode of this disclosure, or the third mode where the stevia substance comprises stevia glycosides, or the third mode where rhamnosyl-containing or xylose-containing steviol glycosides are minor components of the stevia substance, are typical. In particular, embodiments where the stevia substance comprises rebaudioside A and / or steviol glycosides and / or stevia glycosides are typical. In some embodiments, the obtained steviol glycoside comprises about 1% to about 38% rebaudioside A by dry weight. In one embodiment, the obtained steviol glycoside comprises about 11% rebaudioside A. In some embodiments, the obtained steviol glycoside comprises about 10% to about 33% rebaudioside D by dry weight. In one embodiment, the obtained steviol glycoside comprises about 13% rebaudioside D. In some embodiments, the obtained steviol glycoside comprises about 1% to about 5% rebaudioside I by dry weight. In one embodiment, the obtained steviol glycoside comprises about 4% rebaudioside I. In some embodiments, the obtained steviol glycoside comprises about 21% to about 78% rebaudioside M by dry weight. In one embodiment, the obtained steviol glycoside comprises about 60% rebaudioside M. In some embodiments, the obtained steviol glycoside comprises about 0% steviol glycoside and stevioside.

[0113] In a further embodiment, the steviol glycosides obtained by the methods of this disclosure comprise rebaudin A, rebaudin D, rebaudin E, rebaudin I, and / or rebaudin M. Again, embodiments according to the first or second mode of this disclosure, or the third mode where the stevia substance comprises stevia glycosides, or the third mode where rhamnosyl-containing or xylose-containing steviol glycosides are minor components of the stevia substance, are typical. In particular, embodiments where the stevia substance comprises rebaudin A and / or steviol glycosides and / or stevia glycosides are typical. In some embodiments, the obtained steviol glycosides comprise about 0% to about 2% rebaudin A by dry weight. In one embodiment, the obtained steviol glycosides comprise about 1% rebaudin A. In some embodiments, the obtained steviol glycosides comprise about 0% to about 13% rebaudin D by dry weight. In one embodiment, the obtained steviol glycosides comprise about 3% rebaudin D. In some embodiments, the obtained steviol glycoside comprises about 0% to about 10% rebaudioside E on a dry weight basis. In one embodiment, the obtained steviol glycoside comprises about 6% rebaudioside E. In some embodiments, the obtained steviol glycoside comprises about 0% to about 5% rebaudioside I on a dry weight basis. In one embodiment, the obtained steviol glycoside comprises about 4% rebaudioside I. In some embodiments, the obtained steviol glycoside comprises about 65% to about 90% rebaudioside M on a dry weight basis. In one embodiment, the obtained steviol glycoside comprises about 86% rebaudioside M.

[0114] In a further embodiment, the steviol glycosides obtained by the methods of this disclosure comprise rebaudin A, rebaudin D, rebaudin E, rebaudin I, and / or rebaudin M. Again, embodiments according to the first or second mode of this disclosure, or the third mode where the stevia substance comprises stevia glycosides, or the third mode where rhamnosyl-containing or xylose-containing steviol glycosides are minor components of the stevia substance, are typical. In particular, embodiments where the stevia substance comprises rebaudin A and / or steviol glycosides and / or stevia glycosides are typical. In some embodiments, the obtained steviol glycosides comprise about 0% to about 2% rebaudin A by dry weight. In some embodiments, the obtained steviol glycosides comprise about 1% rebaudin A. In some embodiments, the obtained steviol glycosides comprise about 0% to about 17% rebaudin D by dry weight. In one embodiment, the obtained steviol glycosides comprise about 4% rebaudin D. In some embodiments, the obtained steviol glycoside comprises about 0% to about 13% rebaudioside E by dry weight. In one embodiment, the obtained steviol glycoside comprises about 1% rebaudioside E. In some embodiments, the obtained steviol glycoside comprises about 1% to about 4% rebaudioside I by dry weight. In one embodiment, the obtained steviol glycoside comprises about 3% rebaudioside I. In some embodiments, the obtained steviol glycoside comprises about 60% to about 80% rebaudioside M by dry weight. In one embodiment, the obtained steviol glycoside comprises about 75% rebaudioside M.

[0115] When the stevia compound comprises rebaudioside A and / or steviol glycoside and / or stevioside, the one or more steviol glycosides typically obtained comprise rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I. More typically, the one or more steviol glycosides obtained comprise rebaudioside M and at least two further steviol glycosides selected from rebaudioside D, rebaudioside E, and rebaudioside I.

[0116] In one embodiment where the stevia substance comprises rebaudioside A and / or steviol glycoside and / or stevia glycoside, the obtained one or more steviol glycosides comprise rebaudioside M and rebaudioside D, optionally rebaudioside E and optionally rebaudioside I.

[0117] When a stevia substance contains rebaudioside C, one or more steviol glycosides typically obtained contain at least one steviol glycoside selected from rebaudioside K, rebaudioside N2, and rebaudioside H4. For example, when a stevia substance contains (i) rebaudioside A and / or steviol glycoside and (ii) rebaudioside C as a minor component of the stevia substance, one or more steviol glycosides typically obtained contain rebaudioside M, at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I, and at least one further steviol glycoside selected from rebaudioside K, rebaudioside N2, and rebaudioside H4. More typically, in cases where the stevia substance comprises (i) rebaudioside A and / or steviol glycoside and (ii) rebaudioside C as a minor component of the stevia substance, the resulting one or more steviol glycosides comprise rebaudioside M, at least two further steviol glycosides selected from rebaudioside D, rebaudioside E and rebaudioside I, and at least two further steviol glycosides selected from rebaudioside K, rebaudioside N2 and rebaudioside H4.

[0118] When the stevia compound contains rebaudioside C as a minor component of the stevia compound, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is typically from 0.01% to 50% by weight of the total steviol glycosides obtained. More typically, in such an embodiment, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 0.1% to 25% by weight of the total steviol glycosides obtained. Even more typically, in such an embodiment, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 0.5% to 10% by weight. Still more typically, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 1% to 5% by weight.

[0119] Similarly, when a stevia substance contains rebaudioside F, one or more steviol glycosides typically obtained contain at least one β1-2 and / or β1-3 glucosylated derivative of rebaudioside F. For example, when a stevia substance contains (i) rebaudioside A and / or steviol glycoside and (ii) rebaudioside F as a minor component of the stevia substance, one or more steviol glycosides typically obtained contain rebaudioside M, at least one further steviol glycoside selected from rebaudioside D, rebaudioside E and rebaudioside I, and at least one β1-2 and / or β1-3 glucosylated derivative of rebaudioside F. More typically, in cases where the stevia substance comprises (i) rebaudioside A and / or steviol glycoside and (ii) rebaudioside F as a minor component of the stevia substance, the resulting one or more steviol glycosides comprise rebaudioside M, at least two further steviol glycosides selected from rebaudioside D, rebaudioside E and rebaudioside I, and at least two β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F.

[0120] When the stevia compound contains rebaudioside F as a minor component of the stevia compound, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is typically from 0.01% by weight to 50% by weight of the total steviol glycosides obtained. More typically, in such embodiments, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 0.1% by weight to 15% by weight of the total steviol glycosides obtained. Even more typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 0.5% by weight to 10% by weight. Still more typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 1% by weight to 5% by weight.

[0121] In any embodiment where the obtained steviol glycoside comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I, the content of rebaudioside M is typically 10 to 99% by weight of the total steviol glycosides obtained. More typically, the content of rebaudioside M is 20 to 95% by weight of the total steviol glycosides obtained. Even more typically, the content of rebaudioside M is 40 to 90% by weight or 50 to 85% by weight of the total steviol glycosides obtained. Still more typically, the content of rebaudioside M is 60 to 80% by weight of the total steviol glycosides obtained.

[0122] When the obtained steviol glycosides include rebaudioside D, the content of rebaudioside D is typically 0.1 to 99% by weight of the total obtained steviol glycosides. More typically, the content of rebaudioside D is 0.5 to 40% by weight of the total obtained steviol glycosides. Even more typically, the content of rebaudioside D is 2 to 20% by weight of the total obtained steviol glycosides. Still more typically, the content of rebaudioside D is 5 to 15% by weight of the total obtained steviol glycosides.

[0123] In some embodiments where the obtained steviol glycosides comprise rebaudine M and rebaudine D, the content of rebaudine M is 56 to 85% by weight of the total obtained steviol glycosides, and the content of rebaudine D is 2 to 19% by weight of the total obtained steviol glycosides. Typically, in such embodiments, the content of rebaudine M is 58 to 82% by weight of the total obtained steviol glycosides, and the content of rebaudine D is 3 to 17% by weight. More typically, in such embodiments, the content of rebaudine M is 60 to 80% by weight, and the content of rebaudine D is 5 to 15% by weight.

