Glycosylated steviol glycoside composition and method for producing the glycosylated steviol glycoside composition

Abstract

To provide a method for producing glycosylated steviol glycoside compositions useful as sweeteners and flavor modifiers in food and beverage products and the like.SOLUTION: A production method is improved by the use of basic conditions before, during and / or after enzyme-catalyzed glycosylation of a steviol glycoside composition.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 346,148, filed June 6, 2016, the entire disclosure of which is incorporated herein by reference for all purposes.

[0003] The present invention relates to improved methods for producing glycosylated steviol glycoside compositions, the glycosylated steviol glycoside compositions obtained thereby, sweeteners and blends comprising such glycosylated steviol glycoside compositions, and the use of such glycosylated steviol glycosides as sweeteners and flavor modifiers in edible products such as foods and beverages. [Background technology]

[0004] Recently, there has been considerable interest in the development of high intensity sweeteners based on natural products. Such sweeteners could replace sugar in food and beverage products, potentially lowering the sugar and possibly caloric content of such products. Steviol glycosides extracted and isolated from the leaves of the Stevia plant have been the subject of significant research and commercial interest, as most such compounds are many times sweeter than sugar (sucrose). Older varieties of the Stevia plant contain stevioside as the major steviol glycoside component, along with small amounts of rebaudioside A and other minor steviol glycosides. Although stevioside is approximately 100-300 times sweeter than sucrose, its taste quality is considered significantly inferior to that of rebaudioside A. Thus, new varieties of stevia plants with very high levels of rebaudioside A and processing methods of stevia leaves to obtain highly pure (95%+) rebaudioside A have been developed. However, even highly purified rebaudioside A does not have a taste profile that is entirely comparable to sucrose, and therefore, preparing low sugar foods and beverages using only purified rebaudioside A remains a significant challenge.

[0005] Steviol glycosides are characterized by having a glucose molecule attached to a central steviol (terpenoid) moiety. It has previously been discovered that naturally occurring steviol glycosides, such as stevioside and rebaudioside A, can be modified by allowing alpha-glucosyltransferase to react on an aqueous solution containing one or more steviol glycosides and a glucose donor, such as starch or cyclodextrin, to transfer one or more glucose units from the glucose donor to the steviol glycoside(s). The resulting reaction product, commonly referred to as a "glycosylated steviol glycoside," can have modified / improved sensory properties compared to the starting steviol glycoside(s). Examples of such reactions, which may be considered to involve glycosylation or transglycosylation, are described in U.S. Pat. No. 4,219,571 and U.S. Patent Publication No. 2007 / 0082102.

[0006] However, further improvements in steviol glycoside glycosylation technology would be desirable. For example, the yields of glycosylated steviol glycoside(s) obtained in such reactions tend to be low. Furthermore, the flavor and taste of glycosylated steviol glycosides produced using known methods, while likely superior to the flavor and taste of the starting steviol glycoside(s), are still not entirely ideal in that they differ significantly from the organoleptic properties of sucrose. Summary of the Invention [Means for solving the problem]

[0007] Various non-limiting exemplary aspects of the present invention can be summarized as follows: Mode 1: A method for producing a glycosylated steviol glycoside composition comprising, consisting essentially of, or consisting of contacting a starting steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium having a pH of greater than 7.5 and less than or equal to 10 for a time effective to produce a glycosylated steviol glycoside composition.

[0008] Mode 2: The method of mode 1, wherein the starting steviol glycoside composition consists of, consists essentially of, or consists of one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, rebaudioside M(X), stevioside, steviolbioside, rubusoside, and dulcoside A.

[0009] Mode 3: The method of mode 1, wherein the starting steviol glycoside composition is comprised of at least about 50% rebaudioside B on a dry weight basis.

[0010] Mode 4: The method of mode 1, wherein the starting steviol glycoside composition is comprised of at least about 50% rebaudioside A on a dry weight basis.

[0011] Embodiment 5: The method of embodiment 1, wherein the starting steviol glycoside composition is comprised of at least about 50% stevioside on a dry weight basis.

[0012] Embodiment 6: The method of embodiment 1, wherein the starting steviol glycoside composition is comprised of at least about 10% steviolbioside on a dry weight basis.

[0013] Mode 7: The method according to any one of modes 1 to 6, wherein the glucose donor is selected from the group consisting of oligosaccharides, cyclodextrins and polysaccharides.

[0014] Mode 8: The method according to any one of modes 1 to 6, wherein the glucose donor is selected from the group consisting of starch, dextrin, liquefied starch, and combinations thereof.

[0015] Aspect 9: The method according to any one of aspects 1 to 8, wherein the pH is about 8 to about 9.

[0016] Aspect 10: The method according to any one of aspects 1 to 9, wherein the cyclodextrin glycosyltransferase is obtained or derived from Bacillus stearothermophilus, Bacillus macerans, Bacillus circulans, Bacillus alcalophilus, Bacillus halophilus, a Thermoanaerobacter strain, or a Thermoanaerobium strain.

[0017] Aspect 11: The method according to any one of aspects 1 to 9, wherein the cyclodextrin glycosyltransferase is obtained or derived from a strain of Thermoanaerobacter sp.

[0018] Embodiment 12: The method according to any one of embodiments 1 to 9, wherein the cyclodextrin glycosyltransferase is a non-alkalophilic cyclodextrin glycosyltransferase.

[0019] Mode 13: The method according to any one of modes 1 to 12, wherein the contacting is carried out at a temperature of about 20° C. to about 95° C.

[0020] Embodiment 14: The method of any one of embodiments 1 to 13, wherein the contacting is carried out for a period of about 1 hour to about 250 hours.

[0021] Aspect 15: The method according to any one of aspects 1 to 14, wherein the aqueous medium is a buffered aqueous medium.

[0022] Embodiment 16: The method of any of embodiments 1-15, comprising the additional step of contacting the glycosylated steviol glycoside composition with at least one enzyme capable of cleaving bonds between glucose.

[0023] Aspect 17: The method of any one of aspects 1 to 16, wherein the contacting step is carried out under conditions effective to achieve a conversion of 60% to 85%, or 65% to 80%, of the steviol glycosides present in the starting steviol glycoside composition to glycosylated steviol glycosides.