[0124] In any embodiment where the obtained steviol glycosides include rebaudioside E, the content of rebaudioside E is typically 0.5 to 30% by weight of the total obtained steviol glycosides. More typically, the content of rebaudioside E is 2 to 20% by weight of the total obtained steviol glycosides. Even more typically, the content of rebaudioside E is 5 to 15% by weight of the total obtained steviol glycosides.

[0125] In any embodiment where the obtained steviol glycosides comprise rebaudioside I, the content of rebaudioside I is typically 0.1 to 99% by weight of the total obtained steviol glycosides. More typically, the content of rebaudioside I is 0.5 to 25% by weight of the total obtained steviol glycosides. Even more typically, the content of rebaudioside I is 1 to 10% by weight of the total obtained steviol glycosides. Still more typically, the content of rebaudioside I is 1.5 to 5% by weight of the total obtained steviol glycosides.

[0126] In some embodiments, the obtained steviol glycosides comprise rebaudioside A. Rebaudioside A may be present as a glycosylated product or as residual unreacted rebaudioside A from stevia. For example, the content of rebaudioside A may be from 0.1 to 50% by weight of the total obtained steviol glycosides. More typically, in such embodiments, the content of rebaudioside A is from 0.5 to 25% by weight of the total obtained steviol glycosides. Even more typically, the content of rebaudioside A is from 1 to 15% by weight of the total obtained steviol glycosides.

[0127] Alternatively, the obtained steviol glycosides may substantially not contain rebaudioside A. For example, the rebaudioside A content may be <1% by weight of the total steviol glycosides obtained. More typically, in such embodiments, the rebaudioside A content is <0.1% by weight or <0.01% by weight of the total steviol glycosides obtained.

[0128] Typically, the obtained steviol glycosides contain virtually no steviol glycosides. For example, the steviol glycoside content may be <1% by weight of the total steviol glycosides obtained. More typically, the steviol glycoside content is <0.1% by weight or <0.01% by weight of the total steviol glycosides obtained.

[0129] Typically, the obtained steviol glycosides contain virtually no steviosides. For example, the stevioside content may be <1% by weight of the total steviol glycosides obtained. More typically, the stevioside content is <0.1% by weight or <0.01% by weight of the total steviol glycosides obtained.

[0130] Typically, the total steviol glycosides obtained are obtained as part of a product, wherein the total steviol glycoside content of the product is ≥50% on a dry weight basis. Typically, the total steviol glycoside content of the product is ≥60% on a dry weight basis. More typically, the total steviol glycoside content of the product is ≥75% on a dry weight basis. For example, the product may have a total steviol glycoside content of ≥85% or ≥95% on a dry weight basis.

[0131] In some embodiments, the amount of steviol glycosides obtained by the method of this disclosure is at least about 30% higher, on a dry weight basis, than the amount of steviol glycosides in the stevia material. In a further embodiment, the amount of steviol glycosides obtained is at least about 100% higher than the amount of steviol glycosides in the stevia material.

[0132] In some embodiments, the amount of steviol glycosides obtained by the methods of this disclosure is at least about 38% higher, on a dry weight basis, than the amount of steviol glycosides in the extract. In a further embodiment, the amount of steviol glycosides obtained is at least about 100% higher than the amount of steviol glycosides in the extract.

[0133] In some embodiments, the amount of steviol glycosides obtained by the method of this disclosure is at least about 35% higher, on a dry weight basis, than the amount of steviol glycosides in the mother liquor. In a further embodiment, the amount of steviol glycosides is at least about 100% higher than the amount of steviol glycosides in the mother liquor.

[0134] In some embodiments, the steviol glycosides obtained using the methods of this disclosure contain about 60% to about 95% minimally processed stevia.

[0135] In some implementations, based on the conversion of rebaudioside D to rebaudioside M and / or stevia to rebaudioside M, the enzyme mixture increases the enrichment efficiency by 14% to 100%.

[0136] In some embodiments, the steviol glycosides obtained using the methods of this disclosure are purified. In some embodiments, the purified steviol glycosides comprise rebaudioside A, rebaudioside D, rebaudioside M, rebaudioside E, rebaudioside I, steviol glycoside, or stevioside. It will be understood that the methods of this disclosure can facilitate downstream purification techniques, especially because the obtained steviol glycosides can be more easily separated from impurities and from each other than steviol glycosides in the source stevia material.

[0137] In some embodiments, this disclosure provides a method for obtaining a mixture of one or more steviol glycosides, the method comprising the steps of: adding an enzyme mixture to a stevia substance during an extraction process, wherein the stevia substance is an extract, byproduct, or waste product; incubating the enzyme mixture and the stevia substance; and obtaining a mixture of one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0138] The method disclosed herein can be used to catalyze the conversion of unprocessed stevia into various steviol glycosides. In some embodiments, the method is used to obtain a mixture of steviol glycosides comprising rebaudioside M, rebaudioside A, and rebaudioside D. In some embodiments, the method is used to obtain a mixture of steviol glycosides comprising more than 50% rebaudioside M by dry weight.

[0139] A mixture of one or more steviol glycosides produced by the methods of this disclosure may comprise rebaudine M, rebaudine A, rebaudine D, or combinations thereof. In some embodiments, the mixture comprises rebaudine M, rebaudine A, rebaudine D, rebaudine I, and rebaudine E. In some embodiments, the mixture comprises at least 50% rebaudine M by dry weight. In some embodiments, the mixture comprises at least 59% rebaudine M and at least one other steviol glycoside. In some embodiments, the mixture comprises at least 80% rebaudine M and at least one other steviol glycoside. In some embodiments, the mixture comprises at least 15% rebaudine A by dry weight. In some embodiments, the mixture comprises at least 12% rebaudine A. In some embodiments, the mixture comprises at least 15% rebaudine D by dry weight. In some embodiments, the mixture comprises at least 11% rebaudine D. In some embodiments, the mixture comprises about 5% rebaudine I by dry weight. In some embodiments, the mixture contains less than 1% rebaudine E. In some embodiments, the mixture contains at least 94% rebaudine M and at least one other steviol glycoside by dry weight. In some embodiments, the mixture contains about 1% rebaudine I by dry weight. In some embodiments, the mixture contains less than 1% rebaudine A, rebaudine D, or rebaudine E by dry weight. In some embodiments, the mixture contains at least 86% rebaudine M and at least one other steviol glycoside by dry weight. In some embodiments, the mixture contains about 6% rebaudine E by dry weight. In some embodiments, the mixture contains about 4% rebaudine I by dry weight. In some embodiments, the mixture contains about 3% rebaudine I. In some embodiments, the mixture contains less than 1% rebaudine A by dry weight.

[0140] As described above, the method of the first aspect of this disclosure includes the steps of incubating the enzyme mixture and the stevia substance. Typical incubation conditions are given below and are equally applicable to each mode of the first aspect of this disclosure.

[0141] The enzyme mixture and stevia substance are typically incubated in a solvent. Step (a) may therefore include adding a solvent to the stevia substance. The solvent may be a residual solvent from an earlier processing or extraction step, a fresh solvent, or a combination of residual and fresh solvents. Typically, the solvent is an alcoholic or aqueous solvent. For example, the solvent may be water, methanol, ethanol, propanol, or mixtures thereof. More typically, the solvent is water, ethanol, or mixtures thereof. Most typically, the solvent is water.

[0142] When the enzyme mixture and stevia are incubated in a solvent (such as water), the stevia is typically present at a concentration of 0.1 to 300 g / L. More typically, it is present at a concentration of 1 to 250 g / L. More typically, it is present at a concentration of 25 to 200 g / L.

[0143] As described above, the enzyme mixture contains one or more glycosyltransferases. Typically, the enzyme mixture contains one or more glucosyltransferases, such as one or more β1-2 and / or β1-3 glucosyltransferases (i.e., glucosyltransferases capable of forming β1-2 and / or β1-3 glucosyl bonds).

[0144] In one embodiment, the enzyme mixture comprises one or more nucleoside diphosphate (NDP)-dependent glycosyltransferases. For example, the enzyme mixture may comprise one or more NDP-glucose-dependent glycosyltransferases, such as one or more adenosine diphosphate (ADP)-glucose-dependent, cytidine diphosphate (CDP)-glucose-dependent, guanosine diphosphate (GDP)-glucose-dependent, thymidine diphosphate (TDP)-glucose-dependent, uridine diphosphate (UDP)-glucose-dependent, or inosine diphosphate (IDP)-glucose-dependent glycosyltransferases. Suitable examples of such enzymes that may be used according to this disclosure are disclosed in US2020 / 0032227 A1. Typically, the enzyme mixture comprises one or more ADP-glucose-dependent glycosyltransferases.

[0145] Typically, when an enzyme mixture contains one or more NDP-glucose-dependent glycosyltransferases, the enzyme mixture contains one or more NDP-glucose-dependent β1-2 and / or β1-3 glucosyltransferases. For example, an enzyme mixture may contain one or more ADP-glucose-dependent β1-2 and / or β1-3 glucosyltransferases.

[0146] In some embodiments, when the enzyme mixture and stevia substance are incubated in a solvent (such as water), one or more glycosyltransferases are present at a total concentration of 0.01 to 10 g / L. Typically, one or more glycosyltransferases are present at a total concentration of 0.02 to 5 g / L. More typically, one or more glycosyltransferases are present at a total concentration of 0.05 to 2 g / L.

[0147] When the enzyme mixture contains one or more glucosyltransferases, the enzyme mixture and stevia substance are typically incubated in the presence of a glucose source. For example, when the enzyme mixture contains one or more NDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia substance are typically incubated in the presence of an NDP-glucose source. Step (a) may therefore include adding a glucose source or an NDP-glucose source to the stevia substance. It will be understood that when the enzyme mixture and stevia substance are incubated in the presence of an NDP-glucose source, the NDP-glucose source will be suitable for the type of NDP-glucose-dependent glycosyltransferase present. Therefore, for example, when the enzyme mixture contains one or more ADP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of an ADP-glucose source; when the enzyme mixture contains one or more CDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of a CDP-glucose source; when the enzyme mixture contains one or more GDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of a GDP-glucose source; when the enzyme mixture contains one or more TDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of a TDP-glucose source; when the enzyme mixture contains one or more UDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of a UDP-glucose source; and when the enzyme mixture contains one or more IDP-glucose-dependent glycosyltransferases, the enzyme mixture and stevia are typically incubated in the presence of an IDP-glucose source.

[0148] In one embodiment, the NDP-glucose source is NDP-glucose. Step (a) may therefore include adding NDP-glucose to the stevia substance. For example, the NDP-glucose source may be ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose, UDP-glucose, or IDP-glucose. When the enzyme mixture and the stevia substance are incubated in a solvent (such as water) and the NDP-glucose source is NDP-glucose, the NDP-glucose is typically present at a concentration of 0.1 to 200 g / L. More typically, the NDP-glucose is present at a concentration of 1 to 100 g / L. Even more typically, the NDP-glucose is present at a concentration of 25 to 75 g / L.

[0149] In another embodiment, NDP-glucose is produced in situ. For example, in one embodiment, the enzyme mixture further comprises sucrose synthase. Typically, in such an embodiment, the enzyme mixture and stevia substance are incubated in the presence of sucrose and nucleoside diphosphate (NDP) such as ADP, CDP, GDP, TDP, UDP, or IDP (depending on the type of NDP-glucose-dependent glycosyltransferase present). Step (a) may therefore include adding NDP and sucrose to the stevia substance. Suitable examples of sucrose synthases that may be used according to this disclosure are disclosed in US2020 / 0032227 A1.

[0150] When the enzyme mixture contains sucrase synthase and the enzyme mixture and stevia are incubated in a solvent (such as water), the sucrase synthase is typically present at a concentration of 0.01 to 10 g / L. More typically, it is present at a concentration of 0.02 to 2 g / L. Even more typically, it is present at a concentration of 0.05 to 0.5 g / L.

[0151] Typically, when the enzyme mixture contains sucrase and the enzyme mixture and stevia are incubated in a solvent (such as water), sucrose is present at a concentration of 0.1 to 500 g / L. More typically, sucrose is present at a concentration of 1 to 300 g / L. Even more typically, sucrose is present at a concentration of 50 to 250 g / L.

[0152] Typically, when the enzyme mixture contains sucrase synthase and the enzyme mixture and stevia are incubated in a solvent (such as water), NDP is present at a concentration of 0.01 to 10 g / L. More typically, NDP is present at a concentration of 0.05 to 1 g / L. Even more typically, NDP is present at a concentration of 0.1 to 0.5 g / L.

[0153] Optionally, the incubation step is performed in the presence of a chelating agent (such as EDTA). Step (a) may therefore include adding a chelating agent to the stevia substance. Typically, when incubating the enzyme mixture and the stevia substance in a solvent (such as water), the chelating agent is present at a concentration of 0.01 to 10 g / L. More typically, the chelating agent is present at a concentration of 0.02 to 5 g / L. Even more typically, the chelating agent is present at a concentration of 0.05 to 2 g / L.

[0154] When incubating the enzyme mixture and stevia in a solvent (such as water), incubation is typically carried out at a pH of 5 to 9. More typically, incubation is carried out at a pH of 5.5 to 8.5. Even more typically, incubation is carried out at a pH of 6.0 to 8.0. Optionally, a buffer may be used to maintain the desired pH. In one embodiment, the buffer is selected from potassium phosphate or tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl).

[0155] In one implementation, the incubation is carried out at a temperature between 20°C and 90°C. Typically, the incubation is carried out at a temperature between 40°C and 85°C. More typically, the incubation is carried out at a temperature between 50°C and 80°C.

[0156] In some embodiments, the enzyme mixture and stevia substance are incubated for at least 2 hours. In further embodiments, the enzyme mixture and stevia substance are incubated for between 4 and 8 hours. In still other embodiments, the enzyme mixture and extract are incubated for between 6 and 24 hours.

[0157] In some embodiments, the enzyme mixture and mother liquor are incubated for at least 6 hours. In further embodiments, the enzyme mixture and mother liquor are incubated for between 14 and 30 hours.

[0158] Optionally, the method of the first aspect of this disclosure further includes the step of inactivating the enzyme mixture at the end of the incubation period. In one embodiment, the inactivation step includes heat treatment of the incubation mixture to inactivate the enzyme mixture. For example, the incubation mixture may be heated to a temperature of at least 90°C, such as to a temperature in the range of 90-110°C or 95-100°C. Alternatively or additionally, the inactivation step may include quenching the incubation mixture with a suitable acid such as formic acid or acetic acid.

[0159] Residual inactivated enzymes can be removed by filtration, centrifugation, or other suitable separation techniques.

[0160] Optionally, the method of the first aspect of this disclosure further includes a step of removing the solvent at the end of the incubation period, typically after or simultaneously with the step of inactivating the enzyme mixture.

[0161] Optionally, the method of the first aspect of this disclosure further includes the step of purifying one or more steviol glycosides obtained to obtain a purified composition. Any conventional purification technique known to those skilled in the art may be employed, including but not limited to solvent evaporation, drying, precipitation, (re)crystallization, filtration, centrifugation, chromatography, two-phase solvent washing, treatment with ion exchange resins, treatment with activated carbon, and any combination thereof.

[0162] In some embodiments, the purification step produces a solid purified composition. In other embodiments, the purification step produces a liquid purified composition, such as a solution of one or more steviol glycosides.

[0163] Typically, the purification step ensures that the total steviol glycoside content of the purified composition is ≥95% on a dry weight basis. In some embodiments, the total steviol glycoside content of the purified composition is ≥98% or ≥99% on a dry weight basis.

[0164] In cases where one or more steviol glycosides are obtained, comprising a mixture of steviol glycosides, in some embodiments, the purified composition is a purified mixture of steviol glycosides.

[0165] In other embodiments, the purified composition comprises ≥95% by dry weight of a single steviol glycoside, such as rebaudioside M, rebaudioside D, or rebaudioside I. Typically, in such embodiments, the purified composition comprises ≥98% or ≥99% by dry weight of a single steviol glycoside.

[0166] In certain embodiments, the purified composition comprises ≥95% rebaudioside M by dry weight. Typically, in such embodiments, the purified composition comprises ≥98% or ≥99% rebaudioside M by dry weight.

[0167] enzymatic conversion products

[0168] As described above, according to a second aspect of this disclosure, a product comprising one or more steviol glycosides is provided, wherein the product is obtained by or can be obtained by the method of the first aspect of this disclosure. In some embodiments, the product is a crude product obtained or available after the incubation step, optionally after a step of inactivating the enzyme mixture and / or removing the solvent. In other embodiments, the product is a purified composition as described above.

[0169] In some embodiments, this disclosure provides a product comprising one or more steviol glycosides, said one or more steviol glycosides being produced by: adding an enzyme mixture to a stevia substance during an extraction process, said stevia substance being an extract, byproduct, or waste product; incubating said enzyme mixture and stevia substance; and obtaining one or more steviol glycosides from said enzyme mixture and stevia substance.

[0170] A product comprising one or more steviol glycosides produced by the methods of this disclosure may comprise a mixture of 7-15% rebaudioside A, 6-14% rebaudioside D, 0.2-4% rebaudioside E, 1-9% rebaudioside I, and 49-66% rebaudioside M by dry weight, with the remainder being stevia extract. In another embodiment, the product comprises a mixture of 0.2-3% rebaudioside A, 1-8% rebaudioside D, 3-10% rebaudioside E, 1-8% rebaudioside I, and 74-94% rebaudioside M by dry weight, with the remainder being stevia extract. In another embodiment, the product comprises a mixture of 0.2-3% rebaudine A, 0.2-7% rebaudine D, 0.2-3% rebaudine E, 0.2-8% rebaudine I, and 68-97% rebaudine M by dry weight, and the remainder is mother liquor or stevia extract.