[0024] Mode 18: A method for producing a glycosylated steviol glycoside composition comprising, consisting essentially of, or consisting of the steps of: a) contacting a starting steviol glycoside composition with a basic aqueous medium or a basic resin to obtain a base-treated steviol glycoside composition; and b) contacting the base-treated steviol glycoside composition, a glucose donor and a cyclodextrin glycosyltransferase in an aqueous medium for a time effective to produce a glycosylated steviol glycoside composition.

[0025] Aspect 19: The method of aspect 18, wherein the starting steviol glycoside composition is contacted with a basic aqueous medium having a pH of 7.5 to 14.

[0026] Embodiment 20: The method according to embodiment 18 or 19, wherein step a) is carried out at a temperature between 20°C and 100°C.

[0027] Embodiment 21: The method according to any of embodiments 18 to 20, wherein step a) is carried out for a period of 0.5 hours to 250 hours or 1 hour to 250 hours.

[0028] Embodiment 22: The method of any of embodiments 18-21, wherein the starting steviol glycoside composition comprises, consists essentially of, or consists of at least one of rebaudioside A or stevioside.

[0029] Embodiment 23: The method of embodiment 22, wherein step a) is carried out under conditions effective to convert at least a portion of rebaudioside A, if present, to rebaudioside B, and at least a portion of stevioside, if present, to steviolbioside.

[0030] Embodiment 24: The method of any of embodiments 18-23, comprising the additional step of contacting the glycosylated steviol glycoside composition with at least one enzyme capable of cleaving bonds between glucose.

[0031] Aspect 25: A method of improving one or more sensory properties of a glycosylated steviol glycoside composition comprising, consisting essentially of, or consisting of contacting the glycosylated steviol glycoside composition with a basic aqueous medium or a basic resin.

[0032] Aspect 26: The method of aspect 25, wherein the glycosylated steviol glycoside composition is contacted with a basic aqueous medium, and the basic aqueous medium has a pH of 7.5 to 14 or 8 to 14.

[0033] Embodiment 27: The method of embodiment 25 or 26, wherein the contacting step is carried out at a temperature between 20° C. and 100° C.

[0034] Mode 28: The method of any one of modes 25 to 27, wherein the glycosylated steviol glycoside composition consists of, consists essentially of, or consists of at least one of glycosylated rebaudioside A or glycosylated stevioside.

[0035] Embodiment 29. The method of embodiment 28, wherein the contacting step is carried out under conditions effective to convert at least a portion of glycosylated rebaudioside A, if present, to glycosylated rebaudioside B, and at least a portion of glycosylated stevioside, if present, to glycosylated steviolbioside.

[0036] Aspect 30: A glycosylated steviol glycoside composition obtained by any of the methods of aspects 1 to 29.

[0037] Aspect 31: A sweetener composition consisting essentially of, consisting of, or consisting of a) a glycosylated steviol glycoside composition and b) 2-8% rebaudioside B, based on the total dry weight of a) the glycosylated steviol glycoside composition and b) rebaudioside B.

[0038] Aspect 32: The sweetener composition according to aspect 31, wherein the glycosylated steviol glycoside composition is obtainable by the method according to any one of aspects 1 to 29.

[0039] Mode 33: A sweetener composition consisting essentially of, or consisting of, a) a first glycosylated steviol glycoside composition and b) a second glycosylated steviol glycoside composition obtainable by the method of any of modes 18 to 24.

[0040] Aspect 34: The sweetener composition according to aspect 33, wherein the first glycosylated steviol glycoside composition is obtainable by the method of any one of aspects 1 to 17.

[0041] Aspect 35: The sweetener composition of aspect 33 or 34, consisting essentially of, or consisting of, a) 65-95% by weight of the first steviol glycoside composition and b) 5-35% by weight of the second glycosylated steviol glycoside composition, based on the total dry weight of a) the first steviol glycoside composition and b) the second glycosylated steviol glycoside composition.

[0042] Aspect 36: A sweetener composition consisting essentially of, or consisting of, a) a first glycosylated steviol glycoside composition and b) a second glycosylated steviol glycoside composition obtainable by the method of any of aspects 25 to 28.

[0043] Aspect 37: The sweetener composition according to aspect 36, wherein the first glycosylated steviol glycoside composition is obtainable by the method of any one of aspects 1 to 17.

[0044] Aspect 38: The sweetener composition of aspect 36 or 37, which consists of, essentially consists of, or consists of a) 50-95% by weight of the first glycosylated steviol glycoside composition and b) 5-50% by weight of the second glycosylated steviol glycoside composition, based on the total dry weight of a) the first steviol glycoside composition and b) the second glycosylated steviol glycoside composition.

[0045] Mode 39: A glycosylated steviol glycoside composition, comprising glycosylated rebaudioside B, glycosylated steviolbioside, steviolbioside, and rebaudioside B, each of which accounts for 5 to 50% or 10 to 25% of the glycosylated steviol glycoside composition on a dry weight basis.

[0046] Aspect 40: A glycosylated steviol glycoside composition, wherein glycosylated rebaudioside B and glycosylated steviolbioside together account for 2-25% or 5-15% of the glycosylated steviol glycoside composition on a dry weight basis.

[0047] Embodiment 41: A food, beverage product, cosmetic or pharmaceutical comprising, consisting essentially of, or consisting of: i) at least one of a) a glycosylated steviol glycoside composition obtainable by a method of any of embodiments 1-29; b) a sweetener composition of any of embodiments 31-38; or c) a glycosylated steviol glycoside composition of embodiment 39 or 40; and ii) at least one additional food, beverage, cosmetic or pharmaceutical ingredient.

[0048] Mode 42: A method for producing a food, beverage product, cosmetic or pharmaceutical product comprising, consisting essentially of, or consisting of combining at least one of a) a glycosylated steviol glycoside composition obtainable by a method of any of modes 1 to 29, b) a sweetener composition of any of modes 31 to 38, or c) a glycosylated steviol glycoside composition of mode 39 or 40, and ii) at least one additional food, beverage, cosmetic or pharmaceutical ingredient.