[0171] In one embodiment of the second aspect of this disclosure, the product comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I. More typically, the product comprises rebaudioside M and at least two further steviol glycosides selected from rebaudioside D, rebaudioside E, and rebaudioside I. For example, the product may comprise rebaudioside M and rebaudioside D, optionally rebaudioside E, and optionally rebaudioside I.

[0172] In any embodiment where the product comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I, the content of rebaudioside M is typically 10 to 99% by weight of the total steviol glycoside content of the product. More typically, the content of rebaudioside M is 20 to 95% by weight of the total steviol glycoside content of the product. Even more typically, the content of rebaudioside M is 40 to 90% by weight or 50 to 85% by weight of the total steviol glycoside content of the product. Still more typically, the content of rebaudioside M is 60 to 80% by weight of the total steviol glycoside content of the product.

[0173] When the product contains rebaudioside D, the content of rebaudioside D is typically 0.1 to 99% by weight of the total steviol glycoside content of the product. More typically, the content of rebaudioside D is 0.5 to 40% by weight of the total steviol glycoside content of the product. Even more typically, the content of rebaudioside D is 2 to 20% by weight of the total steviol glycoside content of the product. Still more typically, the content of rebaudioside D is 5 to 15% by weight of the total steviol glycoside content of the product.

[0174] According to a third aspect of this disclosure, a composition comprising rebaudioside M and rebaudioside D is provided, wherein the content of rebaudioside M is 56% to 85% by weight of the total steviol glycoside content of the composition, and wherein the content of rebaudioside D is 2% to 19% by weight of the total steviol glycoside content of the composition.

[0175] Typically, according to a third aspect of this disclosure, the content of rebaudioside M is from 58% to 82% by weight of the total steviol glycoside content of the composition. More typically, the content of rebaudioside M is from 60% to 80% by weight of the total steviol glycoside content of the composition. Typically, the content of rebaudioside D is from 3% to 17% by weight of the total steviol glycoside content of the composition. More typically, the content of rebaudioside D is from 5% to 15% by weight of the total steviol glycoside content of the composition.

[0176] In one embodiment of the third aspect of this disclosure, the composition further comprises rebaudioside E and / or rebaudioside I.

[0177] Typically, the compositions of the third aspect of this disclosure are obtained or available through the methods of the first aspect of this disclosure. For example, the compositions may be obtained or available through the methods of the first or second mode of the first aspect of this disclosure, or through the methods of the third mode of the first aspect of this disclosure, wherein the stevia substance comprises stevia glycosides.

[0178] According to a fourth aspect of this disclosure, a composition is provided comprising rebaudioside M and at least one further steviol glycoside selected from rebaudioside K, rebaudioside N2, and / or rebaudioside H4. Typically, the composition is obtained or is available by a method of the third mode of the first aspect of this disclosure, wherein the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside C as a minor component.

[0179] Typically, the content of rebaudioside M is 10 to 99% by weight of the total steviol glycoside content of the composition of the fourth aspect of this disclosure. More typically, the content of rebaudioside M is 20 to 95% by weight of the total steviol glycoside content of the composition. Even more typically, the content of rebaudioside M is 40 to 90% by weight or 50 to 85% by weight of the total steviol glycoside content of the composition. Still more typically, the content of rebaudioside M is 60 to 80% by weight of the total steviol glycoside content of the composition.

[0180] Typically, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 0.01% by weight to 50% by weight of the total steviol glycoside content of the composition of the fourth aspect of this disclosure. More typically, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 0.1% by weight to 25% by weight of the total steviol glycoside content of the composition. Even more typically, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 0.5% by weight to 10% by weight of the total steviol glycoside content of the composition. Still more typically, the combined content of rebaudioside K, rebaudioside N2, and rebaudioside H4 is from 1% by weight to 5% by weight of the total steviol glycoside content of the composition.

[0181] According to a fifth aspect of this disclosure, a composition comprising a β1-2 and / or β1-3 glucosylated derivative of rebaudioside M and at least one rebaudioside F is provided. Typically, the composition is obtained or can be obtained by a method of the third mode of the first aspect of this disclosure, wherein the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside F as a minor component.

[0182] Typically, the content of rebaudioside M is 10 to 99% by weight of the total steviol glycoside content of the composition of the fifth aspect of this disclosure. More typically, the content of rebaudioside M is 20 to 95% by weight of the total steviol glycoside content of the composition. Even more typically, the content of rebaudioside M is 40 to 90% by weight or 50 to 85% by weight of the total steviol glycoside content of the composition. Still more typically, the content of rebaudioside M is 60 to 80% by weight of the total steviol glycoside content of the composition.

[0183] Typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 0.01% by weight to 50% by weight of the total steviol glycoside content of the composition of the fifth aspect of this disclosure. More typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 0.1% by weight to 15% by weight of the total steviol glycoside content of the composition. Even more typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 0.5% by weight to 10% by weight of the total steviol glycoside content of the composition. Still more typically, the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is from 1% by weight to 5% by weight of the total steviol glycoside content of the composition.

[0184] In one embodiment, which also applies to the fourth and fifth aspects of this disclosure, the composition further comprises at least one further steviol glycoside selected from rebaudioside D, rebaudioside E, and rebaudioside I. More typically, the composition further comprises at least two further steviol glycosides selected from rebaudioside D, rebaudioside E, and rebaudioside I. For example, the composition may further comprise rebaudioside D, optionally rebaudioside E, and optionally rebaudioside I.

[0185] When the compositions of the fourth or fifth aspect of this disclosure contain rebaudioside D, the content of rebaudioside D is typically from 0.1% to 99% by weight of the total steviol glycoside content of the composition. More typically, the content of rebaudioside D is from 0.5% to 40% by weight of the total steviol glycoside content of the composition. Even more typically, the content of rebaudioside D is from 2% to 20% by weight of the total steviol glycoside content of the composition. Still more typically, the content of rebaudioside D is from 5% to 15% by weight of the total steviol glycoside content of the composition.

[0186] In the case where the product or composition of any of the second to fifth aspects of this disclosure contains rebaudioside E, the content of rebaudioside E is typically 0.5 to 30% by weight of the total steviol glycoside content of the product or composition. More typically, the content of rebaudioside E is 2 to 20% by weight of the total steviol glycoside content of the product or composition. Even more typically, the content of rebaudioside E is 5 to 15% by weight of the total steviol glycoside content of the product or composition.

[0187] In the case where the product or composition of any of the second to fifth aspects of this disclosure contains rebaudioside I, the content of rebaudioside I is typically 0.1 to 99% by weight of the total steviol glycoside content of the product or composition. More typically, the content of rebaudioside I is 0.5 to 25% by weight of the total steviol glycoside content of the product or composition. Even more typically, the content of rebaudioside I is 1 to 10% by weight of the total steviol glycoside content of the product or composition. Still more typically, the content of rebaudioside I is 1.5 to 5% by weight of the total steviol glycoside content of the product or composition.

[0188] In some embodiments, the product or composition of any of the second to fifth aspects of this disclosure comprises rebaudioside A. Rebaudioside A may be present as a glycosylated product or as residual unreacted rebaudioside A from stevia substances. For example, the content of rebaudioside A may be from 0.1 to 50% by weight of the total steviol glycoside content of the product or composition. More typically, in such embodiments, the content of rebaudioside A is from 0.5 to 25% by weight of the total steviol glycoside content of the product or composition. Even more typically, the content of rebaudioside A is from 1 to 15% by weight of the total steviol glycoside content of the product or composition.

[0189] Alternatively, the product or composition of any of the second to fifth aspects of this disclosure may substantially not contain rebaudioside A. For example, the content of rebaudioside A may be <1% by weight of the total steviol glycoside content of the product or composition. More typically, in such embodiments, the content of rebaudioside A is <0.1% by weight or <0.01% by weight of the total steviol glycoside content of the product or composition.

[0190] Typically, the products or compositions of any of the second to fifth aspects of this disclosure substantially do not contain steviol glycosides. For example, the steviol glycoside content may be <1% by weight of the total steviol glycoside content of the product or composition. More typically, the steviol glycoside content is <0.1% by weight or <0.01% by weight of the total steviol glycoside content of the product or composition.

[0191] Typically, the products or compositions of any of the second to fifth aspects of this disclosure substantially do not contain steviol glycosides. For example, the steviol glycoside content may be <1% by weight of the total steviol glycoside content of the product or composition. More typically, the steviol glycoside content is <0.1% by weight or <0.01% by weight of the total steviol glycoside content of the product or composition.

[0192] Typically, the total steviol glycoside content of the product or composition of any of the second to fifth aspects of this disclosure is ≥50% on a dry weight basis. More typically, the total steviol glycoside content of the product or composition is ≥60% on a dry weight basis. Even more typically, the total steviol glycoside content of the product or composition is ≥75% on a dry weight basis. For example, the product or composition may have a total steviol glycoside content of ≥85% or ≥95% on a dry weight basis.

[0193] In some embodiments of any of the second to fifth aspects of this disclosure, the product or composition is a solid. In other embodiments, the product or composition is a liquid, typically an aqueous solution.

[0194] Typically, according to any of the second to fifth aspects of this disclosure, the product or composition is suitable for human consumption. Typically, according to any of the second to seventh aspects of this disclosure, the product or composition is suitable for use as a sweetener.

[0195] application

[0196] Products and compositions produced by the methods of this disclosure can be blended with further components suitable for human and / or animal consumption to produce sweetener compositions, foods, beverages, etc. One or more steviol glycosides produced by the methods of this disclosure can be at least partially purified before use in the product.

[0197] Therefore, a sixth aspect of this disclosure provides a method for preparing a sweetener composition, the method comprising the steps of obtaining one or more steviol glycosides by the method of a first aspect of this disclosure, and blending the obtained steviol glycosides with one or more auxiliary sweeteners. Similarly, a seventh aspect of this disclosure provides a method for preparing a sweetener composition, the method comprising the step of blending a product or composition of any of the second to fifth aspects of this disclosure with one or more auxiliary sweeteners. A corresponding eighth aspect of this disclosure provides a sweetener composition comprising a product or composition of any of the second to fifth aspects of this disclosure and one or more auxiliary sweeteners. Typically, according to the sixth, seventh, and eighth aspects of this disclosure, one or more auxiliary sweeteners are selected from stevia extract, steviol glycosides, glycosylated steviol glycosides, monk fruit extract, mogroside, glycosylated mogroside, sucrose, glucose, fructose, high-fructose corn syrup, sucralose, saccharin, acesulfame K, sodium saccharin, aspartame, neotame, and adventitia. In some embodiments, one or more auxiliary sweeteners are selected from monk fruit extract, mogroside, glycosylated mogroside, sucrose, glucose, fructose, high-fructose corn syrup, sucralose, saccharin, acesulfame K, sodium saccharin, aspartame, neotame, and adventitia. Typically, according to a sixth aspect of this disclosure, the obtained steviol glycoside is blended with one or more auxiliary sweeteners at a dry weight ratio of 5:95 to 95:5. Typically, according to seventh and eighth aspects of this disclosure, the dry weight ratio of one or more auxiliary sweeteners to the product or composition of any of the second to fifth aspects of this disclosure is 5:95 to 95:5.

[0198] In some embodiments, the product comprising one or more steviol glycosides produced by the methods of this disclosure is a consumer product intended for human and / or animal consumption. In some embodiments, the consumer product is a food, beverage, pharmaceutical, nutritional supplement, or nutritional health product.

[0199] A ninth aspect of this disclosure provides a method for preparing a food or beverage, the method comprising the steps of: obtaining one or more steviol glycosides by the method of a first aspect of this disclosure; and blending the obtained steviol glycosides with a food, beverage, or an ingredient thereof. Similarly, a tenth aspect of this disclosure provides a method for preparing a food or beverage, the method comprising the step of blending a product or composition of any of the second to fifth aspects of this disclosure with a food, beverage, or an ingredient thereof. A corresponding eleventh aspect of this disclosure provides a food or beverage comprising a product or composition of any of the second to fifth aspects of this disclosure.

[0200] The twelfth aspect of this disclosure provides a method for preparing a food or beverage, the method comprising the steps of: preparing a sweetener composition by the method of the sixth or seventh aspect of this disclosure, and blending the sweetener composition with a food, beverage, or an ingredient thereof. Similarly, the thirteenth aspect of this disclosure provides a method for preparing a food or beverage, the method comprising the step of blending a sweetener composition of the eighth aspect of this disclosure with a food, beverage, or an ingredient thereof. A corresponding fourteenth aspect of this disclosure provides a food or beverage comprising the sweetener composition of the eighth aspect of this disclosure.

[0201] Typically, the food or beverage obtained from any of the ninth to fourteenth aspects of this disclosure contains 0.0001 to 1% by weight of steviol glycosides.

[0202] In some implementations, when the consumer product is food, the food is confectionery, desserts, cereals, baked goods, frozen dairy products, meat, dairy products, condiments, snack bars, nutrition bars, soups, seasonings, mixed ingredients, prepared foods, baby food, weight-loss foods, syrups, food coatings, dried fruits, sauces, gravy, jams, or jellies.

[0203] In some implementations, when the consumer product is a beverage, the beverage is an alcoholic beverage, a carbonated soft drink, a fruit juice, tea, or a smoothie.

[0204] The fifteenth aspect of this disclosure provides a method for preparing a pharmaceutical product, nutritional product, or nutritional supplement, the method comprising the steps of: obtaining one or more steviol glycosides by the method of the first aspect of this disclosure, and blending the obtained steviol glycoside with a pharmaceutical product, nutritional product, nutritional supplement, or a precursor thereof. Similarly, the sixteenth aspect of this disclosure provides a method for preparing a pharmaceutical product, nutritional product, or nutritional supplement, the method comprising the step of blending a product or composition of any of the second to fifth aspects of this disclosure with a pharmaceutical product, nutritional product, nutritional supplement, or a precursor thereof. A corresponding seventeenth aspect of this disclosure provides a pharmaceutical product, nutritional product, or nutritional supplement, wherein the pharmaceutical product, nutritional product, or nutritional supplement comprises a product or composition of any of the second to fifth aspects of this disclosure.

[0205] The eighteenth aspect of this disclosure provides a method for preparing a pharmaceutical product, nutritional product, or nutritional supplement, the method comprising the steps of: preparing a sweetener composition by the method of the sixth or seventh aspect of this disclosure; and blending the sweetener composition with a pharmaceutical product, nutritional product, nutritional supplement, or a precursor thereof. Similarly, the nineteenth aspect of this disclosure provides a method for preparing a pharmaceutical product, nutritional product, or nutritional supplement, the method comprising the step of blending the sweetener composition of the eighth aspect of this disclosure with a pharmaceutical product, nutritional product, nutritional supplement, or a precursor thereof. The corresponding twentieth aspect of this disclosure provides a pharmaceutical product, nutritional product, or nutritional supplement, wherein the pharmaceutical product, nutritional product, or nutritional supplement comprises the sweetener composition of the eighth aspect of this disclosure.

[0206] Typically, the pharmaceutical products, nutritional products or nutritional supplements obtained from any of the fifteenth to twentieth aspects of this disclosure contain 0.0001 to 5% by weight of steviol glycosides.

[0207] In some implementations, the product is a nutritional supplement.

[0208] Various exemplary embodiments of the compositions and methods according to this disclosure are now described by the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. In fact, various modifications to this disclosure other than those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples, and all such modifications fall within the scope of the appended claims.

[0209] Example

[0210] The following examples illustrate specific implementations of this disclosure and their various uses. They are given for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way.

[0211] In each of the following enzyme incubation steps, an aqueous solution of stevia material buffered to pH 6.5 (e.g., early extract or mother liquor product) is incubated at 65°C for a specified period of time in the presence of ADP-glucose-dependent glucosyltransferase (concentrations of 0.1 and 0.4 g / L), sucrase synthase (concentration of 0.1 g / L), sucrose (concentration of 150 g / L), ADP (concentration of 0.1 to 0.5 g / L), and EDTA (concentration of 1.9 g / L).

[0212] Example 1: Early Stevia Extract and Reaction

[0213] 1.0 hour

[0214] Early extracts from stevia production contain the glycosides detailed in Table 1. Early extracts are minimally processed stevia extracts containing 60-95% total steviol glycosides by dry weight. In this example, the early extract contains 83.8% total steviol glycosides by dry weight.

[0215] Table 1.

[0216]

[0217]

[0218] 2.2, 5 and 7 hours

[0219] The early extract was dissolved in water to obtain a solution with a total steviol glycoside content of 8.03 g / 100 mL. After 2, 5, and 7 hours of enzyme reaction, the early extract from Table 1 was converted into the glycosides detailed in Table 2. The mass of the stevia components increased due to the increase in molecular weight of the steviol glycosides during enzymatic biotransformation. After 2, 5, and 7 hours of biotransformation, the mass of the reacted steviol glycosides increased by approximately 18%, 27%, and 31% compared to the original extract, respectively.

[0220] Table 2.

[0221]

[0222] Example 2: Mother liquor products and reactions

[0223] 1.0 hour

[0224] The mother liquor is a byproduct stream obtained from the crystallization of stevia feedstock, containing 40-95% total steviol glycosides by dry weight. In this embodiment, a batch of mother liquor containing 77.8% total steviol glycosides by dry weight was dissolved in water to obtain a solution with a total Reb M (Rebaudioside M) convertible content of 4.89 g / 100 mL. This solution contains the steviol glycosides detailed in Table 3.

[0225] Table 3.