[0049] Aspect 43: Use of at least one of a) a glycosylated steviol glycoside composition obtained by the method of any one of aspects 1 to 29, b) a sweetener composition of any one of aspects 31 to 38, or c) a glycosylated steviol glycoside composition of aspect 39 or 40 in a food, beverage, cosmetic or pharmaceutical product. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 is a process flow diagram that diagrammatically illustrates various methods according to the present invention and how such methods may be implemented in relation to one another. [Diagram 2] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Diagram 3] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Figure 4] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Diagram 5] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Figure 6] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Figure 7] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Figure 8] 2-8 are LC-MS chromatograms of various steviol glycoside and glycosylated steviol glycoside compositions as further described in the Examples herein. [Figure 9] FIG. 9 illustrates the format of a test sample evaluation form used in connection with obtaining certain test performance data described in the Examples herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] As used herein, the phrase "steviol glycoside" refers to steviol glycoside compounds (steviol glycosides) found in the stevia plant, such as rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M (sometimes also referred to as rebaudioside X), rubusoside, dulcoside, or stevioside. In naturally occurring steviol glycosides, the steviol glucose units are linked in a beta configuration.

[0052] As used herein, "glycosylated steviol glycoside" refers to a glycosylated steviol glycoside (e.g., one, two, three or more glucose moieties are added to a steviol glycoside via a covalent glycosidic bond) at one or more positions. In particular, a glycosylated steviol glycoside is a steviol glycoside in which one or more glucose moieties or alpha 1-4 linked glucose polymers (e.g., maltose, maltotriose, maltotetraose) are introduced into the parent steviol glycoside molecule by 1-4 linkages to a sugar moiety on the parent steviol glycoside. In a glycosylated steviol glycoside, the glucose unit(s) added by the enzymatic glycosylation reaction are linked in alpha configuration. Thus, a glycosylated steviol glycoside has a structure that is different from a naturally occurring steviol glycoside.

[0053] As used herein, the phrase "glycosylated steviol glycoside composition(s)" refers to a composition that contains one or more glycosylated steviol glycosides, but may further contain one or more substances other than glycosylated steviol glycosides (e.g., unreacted steviol glycoside(s) and / or unreacted glucose donor and / or carbohydrate products derived from the glucose donor).

[0054] The present invention includes methods corresponding to at least three main embodiments, generally referred to as embodiment A, embodiment B, and embodiment C, and described in more detail below. Such main embodiments can be practiced independently or in combination with each other. Embodiment A

[0055] Embodiment A involves a method for producing a glycosylated steviol glycoside composition comprising contacting a starting steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium having a pH of greater than 7.5 and less than or equal to 10 for a time effective to produce a glycosylated steviol glycoside composition. It has been discovered that performing glycosylation under such conditions provides one or more of the following advantages compared to CGTase-catalyzed glycosylation at lower pH values: increased glycosylated steviol glycoside yields, glycosylated steviol glycoside compositions with improved taste, and / or glycosylated steviol glycoside compositions with increased sweetness. Starting Steviol Glycoside Composition

[0056] The starting steviol glycoside composition can include one or more steviol glycosides, which can be natural steviol glycosides (i.e., steviol glycosides naturally found in the leaves of the Stevia plant) and / or non-natural steviol glycosides (i.e., steviol glycosides not found in Stevia leaves or other natural sources). The starting steviol glycoside composition can be characterized as a composition having one or more attributes, particularly one or more sensory properties, identified as needing improvement. For example, the attributes needing improvement can be selected from the group consisting of bitterness, sweet aftertaste, sweetness intensity, licorice flavor, astringency, and combinations thereof. The starting steviol glycoside composition can include one or more substances other than steviol glycosides, particularly one or more non-steviol glycosides present in Stevia plant material along with steviol glycosides, extracts obtained or produced from Stevia plant material, or synthetically produced steviol glycoside compositions. However, in various advantageous embodiments of the invention, the starting steviol glycoside composition is comprised of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% by weight total steviol glycoside(s) on a dry solids basis.

[0057] Exemplary steviol glycosides suitable for use in the starting steviol glycoside composition can be selected from the group consisting of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside X(M), rebaudioside N, rebaudioside O, dulcoside A, steviolbioside, rubusoside, and combinations thereof. The starting steviol glycoside composition can be an extract of steviol glycosides from the leaves of the stevia plant, including any of the known varieties of stevia plants (natural and hybrids). Such extracts are generally mixtures of different steviol glycosides (depending on the variety of the stevia plant from which the extract is obtained), with stevioside and rebaudioside A typically being the most abundant steviol glycosides. In one embodiment, the starting steviol glycoside composition can include 10-70% stevioside and 20-70% rebaudioside A, where the total amount of stevioside and rebaudioside A is 70% or more, by dry weight, and other steviol glycosides can optionally be present to provide a total steviol glycoside content of 90% or more, or 95% or more, by dry weight. For example, the starting steviol glycoside composition can be an extract that is comprised of (by dry weight) 28-30% stevioside, 50-55% rebaudioside A, 9-12% rebaudioside C, 1-3% rebaudioside F, and other steviol glycosides, resulting in a total steviol glycoside content of at least 90% or at least 95%. In another embodiment, the starting steviol glycoside composition can be an extract consisting of (on a dry weight basis) 25-30% stevioside, 55-65% rebaudioside A, and other steviol glycosides, resulting in a total steviol glycoside content of at least 95%. In yet another embodiment, the starting steviol glycoside composition can be an extract consisting of (on a dry weight basis) 55-65% rebaudioside A, 20-30% stevioside, and a total of 3-8% rebaudioside C and dulcoside A, resulting in a total steviol glycoside content of at least 90%.Alternatively, stevia extracts having different ratios of steviol glycosides as well as highly purified (e.g., at least 80%, at least 85%, at least 90%, or at least 95% pure) steviol glycosides such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside X(M), rebaudioside N, rebaudioside O, dulcoside A, steviolbioside, or rubusoside can be used.