[0226] Reb M convertibles by weight %* Lebodiin A 44.30％ Lebodiin D 4.37％ Lebodiin M 2.03％ Lebodiin E 0.92％ Lebodiin I 0％ Stevioside 46.56％ Betaine 1.82％

[0227] *The weight percentage of Reb M convertibles = the weight of each of the described steviol glycosides relative to the total content of Rebaudioside A + Rebaudioside D + Rebaudioside M + Rebaudioside E + Rebaudioside I + Stevioside + Stevia.

[0228] 2.14 hours

[0229] After 14 hours of reaction with the enzyme, the mother liquor product from Table 3 was converted into the glycosides detailed in Table 4. Due to the mass increase associated with this reaction, the amount of steviol glycosides after the reaction increased by approximately 37% compared to the original mother liquor.

[0230] Table 4.

[0231] % by weight of Reb M convertible Lebodiin A 1.06％ Lebodiin D 3.99％ Lebodiin M 77.58％ Lebodiin E 13.64％ Lebodiin I 3.73％ Stevioside 0％ Betaine 0％

[0232] Example 3: Purification of early stevia extract

[0233] The final product from the reaction shown in Example 1 can be purified by carbon treatment, adsorption-desorption resin, and drying, with an expected yield of 108% relative to the original steviol glycoside content. Further purification can be achieved by solvent washing to obtain a yield of 90% relative to the original steviol glycoside.

[0234] Example 4: Purification of Mother Liquor Product

[0235] The substance from the reaction shown in Example 2 can be purified by filtration and dried, with an expected yield of 104% relative to the original steviol glycoside content.

[0236] Example 5: Glycosylation

[0237] Rebaudioside C was glycosylated using one or both of ADP, sucrase synthase, sucrose, and β1-2 and β1-3 ADP-glucose-dependent glycosyltransferases. After 24 hours of exposure to the enzymatic reaction conditions, rebaudioside C was almost completely converted to rebaudioside K or rebaudioside N2. However, conversion to rebaudioside H4 was relatively slow. Conversion of rebaudioside C to a derivative containing 5 glucosyl residues and 1 rhamnosyl residue resulted in a 34.1% increase in mass.

[0238] Figures 5A-5C The steviol glycosides in the substances shown include steviol glycosides having one xylose residue and four glucose residues linked to the steviol core (i.e., rebaudioside F). The products from this reaction are shown in LC-MS as steviol glycosides containing glycosylated xylose. The three products formed contain five glucose residues and one xylose residue. This indicates the existence of multiple xylose substitution modes, where the glucose residues fill the other available 2 and 3 positions of the glucose residue directly linked to steviol. The conversion of rebaudioside F to a derivative containing five glucose residues and one xylose residue resulted in a 34.6% increase in mass.

[0239] Example 6: Mother liquor products and reactions

[0240] 1.0 hour

[0241] A batch of mother liquor containing 62.3% total steviol glycosides by dry weight was dissolved in water to obtain a solution with a total steviol glycoside content of 8.02 g / 100 mL. The solution contained the steviol glycosides detailed in Table 5.

[0242] Table 5.

[0243] Total steviol glycosides content (%) Lebodiin A 38.14％ Lebodiin D 4.3％ Lebodiin M 5.91％ Lebodiin I 0.26％ Stevioside 16.24％ Betaine 1.75％

[0244] 2.27 hours

[0245] After 27 hours of reaction with the enzyme, the mother liquor product from Table 4 was converted into the glycosides detailed in Table 6.

[0246] Table 6.

[0247] Total steviol glycosides content (%) Lebodiin A 0％ Lebodiin D 8.51％ Lebodiin M 71.63％ Lebodiin I 1.93％ Stevioside 0％ Betaine 0％

[0248] Example 7: Purification of Mother Liquor Product

[0249] The material from the reaction shown in Example 6 can be purified by filtration and dried to obtain a product containing 92.68% Reb M and 101.96% total steviol glycosides by dry weight.

[0250] Example 8: Sensory evaluation of the separated products of Example 1.

[0251] After 7 hours, the substance from Example 1 was separated and dried to obtain a product containing 12.4% Reb A, 13.01% Reb D, 58.8% Reb M, and 96.4% total steviol glycosides, all percentages on a dry weight basis. Sensory evaluation of this substance was compared with commercial RA60-TSG95 and RA97 samples. The results are shown in Table 7. Samples were prepared in neutral water at a concentration of 500 ppm. The sweetness intensity, off-flavor, and palatability of these solutions were evaluated by a team of eight trained specialists. Sweetness intensity was compared with sucrose standards (sucrose equivalent, SEV), while other attributes were rated using a 0-7 scale.

[0252] Table 7

[0253] ppm SEV palatability Odor Enzyme conversion products 500 9.8 4.3 1.6 Lebodiin A60 500 6.6 3.6 2.4 Lebodiin A97 500 7.7 3.5 2.8

[0254] It can be seen that at a concentration of 500 pm, the product prepared according to this disclosure produces excellent sweetness and palatability, and has less off-flavor than commercially available products.

[0255] Invention entries:

[0256] Article 1: A method for obtaining one or more steviol glycosides, said method comprising the following steps:

[0257] (a) An enzyme mixture is added to a stevia substance during the extraction process, wherein the stevia substance is an extract, a by-product, or a waste product;

[0258] (b) Incubate the enzyme mixture and stevia substance; and

[0259] (c) Obtain one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0260] Article 2: According to the method of Article 1, the enzyme mixture comprises glycosyltransferase and sucrose synthase.

[0261] Article 3: The method according to Article 1, wherein the stevia substance comprises an extract.

[0262] Article 4: The method according to Article 1, wherein the stevia substance comprises waste products.

[0263] Article 5: According to the method of Article 4, the waste product comprises a mother liquor byproduct stream obtained from the crystallization of the stevia stream.

[0264] Article 6: According to the method of Article 5, the mother liquor contains about 40% to about 95% of steviol glycosides on a dry weight basis.

[0265] Article 7: According to the method of Article 1, the steviol glycoside comprises one or more of rebaudioside A, rebaudioside D, rebaudioside M, rebaudioside E, rebaudioside I, steviol glycoside and / or stevioside.

[0266] Article 8: The method according to Article 7, wherein the steviol glycoside comprises rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside I and / or rebaudioside M.

[0267] Article 9: According to the method of Article 8, the steviol glycoside contains about 1% to about 38% of rebaudioside A.

[0268] Article 10: According to the method of Article 9, wherein the steviol glycoside comprises about 11% rebaudioside A.

[0269] Article 11: The method according to Article 8, wherein the steviol glycoside comprises about 10% to about 33% of rebaudioside D.

[0270] Article 12: According to the method of Article 11, the steviol glycoside contains about 13% rebaudioside D.

[0271] Article 13: According to the method of Article 8, the steviol glycoside contains about 1% to about 5% of rebaudioside I.

[0272] Article 14: The method according to Article 13, wherein the steviol glycoside comprises about 4% rebaudioside I.

[0273] Article 15: According to the method of Article 8, the steviol glycoside contains about 21% to about 78% of rebaudioside M.

[0274] Article 16: According to the method of Article 15, the steviol glycoside contains about 60% rebaudioside M.

[0275] Article 17: According to the method of Article 1, the steviol glycosides contained in the method comprised about 0% steviol glycosides and steviosides.

[0276] Article 18: The method according to Article 3, wherein the steviol glycoside comprises rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside I and / or rebaudioside M.

[0277] Article 19: The method according to Article 18, wherein the steviol glycoside comprises about 0% to about 2% rebaudioside A.

[0278] Article 20: The method according to Article 19, wherein the steviol glycoside comprises about 1% rebaudioside A.

[0279] Article 21: The method according to Article 18, wherein the steviol glycoside comprises about 0% to about 13% of rebaudioside D.

[0280] Article 22: The method according to Article 21, wherein the steviol glycoside comprises about 3% rebaudioside D.

[0281] Article 23: The method according to Article 18, wherein the steviol glycoside comprises about 0% to about 10% of rebaudioside E.

[0282] Article 24: The method according to Article 23, wherein the steviol glycoside comprises about 6% rebaudioside E.

[0283] Article 25: The method according to Article 18, wherein the steviol glycoside comprises about 0% to about 5% of rebaudioside I.

[0284] Article 26: The method according to Article 25, wherein the steviol glycoside comprises about 4% rebaudioside I.

[0285] Article 27: The method according to Article 18, wherein the steviol glycoside comprises about 65% to about 90% of rebaudioside M.

[0286] Article 28: The method according to Article 27, wherein the steviol glycoside comprises about 86% rebaudioside M.

[0287] Article 29: The method according to Article 5, wherein the steviol glycoside comprises rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside I and / or rebaudioside M.

[0288] Article 30: The method according to Article 29, wherein the steviol glycoside comprises about 0% to about 2% of rebaudioside A.

[0289] Article 31: The method according to Article 30, wherein the steviol glycoside comprises about 1% rebaudioside A.