[0058] For example, the starting steviol glycoside composition may be comprised of at least about 50%, at least 60%, at least 70%, at least 80%, or at least 90% rebaudioside B on a dry weight basis. In other embodiments, the starting steviol glycoside composition may be comprised of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% rebaudioside A on a dry weight basis. In further embodiments, the starting steviol glycoside composition may be comprised of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% stevioside on a dry weight basis. According to additional embodiments, the starting steviol glycoside composition may be comprised of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% steviolbioside on a dry weight basis.

[0059] Also suitable for use as the starting steviol glycoside composition is a steviol glycoside composition that has been base-treated according to embodiment B of the present invention. Glucose Donor

[0060] Glucose donors suitable for use in the present invention include any of the oligomeric, polymeric and cyclic forms of glucose that can be reacted in the presence of a CGTase to cleave and transfer one or more glucose units to the steviol glycoside molecules present in the starting steviol glycoside composition. Suitable glucose donors include, for example, but are not limited to, starch (including starches from different sources such as wheat, corn, potato, tapioca and sago), liquefied starch (including starches that have undergone partial hydrolysis catalyzed by acid and / or enzymes such as amylase), dextrins (including maltodextrin), cyclodextrins, and the like, and combinations thereof. In one advantageous embodiment, the glucose donor is a maltodextrin having a dextrose equivalent (DE) value of about 0.5 to about 10.

[0061] In one embodiment of the invention, a suitable glucose donor composition is produced by treating starch or other suitable polysaccharide with a mixture of amylase and CGTase under conditions effective to liquefy the polysaccharide. For example, starch and water can be combined to form a starch suspension to which α-amylase and CGTase are added. The resulting mixture can then be incubated at a suitable temperature (e.g., from about 50° C. to about 90° C. or from about 70° C. to about 90° C.) for a time (e.g., from about 0.2 hours to about 6 hours) effective to liquefy the starch (thereby reducing its molecular weight and increasing its DE value). The amylase is then inactivated by any suitable method, such as low pH heat treatment, to produce a glucose donor composition, which is then combined with the starting steviol glycoside composition and an additional portion of CGTase and subjected to glycosylation conditions.

[0062] In various embodiments of the invention, an amount of glucose donor effective to provide a weight ratio of glucose donor to steviol glycoside (on a dry weight basis) of about 0.5:1 to about 2:1 is used in the glycosylation reaction. In other embodiments, the weight ratio of glucose donor:steviol glycoside can be 1:1 to 2:1 or 1.5:1 to 2:1. CGTase

[0063] The cyclodextrin glycosyltransferase (CGTase) can be any enzyme known in the art capable of catalyzing the addition of glucose units to steviol glycosides. A combination of different CGTase enzymes can be utilized. CGTases can be produced by mesophilic, thermophilic, alkalophilic and halophilic Bacillus. Suitable CGTase enzymes can be cultured or derived, for example, from Bacillus stearothermophilus, Bacillus macerans, Bacillus circulans, Bacillus alcalophilus and / or Bacillus halophilus, as well as from strains of Thermoanaerobacter or Thermoanaerobium. Examples of such CGTases include those that are natural or recombinant enzymes derived, for example, from the above-mentioned microbial species. For example, CGTase can be produced by inoculating a sterilized culture medium with a suitable Bacillus species, culturing the inoculated medium at a temperature of about 20° C. to about 90° C. for about 12 to 48 hours, preferably with aeration and agitation, filtering the resulting culture medium to separate the Bacillus cells, and further concentrating the cell-free permeate, for example, using ultrafiltration. Such enzymes can also be used in conjunction with one or more amylases to liquefy starch to provide a glucose donor suitable for use in glycosylation reactions. The CGTase enzyme(s) can be in the form of a cell-free broth, a concentrated liquid cell-free broth, a spray-dried or freeze-dried cell-free broth, or a highly purified protein. Free enzyme as well as immobilized enzyme preparations can be used. In one embodiment of the present invention, the CGTase is immobilized (e.g., by gel capture, adsorption, or covalent attachment) to a suitable support. A suitable CGTase can also be produced by isolating the CGTase from a selected microorganism (e.g., a species or strain of Thermoanaerobacter) and cloning it into a suitable host microorganism, such as E. coli, where the CGTase is expressed. Suitable CGTases can be obtained from commercial sources, such as, for example, Novozymes and Amano.

[0064] In one embodiment of the invention, the CGTase enzyme is cultured or derived from a non-alkalophilic organism (i.e. an organism that is not an alkalophilic organism, which is understood to mean an organism that exhibits optimal growth at a pH of 9 or higher). Thus, the CGTase enzyme of a preferred embodiment of the invention is cultured or derived from an organism that exhibits optimal growth at a pH of less than 9. In a particular embodiment, the CGTase enzyme is cultured or derived from a strain of Thermoanaerobacter, such as Thermoanaerobacter sp. ATCC 53627. Thus, preferably, the CGTase enzyme is cultured or derived from a strain of Thermoanaerobacter, such as Thermoanaerobacter sp. ATCC 53627, which is marketed by Novozymes under the trade name Toruzym. (登録商標) A Thermoanaerobacter CGTase, such as the CGTase enzyme commercially available in 3.0 L, is used in the glycosylation reactions of the present invention. In certain embodiments of the present invention, glycosylation of the starting steviol glycoside composition is carried out using a CGTase having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of the CGTase having GenBank Accession Number Z35484 (see Joergensen et al., Cloning and nucleotide sequence of a thermostable cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 and its expression in Escherichia coli "Biotechnol. Lett. 19, 1027-1031 (1997)).