[0290] Article 32: The method according to Article 29, wherein the steviol glycoside comprises about 0% to about 17% of rebaudioside D.

[0291] Article 33: The method according to Article 32, wherein the steviol glycoside comprises about 4% rebaudioside D.

[0292] Article 34: The method according to Article 29, wherein the steviol glycoside comprises about 0% to about 13% of rebaudioside E.

[0293] Article 35: The method according to Article 34, wherein the steviol glycoside comprises about 1% rebaudioside E.

[0294] Article 36: The method according to Article 29, wherein the steviol glycoside comprises about 1% to about 4% of rebaudioside I.

[0295] Article 37: The method according to Article 36, wherein the steviol glycoside comprises about 3% rebaudioside I.

[0296] Article 38: The method according to Article 29, wherein the steviol glycoside comprises about 60% to about 80% of rebaudioside M.

[0297] Article 39: The method according to Article 38, wherein the steviol glycoside comprises about 75% rebaudioside M.

[0298] Article 40: The method of Article 1, wherein the enzyme mixture and stevia substance are incubated for at least 2 hours.

[0299] Article 41: The method according to Article 40, wherein the enzyme mixture and stevia substance are incubated for between 4 and 8 hours.

[0300] Article 42: The method according to Article 3, wherein the enzyme mixture and extract are incubated for between 4 and 24 hours.

[0301] Article 43: The method of Article 5, wherein the enzyme mixture and mother liquor are incubated for at least 6 hours.

[0302] Article 44: The method according to Article 43, wherein the enzyme mixture and mother liquor are incubated for between 14 and 30 hours.

[0303] Article 45: The method according to Article 1, wherein the amount of steviol glycosides is at least about 30% higher than the amount of steviol glycosides in the stevia substance.

[0304] Article 46: The method according to Article 45, wherein the amount of steviol glycosides is at least about 100% higher than the amount of steviol glycosides in the stevia substance.

[0305] Article 47: The method according to Article 3, wherein the amount of steviol glycosides is at least about 38% higher than the amount of steviol glycosides in the extract.

[0306] Article 48: The method according to Article 47, wherein the amount of steviol glycosides is at least 100% higher than the amount of steviol glycosides in the extract.

[0307] Article 49: According to the method of Article 5, the amount of steviol glycosides is at least about 35% higher than the amount of steviol glycosides in the mother liquor.

[0308] Article 50: The method according to Article 49, wherein the amount of steviol glycosides is at least about 100% higher than the amount of steviol glycosides in the mother liquor.

[0309] Article 51: The method according to Article 1, wherein the steviol glycoside comprises about 60% to about 95% of minimally processed stevia.

[0310] Article 52: According to the method of Article 1, wherein based on the conversion of rebaudioside D to rebaudioside M and / or stevia to rebaudioside M, the enzyme mixture will increase the mass enrichment efficiency by 14% to 100%.

[0311] Article 53: The method according to Article 1 further includes purifying the said steviol glycoside.

[0312] Article 54: According to the method of Article 53, at least 85% of the purified steviol glycosides comprise rebaudioside A, rebaudioside D, rebaudioside M, rebaudioside E, rebaudioside I, steviol glycoside or stevioside.

[0313] Article 55: A method for obtaining a mixture of one or more steviol glycosides, the method comprising the following steps:

[0314] (a) An enzyme mixture is added to a stevia substance during the extraction process, wherein the stevia substance is an extract, a byproduct, or a waste product;

[0315] (b) Incubate the enzyme mixture and stevia substance; and

[0316] (c) Obtain a mixture of one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0317] Article 56: A mixture of one or more steviol glycosides produced by the method of Article 55, wherein the mixture comprises rebaudioside M, rebaudioside A, rebaudioside D, or a combination thereof.

[0318] Article 57: The mixture according to Article 56, wherein the mixture contains at least 50% lebodiin M.

[0319] Article 58: The mixture according to Article 56, wherein the mixture comprises at least 80% rebaudioside M and at least one other steviol glycoside.

[0320] Article 59: The mixture according to Article 56, wherein the mixture contains at least 15% rebaudioside A.

[0321] Article 61: A mixture according to Article 56, wherein the mixture contains at least 15% rebaudioside D.

[0322] Article 62: A method for obtaining a purified mixture of one or more steviol glycosides, the method comprising the following steps:

[0323] (a) An enzyme mixture is added to a stevia substance during the extraction process, wherein the stevia substance is an extract, a byproduct, or a waste product;

[0324] (b) Incubate the enzyme mixture and stevia substance;

[0325] (c) Obtaining a mixture of one or more steviol glycosides from the enzyme mixture and the steviol substance;

[0326] (d) Purify the mixture of one or more steviol glycosides.

[0327] Article 63: A purified mixture of one or more steviol glycosides produced by the method of Article 62, wherein the mixture comprises rebaudioside M, rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside I, or a combination thereof.

[0328] Article 64: A purified mixture according to Article 63, wherein the purified mixture contains at least 50% rebaudioside M.

[0329] Article 65: A purified mixture according to Article 64, wherein the purified mixture comprises at least 50% rebaudioside M and at least one other steviol glycoside.

[0330] Article 66: A purified mixture according to Article 63, wherein the purified mixture contains at least 10% rebaudioside A.

[0331] Article 67: A purified mixture according to Article 63, wherein the purified mixture contains at least 10% rebaudioside D.

[0332] Article 68: A purified mixture according to Article 64, wherein the purified mixture contains at least 70% rebaudioside M.

[0333] Article 69: A purified mixture according to Article 68, wherein the purified mixture comprises at least 90% rebaudioside M and at least one other steviol glycoside.

[0334] Article 70: The purified mixture according to Article 65, wherein the at least one other steviol glycoside constitutes less than 5% of the mixture.

[0335] Article 71: A product comprising one or more steviol glycosides produced by means of:

[0336] (a) An enzyme mixture is added to a stevia substance during the extraction process, wherein the stevia substance is an extract or a waste product;

[0337] (b) Incubate the enzyme mixture and stevia substance; and

[0338] (c) Obtain one or more steviol glycosides from the enzyme mixture and the stevia substance.

[0339] Article 72: Products pursuant to Article 71, wherein the product is a consumer product.

[0340] Article 73: Consumer goods pursuant to Article 72, wherein the consumer goods are food, beverages, medicines, nutritional products or nutritional supplements.

[0341] Article 74: Consumer products pursuant to Article 73, wherein the food products mentioned are confectionery products, dessert products, cereal products, baked goods, frozen dairy products, meat products, dairy products, condiments, snack bars, nutrition bars, soups, seasonings, mixed ingredients, prepared foods, baby foods, weight-loss foods, syrups, food coatings or frostings, dried fruits, sauces, gravy or jam or jelly.

[0342] Article 75: Beverages pursuant to Article 73, wherein the beverage is an alcoholic beverage, a carbonated soft drink, a fruit juice, tea, or a smoothie.

[0343] Article 76: The product according to Article 71, wherein the one or more steviol glycosides are at least partially purified.

[0344] Article 77: The product according to Article 71, wherein the one or more steviol glycosides comprises a mixture of 7-15% rebaudioside A, 6-14% rebaudioside D, 0.2-4% rebaudioside E, 1-9% rebaudioside I, 49-66% rebaudioside M, and the remainder is a stevia extract.

[0345] Article 78: The product according to Article 71, wherein the one or more steviol glycosides comprises a mixture of 0.2-3% rebaudioside A, 1-8% rebaudioside D, 3-10% rebaudioside E, 1-8% rebaudioside I, 74-94% rebaudioside M, and the remainder is a stevia extract.

[0346] Article 79: The product according to Article 71, wherein the one or more steviol glycosides comprises a mixture of 0.2-3% rebaudioside A, 0.2-7% rebaudioside D, 0.2-3% rebaudioside E, 0.2-8% rebaudioside I, and 68-97% rebaudioside M, and the remainder is a mother liquor.

Claims

1. A method for obtaining one or more steviol glycosides, the method comprising the following steps: (a) Add the enzyme mixture to the stevia substance; (b) Incubate the enzyme mixture and stevia substance; and (c) Obtaining one or more steviol glycosides from the enzyme mixture and the steviol substance; The enzyme mixture contains one or more nucleoside-glucose-dependent glycosyltransferases.

2. The method according to claim 1, wherein the total steviol glycoside content of the stevia substance is ≤90% on a dry weight basis.

3. The method according to claim 2, wherein the total steviol glycoside content of the stevia substance is 60% to 85% on a dry weight basis.

4. The method according to claim 2 or claim 3, wherein the stevia substance comprises rebaudioside A, steviol glycoside, optionally one or more other steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M and stevia glycoside, and optionally one or more further steviol glycosides.

5. The method of claim 4, wherein the one or more steviol glycosides obtained comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E and rebaudioside I.