[0065] Toruzyme (登録商標)Previously published literature on the glycosylation of stevioside using Thermoanaerobacter CGTases such as 3.0L CGTase clearly demonstrated that the optimal pH for glycosylation was around 5.5, and there was a significant decrease in the conversion rate of stevioside when the pH was increased to 7 (Li et al., "Transglycosylation of stevioside to improve the edulcorant quality by lower substitution using cornstarch hydrolyzate and CGTase" Food Chem. 138, 2013 2064-2069). A similar pH optimum was found by Mathew et al., who showed that a pH of approximately 5.5 was optimal for multiple substrates and such enzymes, as reported in the literature ["Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp. Biochem. Eng. J. 79,2013,187-193"]. Furthermore, when used to produce cyclodextrins from starch, Toruzym (登録商標) Toruzyme 3.0L was found to have an optimal operating pH of 5, with activity decreasing as the pH was increased from pH 6 to 10. Thus, Toruzyme 3.0L was found to be effective across a broad range of reactions, including the glycosylation of steviol glycosides. (登録商標) It has been demonstrated that the predicted optimum pH for CGTases such as Toruzyme 3.0L is in the range of 5-6. Therefore, glycosylation of steviol glycoside compositions can be achieved using Toruzyme 3.0L in aqueous media having a pH greater than 7.5 and less than or equal to 10. (登録商標)The inventors' findings that certain benefits are in fact achieved by operating in the presence of a CGTase and a glucose donor, such as 3.0L, are unexpected and could not reasonably be predicted based on the prior art knowledge. Such benefits may include, but are not limited to, improved yield (i.e., increased yield of glycosylated steviol glycosides in a given period of time), improved taste quality (i.e., the resulting glycosylated steviol glycoside composition has better organoleptic properties), and / or improved degree of glycosylation (i.e., production of a glycosylated steviol glycoside composition in which a higher number of glucose moieties are added to the starting steviol glycoside(s)).

[0066] In various embodiments of the invention, the amount of CGTase used in the glycosylation reaction can be from about 0.02 to about 10 units of CGTase (on a solids basis) per gram of glucose donor. Glycosylation conditions

[0067] As mentioned above, glycosylation of the starting steviol glycoside composition is carried out by contacting the starting steviol glycoside composition with a glucose donor in the presence of at least one CGTase in an aqueous medium. It has been discovered that the yield of glycosylated product obtained within 24 hours can be significantly increased by providing an aqueous medium having a weakly basic pH, e.g., a pH greater than 7.5 but less than or equal to 10, a pH between 8 and 10, a pH between 8 and 9, or a pH of about 8.5. Such results are particularly evident when the CGTase used is Toruzyme. (登録商標)This was surprising since it was customary to carry out the CGTase-catalyzed glycosylation of steviol glycosides at pH values ​​that are weakly acidic for non-alkalophilic CGTases such as 3.0 L (e.g., pH 5.5-6.5). The pH of the aqueous medium can be adjusted to the desired value using any suitable base or combination of suitable bases; suitable bases can include strong bases as well as weak bases. The base can be an inorganic base (e.g., an alkali metal hydroxide, carbonate or bicarbonate) or an organic base (e.g., an organic amine, a conjugate base of an organic acid). In one embodiment, the base or bases are solubilized in the aqueous medium. The aqueous medium may be buffered or unbuffered at a selected basic pH. In one embodiment of the present invention, at least a portion of the base required to impart the desired pH to the aqueous medium is provided by the base present in the reaction product obtained by base-treating the starting steviol glycoside composition according to embodiment B of the present invention described elsewhere herein. That is, a base-treated steviol glycoside composition can be used as the starting steviol glycoside composition in embodiment A of the present invention.

[0068] The aqueous medium is composed of water, but may further be composed of one or more solvents other than water. Typically, such additional solvent(s), if present, are miscible with water and are present in a relatively small amount compared to the amount of water (e.g., the amount of solvent other than water may be less than 20% by weight, less than 10% by weight, or less than 5% by weight, based on the total weight of water and solvent). In certain embodiments, the aqueous medium does not contain any solvent other than water.

[0069] The temperature at which the glycosylation reaction is carried out is not believed to be critical, but typically glycosylation is carried out within a temperature range of room temperature (e.g., 20° C.) to about 95° C. In one embodiment, the glycosylation reaction temperature is from about 45° C. to about 70° C. In another embodiment, the glycosylation reaction temperature is from about 45° C. to about 55° C. Contacting of the reactants is carried out for a period of time effective to achieve the desired conversion of the starting steviol glycoside to glycosylated steviol glycoside. For example, in certain embodiments of the invention, 60%-85% or 65%-80% conversion of the starting steviol glycoside is achieved; the extent of conversion can be readily monitored by HPLC and / or LC-MS analytical methods. The content of glycosylated steviol glycosides and the content of unreacted steviol glycosides in a glycosylated steviol glycoside composition (obtained as a glycosylation reaction product) can be measured according to the procedures described on pages 257-258 of the 8th edition of Japan's Specifications and Standards for Food Additives published in 2009 by the Ministry of Health, Labor and Welfare of Japan.

[0070] In general, reaction times of 1 hour to 250 hours or 1 hour to 168 hours may be appropriate depending on temperature, activity of CGTase, composition of the starting steviol glycoside composition, enzyme concentration, and other factors. In one embodiment, the reaction time is about 12 to about 24 hours. When the starting steviol glycoside composition includes stevioside, for example, the resulting reaction mixture may be a relatively complex mixture of mono-, di-, tri-, and more highly glycosylated products at both the 19-O-glucosyl unit and the terminal glucosyl unit of the 13-O-sophorosyl moiety.

[0071] According to certain embodiments, the glycosylation reaction is carried out under conditions effective to achieve only partial conversion of the steviol glycosides present in the starting material. For example, the glycosylation reaction can be stopped once about 60-85% of the starting steviol glycosides have been reacted (i.e., glycosylated at one or more glucose moieties) and about 15-40% of the starting steviol glycosides remain unreacted in the reaction mixture (wherein the total amount of glycosylated steviol glycosides + unreacted steviol glycosides = 100%). According to another embodiment, the glycosylation is carried out until about 65-80% of the starting steviol glycosides have been reacted (i.e., glycosylated at one or more glucose moieties) and about 20-35% of the starting steviol glycosides remain unreacted in the reaction mixture (wherein the total amount of glycosylated steviol glycosides + unreacted steviol glycosides = 100%).

[0072] In various embodiments of the invention, the glycosylation reaction can be carried out in water (initial pH of about 8 to about 8.4) using about 4 to about 6 wt% starting steviol glycoside composition and about 8 to about 12 wt% dextrin (the weight ratio of dextrin to starting steviol glycoside is about 1.5:1 to about 2.5:1). The mixture is incubated with CGTase at about 40° C. to about 60° C. for about 0.5 to about 30 hours to obtain a reaction product containing the desired glycosylated steviol glycoside. The enzyme is then inactivated and the reaction product is purified using adsorption resins, carbon filtration and ion exchange.