6. The method according to claim 4 or claim 5, wherein the total content of rebaudioside A and steviol glycosides in the stevia substance is 50 to 95% by weight of the total steviol glycoside content of the stevia substance.

7. The method according to any one of claims 1 to 5, wherein the stevia substance comprises rebaudioside A, steviol glycoside, and rebaudioside D.

8. The method of claim 7, wherein the stevia substance further comprises one or more additional steviol glycosides selected from rebaudioside E, rebaudioside I, rebaudioside M and stevia glycoside, and optionally one or more further steviol glycosides.

9. The method according to claim 7 or claim 8, wherein the one or more steviol glycosides obtained comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E and rebaudioside I.

10. The method according to any one of claims 7 to 9, wherein the total content of rebaudioside A and steviol glycosides in the stevia substance is 50 to 95% by weight of the total steviol glycoside content of the stevia substance.

11. The method according to any one of claims 7 to 10, wherein the content of rebaudine A is 20 to 80% by weight of the total steviol glycosides of the stevia substance, the content of steviol glycoside is 10 to 60% by weight of the total steviol glycosides of the stevia substance, and the content of rebaudine D is 0.5 to 15% by weight of the total steviol glycosides of the stevia substance.

12. The method according to claim 11, wherein the content of rebaudine A is 35 to 60% by weight of the total steviol glycosides of the stevia substance, the content of steviol glycoside is 15 to 50% by weight of the total steviol glycosides of the stevia substance, and the content of rebaudine D is 2 to 5% by weight of the total steviol glycosides of the stevia substance.

13. The method according to any one of claims 2 to 12, wherein: (i) The stevia substance comprises rebaudioside E, and the content of rebaudioside E is 0.1 to 5% by weight of the total steviol glycoside content of the stevia substance; and / or (ii) The stevia substance comprises rebaudioside I, and the content of rebaudioside I is 0.05 to 2% by weight of the total steviol glycoside content of the stevia substance; and / or (iii) The stevia substance comprises rebaudioside M, and the content of rebaudioside M is 0.1 to 15% by weight of the total steviol glycoside content of the stevia substance; and / or (iv) The stevia substance comprises stevia glycosides, and the content of stevia glycosides is 0.1 to 5% by weight of the total steviol glycoside content of the stevia substance.

14. The method according to claim 1, wherein the stevia substance comprises steviol glycosides and / or rhamnosyl-containing steviol glycosides and / or xylosyl-containing steviol glycosides.

15. The method of claim 14, wherein the stevia substance comprises stevia glycoside, and the one or more stevia glycosides obtained comprise rebaudioside M and at least one further stevia glycoside selected from rebaudioside D, rebaudioside E and rebaudioside I.

16. The method of claim 14 or claim 15, wherein the stevia substance comprises rebaudioside C, and the one or more stevia glycosides obtained comprise at least one stevia glycoside selected from rebaudioside K, rebaudioside N2 and rebaudioside H4.

17. The method according to any one of claims 14 to 16, wherein the stevia substance comprises rebaudioside F, and the one or more stevia glycosides obtained comprise at least one β1-2 and / or β1-3 glucosylated derivative of rebaudioside F.

18. The method according to any one of claims 14 to 17, wherein: (i) The stevia substance contains ≥75% stevia glycosides on a dry weight basis; or (ii) The stevia substance contains ≥75% rhamnosyl-containing steviol glycosides on a dry weight basis; or (iii) The stevia substance contains ≥75% xylose-containing steviol glycosides on a dry weight basis.

19. The method according to any one of claims 14 to 17, wherein the stevia substance contains 0.01% to 10% by weight of the total steviol glycoside content of the stevia substance.

20. The method according to any one of claims 14 to 17, wherein the content of rebaudioside C of the stevia substance is from 0.1% to 25% by weight of the total steviol glycoside content of the stevia substance.

21. The method according to any one of claims 14 to 17, wherein the content of rebaudioside F in the stevia substance is 0.1% to 15% by weight of the total steviol glycoside content of the stevia substance.

22. The method according to any one of claims 19 to 21, wherein the stevia substance further comprises rebaudioside A, steviol glycoside, optionally one or more additional steviol glycosides selected from rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M and stevia glycoside, and optionally one or more further steviol glycosides.

23. The method of claim 22, wherein the one or more steviol glycosides obtained comprises rebaudioside M and at least one further steviol glycoside selected from rebaudioside D, rebaudioside E and rebaudioside I.

24. The method according to claim 22 or claim 23, wherein the total content of rebaudioside A and steviol glycosides in the stevia substance is 50 to 95% by weight of the total steviol glycoside content of the stevia substance.

25. The method according to any one of claims 1 to 26, wherein the stevia substance is an extract, byproduct, or waste product from the stevia extraction process.

26. The method according to any one of claims 1 to 2526, wherein the enzyme mixture comprises one or more NDP-glucose-dependent β1-2 and / or β1-3 glucosyltransferases.

27. The method according to any one of claims 1 to 26, wherein the enzyme mixture further comprises sucrose synthase, and wherein the enzyme mixture and the stevia substance are incubated in the presence of sucrose and nucleoside diphosphate (NDP).

28. A product comprising one or more steviol glycosides, wherein the product is obtained or is obtainable by the method according to any one of claims 1 to 27.

29. The product according to claim 28, wherein the product comprises, on a dry weight basis, a mixture of 7-15% rebaudine A, 6-14% rebaudine D, 0.2-4% rebaudine E, 1-9% rebaudine I, and 49-66% rebaudine M, and the remainder is stevia extract.

30. The product according to claim 28, wherein the product comprises, on a dry weight basis, 0.2-3% of rebaudine A, 1-8% of rebaudine D, 3-10% of rebaudine E, 1-8% of rebaudine I, 74-94% of rebaudine M, and the remainder being stevia extract.

31. The product according to claim 28, wherein the product comprises, on a dry weight basis, 0.2-3% of rebaudine A, 0.2-7% of rebaudine D, 0.2-3% of rebaudine E, 0.2-8% of rebaudine I, and 68-97% of rebaudine M, and the remainder is mother liquor or stevia extract.

32. A composition comprising rebaudine M and rebaudine D, wherein the content of rebaudine M is 56% to 85% by weight of the total steviol glycosides content of the composition, and wherein the content of rebaudine D is 2% to 19% by weight of the total steviol glycosides content of the composition.

33. The composition according to claim 32, wherein the content of rebaudioside M is 60% to 80% by weight of the total steviol glycoside content of the composition, and wherein the content of rebaudioside D is 5% to 15% by weight of the total steviol glycoside content of the composition.

34. A composition comprising rebaudioside M and at least one further steviol glycoside selected from rebaudioside K, rebaudioside N2 and rebaudioside H4.

35. The composition according to claim 34, wherein the content of rebaudioside M is 20 to 95% by weight of the total steviol glycoside content of the composition, and the combined content of rebaudioside K, rebaudioside N2 and rebaudioside H4 is 0.1% to 25% by weight of the total steviol glycoside content of the composition.

36. A composition comprising rebaudioside M and at least one β1-2 and / or β1-3 glucosylated derivative of rebaudioside F.

37. The composition according to claim 36, wherein the content of rebaudioside M is 20 to 95% by weight of the total steviol glycoside content of the composition, and the combined content of the β1-2 and / or β1-3 glucosylated derivatives of rebaudioside F is 0.1% to 15% by weight of the total steviol glycoside content of the composition.

38. The composition according to any one of claims 34 to 37, wherein the composition further comprises rebaudioside D.

39. The composition according to any one of claims 32 to 38, wherein the composition further comprises rebaudioside E and / or rebaudioside I.

40. The composition according to any one of claims 32 to 39, wherein the stevioside content is <0.1% by weight of the total steviol glycoside content of the composition, and wherein the stevioside content is <0.1% by weight of the total steviol glycoside content of the product or composition.

41. A method for preparing a sweetener composition, the method comprising the steps of: obtaining one or more steviol glycosides by the method according to any one of claims 1 to 27, and blending the obtained steviol glycosides with one or more auxiliary sweeteners.

42. A sweetener composition comprising the product or composition according to any one of claims 28 to 40 and one or more auxiliary sweeteners.

43. A method for preparing a food or beverage, the method comprising the steps of: obtaining one or more steviol glycosides by the method according to any one of claims 1 to 27, and blending the obtained steviol glycosides with the food, the beverage or its components.

44. A food or beverage comprising the product or composition according to any one of claims 28 to 40.

45. A method for preparing a pharmaceutical product, a nutritional product, or a nutritional supplement, the method comprising the steps of: obtaining one or more steviol glycosides by the method according to any one of claims 1 to 27, and blending the obtained steviol glycosides with the pharmaceutical product, the nutritional product, the nutritional supplement, or a precursor thereof.

46. ​​A pharmaceutical product, nutritional product, or nutritional supplement, wherein the pharmaceutical product, nutritional product, or nutritional supplement comprises a product or composition according to any one of claims 28 to 40.

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