[0073] Once the desired degree of conversion has been achieved, which can be measured using standard high performance liquid chromatography (HPLC) or liquid chromatography-mass spectroscopy (LC-MS) techniques, the reaction product can be further treated or processed. For example, the CGTase can be inactivated (e.g., by heat treatment, in which the reaction product is heated to a temperature effective to inactivate the CGTase in terms of its ability to catalyze further glycosylation). In other embodiments, the reaction product can be further treated with base according to embodiment C described herein. In another embodiment, the glycosylated steviol glycoside composition can be treated with additional, different types of enzymes, particularly amylases or other enzymes capable of cleaving bonds between glucose (e.g., maltogenic amylases, such as maltogenic amylases derived from Bacillus Subtilis). For example, α-amylase can be combined with the glycosylated reaction product, and the resulting mixture can be incubated at about 55° C. to about 95° C. for about 6 to 24 hours, after which the α-amylase can be inactivated (e.g., using heat at low pH). In another embodiment, an amylase, such as a maltogenic amylase, is combined with the glycosylation reaction product and the resulting mixture is incubated at about 25° C. to about 40° C. for about 12 hours to about 36 hours. Such further enzymatic treatment serves to modify the compositional profile of the glycosylated steviol glycoside composition, potentially resulting in further enhancement of the taste profile of the composition. The glycosylated steviol glycoside composition may also, or alternatively, be subjected to one or more of the further processing steps described herein (e.g., in the section entitled "Purification of Glycosylated Steviol Glycoside Compositions"). Embodiment B

[0074] Embodiment B of the present invention provides a method for producing a glycosylated steviol glycoside composition comprising the steps of: a) contacting a starting steviol glycoside composition with a basic aqueous medium or a basic resin to obtain a base-treated steviol glycoside composition; and b) contacting the base-treated steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium for a time effective to produce a glycosylated steviol glycoside composition. Embodiment B can be practiced in combination with either or both of embodiment A and embodiment C, but alternatively, can be practiced alone or in combination with other processing steps known in the glycosylated steviol glycoside art. By treating the starting steviol glycoside composition with a base in the manner described herein, the taste of the glycosylated steviol glycoside composition resulting from the steviol glycoside composition can be improved by reducing off-flavors, bitterness, lingering aftertaste, etc. (thereby making the glycosylated steviol glycoside composition taste more similar to sucrose) and / or by increasing the sweetness intensity of the glycosylated steviol glycoside composition.

[0075] The starting steviol glycoside composition can correspond to any of the starting steviol glycoside compositions mentioned above in connection with embodiment A. However, in certain aspects of embodiment B, the starting steviol glycoside composition contains at least one of rebaudioside A and stevioside, or both rebaudioside A and stevioside. One or both of rebaudioside A and stevioside can be the steviol glycoside(s) present predominantly (≧50%, ≧60%, ≧70%, ≧80%, ≧90% by weight) in the starting steviol glycoside composition. For example, rebaudioside A and stevioside together can account for ≧50%, ≧60%, ≧70%, ≧80%, ≧90% by weight of the steviol glycosides present in the starting steviol glycoside composition.

[0076] The pH of the basic aqueous medium may be greater than 7.5, at least 8 or at least 9 and not greater than 14 in various embodiments of the invention. For example, the basic aqueous medium may have a pH of 8 to 14, 9 to 14 or about 10 to about 14. The pH of the aqueous medium may be adjusted to the desired value using any suitable base or combination of suitable bases; suitable bases may include strong bases as well as weak bases. The base may be an inorganic base (e.g., an alkali metal hydroxide, carbonate or bicarbonate) or an organic base. In one embodiment, the base or bases are solubilized in the aqueous medium. The aqueous medium may be buffered or unbuffered at the selected basic pH.

[0077] In embodiments of the invention in which the starting steviol glycoside composition is contacted with a basic resin, the basic resin is, for example, contacted with a weakly basic or (preferably) strongly basic polymeric resin (e.g., cross-linked polystyrene) having attached basic functional groups (such as primary, secondary and / or tertiary amino groups or quaternary ammonium groups). Cross-linked polystyrene sulfonate (e.g., polystyrene cross-linked and sulfonated with divinylbenzene) in which the sulfonic acid groups contain quaternary ammonium cations such as trimethylammonium constitutes one type of strongly basic resin. Polyethyleneamine is an example of a suitable weakly basic resin. The starting steviol glycoside composition can dissolve in an aqueous medium when contacted with the basic resin.

[0078] The temperature at which the base treatment of the starting steviol glycoside composition is carried out is not believed to be critical, but typically the treatment is carried out within a temperature range of room temperature (e.g., 20° C.) to about 100° C. For example, the base treatment temperature can be from about 40° C. to about 60° C. The base treatment is carried out for a time effective to achieve the desired conversion of the starting steviol glycosides, which can involve removing one or more glucose units from individual steviol glycosides (particularly removing a glucose unit at the C-19 position), thereby converting at least a portion of the steviol glycosides to other steviol glycosides. For example, if rebaudioside A is present in the starting steviol glycoside composition, at least a portion of the rebaudioside A can be converted to rebaudioside B, and if stevioside is present, at least a portion of the stevioside can be converted to steviolbioside. Rebaudioside B and steviolbioside are reportedly less sweet than rebaudioside A and stevioside, respectively, and steviolbioside in particular can be characterized as having a poor taste profile and low sweetness intensity. However, the glycosylation products of these compounds (rebaudioside B and steviolbioside) have now unexpectedly been found to be sweeter than the glycosylation products obtained upon glycosylation of the "parent" steviol glycosides (rebaudioside A and stevioside), respectively.

[0079] In general, reaction times of 1 hour to 168 hours may be appropriate depending on temperature, pH, base concentration, base identity, and other factors. In a particular embodiment of the invention, the pH of the aqueous medium is adjusted to about 13 to about 14 using sodium hydroxide, and the mixture is heated at about 40° C. to about 60° C. for about 18 to about 30 hours.

[0080] The resulting base-treated steviol glycoside composition is then reacted with a suitable glucose donor in the presence of a CGTase and glycosylated. Prior to glycosylation, the base-treated steviol glycoside composition can optionally be subjected to one or more processing or purification steps, such as neutralization / acidification, precipitation, and the like. The suitable glucose donor and CGTase can be any of those previously described in connection with embodiment A. The glycosylation conditions can be the same as those described above. In various embodiments of the invention, the pH of the aqueous medium can be greater than 7.5 (according to embodiment A). However, in other embodiments, the pH of the aqueous medium is 7.5 or less (e.g., 5.5-7.5 or 5.5-6.5). The glycosylated steviol glycoside composition obtained as the reaction product can then be subjected to one or more further processing and / or purification steps as described elsewhere herein or known in the art to provide an end product suitable for use as a component of a consumable composition. Embodiment C

[0081] The present invention also provides a method for improving one or more sensory properties of a glycosylated steviol glycoside composition, comprising contacting the glycosylated steviol glycoside composition with a basic aqueous medium or a basic resin. The method can be carried out with any glycosylated steviol glycoside composition, i.e., any composition obtained by glycosylation of a steviol glycoside composition. For example, the glycosylated steviol glycoside composition can be a glycosylated steviol glycoside composition produced by glycosylation of a glycosylated steviol glycoside composition produced according to embodiment A or a base-treated steviol glycoside composition produced according to embodiment B of the present invention. Alternatively, the base-treated glycosylated steviol glycoside composition can be produced using any other glycosylation method known in the art. By processing in the manner described herein, the taste of the glycosylated steviol glycoside composition can be improved by reducing off-flavors, bitterness, lingering aftertaste, etc. (which may make the glycosylated steviol glycoside composition taste more similar to sucrose) and / or by increasing the sweetness intensity of the glycosylated steviol glycoside composition.

[0082] The pH of the basic aqueous medium may be greater than 7.5, at least 8, or at least 9, or at least 10, and no greater than 14, in various embodiments of the invention. For example, the basic aqueous medium may have a pH of 8 to 14, or 8.5 to 12, or 9 to 11, or about 10. The pH of the aqueous medium may be adjusted to the desired value using any suitable base or combination of suitable bases; suitable bases may include strong bases as well as weak bases. The base may be an inorganic base (e.g., an alkali metal hydroxide, carbonate or bicarbonate) or an organic base. In one embodiment, the base or bases are solubilized in the aqueous medium. The aqueous medium may be buffered or unbuffered at the selected basic pH.

[0083] In embodiments of the invention in which the glycosylated steviol glycoside composition is contacted with a basic resin, the basic resin is contacted with, for example, a weakly basic or strongly basic polymeric resin (e.g., cross-linked polystyrene) having attached basic functional groups (such as primary, secondary and / or tertiary amino groups or quaternary ammonium groups). Cross-linked polystyrene sulfonate (e.g., polystyrene cross-linked and sulfonated with divinylbenzene) in which the sulfonic acid groups contain quaternary ammonium cations such as trimethylammonium constitutes one type of strongly basic resin. Polyethyleneamine is an example of a suitable weakly basic resin. The glycosylated steviol glycoside composition can dissolve in an aqueous medium when contacted with a basic resin.

[0084] The temperature at which the base treatment of the glycosylated steviol glycoside composition is carried out is not believed to be critical, but typically the treatment is carried out within a temperature range of room temperature (e.g., 20° C.) to about 100° C. For example, the temperature can be from about 40° C. to about 60° C. or 50° C. The base treatment is carried out for a time effective to achieve the desired conversion of the starting glycosylated steviol glycoside, possibly involving conversion of glycosylated rebaudioside A or glycosylated stevioside to glycosylated rebaudioside B or glycosylated steviolbioside, respectively, and / or shortening of the glucose chain added onto the glycosylated steviol glycoside product. In general, reaction times of 1 hour to 168 hours (e.g., about 12 hours to about 36 hours or about 18 hours to about 30 hours) can be appropriate depending on the temperature, pH, base concentration, identity of the base, and other factors. For example, when sodium hydroxide is used as the base and the aqueous medium has a pH of about 9 to about 11, the reaction temperature can be about 40° C. to about 60° C., and the reaction time can be about 18 to about 30 hours.

[0085] The glycosylated steviol glycoside composition thereby obtained as the reaction product can then be subjected to one or more further processing and / or purification steps as described elsewhere herein or known in the art to provide an end product suitable for use as a component of a consumable composition. Purification of Glycosylated Steviol Glycoside Compositions

[0086] Glycosylated steviol glycoside compositions produced according to any of the embodiments of the present invention can be further processed and / or purified prior to being incorporated into or used as an ingredient in food, beverage products, cosmetics and pharmaceuticals, as described in more detail below.

[0087] Suitable purification / processing techniques include, but are not limited to, enzyme inactivation, neutralization or other pH adjustment, filtration, sterilization, decolorization, desalting, fractionation by chromatography or the like, drying, concentration, precipitation, crystallization, adsorbent (e.g., polymeric adsorbents such as macroporous adsorbents and hydrophobic resins, activated carbon), ion exchange resin treatment, and the like, and combinations thereof. When the glycosylated steviol glycoside composition is obtained in the form of an aqueous solution (e.g., a syrup), it may be used as such or may optionally be converted to a dry form, for example, by spray drying. The glycosylated steviol glycoside composition may be combined with one or more other ingredients (e.g., sweeteners or other flavor modifiers) before or after drying.

[0088] In certain embodiments of the invention, a glycosylated steviol glycoside composition is processed and / or purified using one or more of the methods described above such that its steviol glycoside content (including both unreacted starting steviol glycoside(s) and glycosylated products of the starting steviol glycoside(s), if present) is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% on a dry weight basis. The amount of glycosylated steviol glycoside produced can be quantified by measuring the reduction in the amount of starting steviol glycoside using HPLC.

[0089] Depending on the further processing / purification conditions used, the resulting glycosylated steviol glycoside composition may contain small amounts of residual glucose donor and / or carbohydrate products derived from the glucose donor (e.g., maltodextrins when the glucose donor is starch). The composition may also, or alternatively, contain a portion of the steviol glycosides present in the starting steviol glycoside composition (e.g., unreacted steviol glycosides).

[0090] In various embodiments of the invention, glycosylated steviol glycoside compositions are processed and / or purified to provide compositions intended or suitable for use as ingredients in consumables (e.g., food or beverage products) as sweeteners and / or flavoring agents. For example, such glycosylated steviol glycoside compositions can contain, on a dry solids basis, 15%-40% by weight unreacted steviol glycosides and 60%-85% by weight glycosylated steviol glycosides, where the total weight of unreacted steviol glycosides and glycosylated steviol glycosides is at least 90% of the total weight of the glycosylated steviol glycoside composition, on a dry weight basis. In another embodiment, a glycosylated steviol glycoside composition is provided that comprises 20%-35% by weight unreacted steviol glycosides and 65%-80% by weight glycosylated steviol glycosides on a dry solids basis, wherein the total weight of unreacted steviol glycosides and glycosylated steviol glycosides is at least 95% of the total weight of the glycosylated steviol glycoside composition on a dry weight basis.

[0091] Certain embodiments provide glycosylated steviol glycoside compositions that contain glycosylated rebaudioside B, glycosylated steviolbioside, steviolbioside and rebaudioside B. Such components together can comprise, for example, 5-50% or 10-25% of the glycosylated steviol glycoside composition on a dry weight basis. The remainder of the glycosylated steviol glycoside composition may be composed primarily or entirely of glycosylated rebaudioside B, glycosylated steviolbioside, one or more steviol glycosides other than steviolbioside and rebaudioside B and glycosylated steviol glycosides (e.g., rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M (also referred to as rebaudioside X), rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, stevioside, and glycosylated derivatives thereof). For example, the total weight of steviol glycosides and glycosylated steviol glycosides in the glycosylated steviol glycoside composition can be at least 85%, at least 90%, or at least 95% of the glycosylated steviol glycoside composition on a dry weight basis. In addition to the glycosylated steviol glycosides and steviol glycosides described above, the glycosylated steviol glycoside composition can contain relatively minor amounts of residual glucose donors and / or carbohydrate products derived from the glucose donors (e.g., less than 5% or less than 10% total residual glucose donors and / or carbohydrate products derived from the glucose donors on a dry weight basis). The glycosylated steviol glycoside compositions described above have been found to have particularly advantageous organoleptic properties.

[0092] Further embodiments provide glycosylated steviol glycoside compositions containing glycosylated rebaudioside B and glycosylated steviolbioside. Such components together can comprise, for example, 2%-25% or 5%-15% of the glycosylated steviol glycoside composition on a dry weight basis. The remainder of the glycosylated steviol glycoside composition can be primarily or entirely composed of one or more steviol glycosides and glycosylated steviol glycosides other than glycosylated rebaudioside B and glycosylated steviolbioside (e.g., rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I ... The glycosylated steviol glycoside composition may be comprised of rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M (also referred to as rebaudioside X), rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, steviolbioside, stevioside, and glycosylated derivatives thereof, other than glycosylated rebaudioside B and glycosylated steviolbioside. For example, the total weight of steviol glycosides and glycosylated steviol glycosides in the glycosylated steviol glycoside composition, on a dry weight basis, can be at least 85%, at least 90%, or at least 95% of the glycosylated steviol glycoside composition. In addition to the glycosylated steviol glycosides and steviol glycosides mentioned above, the glycosylated steviol glycoside composition can contain relatively minor amounts of residual glucose donors and / or carbohydrate products derived from the glucose donors (e.g., less than 5% or less than 10% total residual glucose donors and / or carbohydrate products derived from the glucose donors, on a dry weight basis). The glycosylated steviol glycoside compositions described above have been found to have particularly advantageous organoleptic properties. Further Description of Exemplary Process Steps

[0093] Aspects of the present invention can be further described with reference to Figure 1, which illustrates in schematic form a series of process steps that may be performed to convert a starting steviol glycoside composition into a glycosylated steviol glycoside composition suitable for use (e.g., as a sweetener or flavoring agent) in a consumable product, such as a food, beverage product, cosmetic, or pharmaceutical. In Figure 1, the numbers of the individual steps are for reference purposes only and do not imply that such steps are performed in numerical order or that all steps referred to are necessarily performed. However, at least one of step 1 (embodiment B), step 3 (embodiment A), or step 8 (embodiment C) is performed in accordance with the present invention.

[0094] In step 1, the starting steviol glycoside composition can be optionally base-treated according to embodiment B of the present invention. The base-treated steviol glycoside composition can then be contacted with a glucose donor in the presence of a CGTase under conditions effective to glycosylate at least a portion of the steviol glycosides present in the base-treated steviol glycoside composition (steps 3 and 4). Such base treatment has been found to be useful in improving one or more sensory properties of the final glycosylated steviol composition, at least when certain types of steviol glycosides are present in the starting steviol glycoside composition. Alternatively (not shown in FIG. 1), the glycosylation reaction can be carried out with a starting steviol glycoside composition that has not been treated with base according to embodiment B. In step 3, which corresponds to embodiment A of the present invention, glycosylation is carried out using an aqueous medium having a pH greater than 7.5 (e.g., a pH of at least 8). It has been discovered that glycosylation under such higher pH conditions results in improved yield, taste quality and / or extent of glycosylation. Alternatively, conventional pH conditions (e.g., pH=5.5-7.5) can be used as shown in step 4.

[0095] The glucose donor used in steps 3 or 4 can be an unmodified or untreated oligosaccharide, such as dextrin or starch, or a polysaccharide. However, it may be advantageous to pre-treat the glucose donor by contacting it with an amylase or a mixture of amylase and CGTase, as shown in step 2, and then subjecting it to a liquefaction step in which the amylase is inactivated (e.g. by low pH heat treatment).

[0096] Once the glycosylation reaction of steps 3 or 4 has proceeded to the desired conversion of the starting steviol glycoside composition (which may be monitored by HPLC or LC-MS once specifications have been set, and such specifications are determined using sensory analysis of the resulting product), the CGTase may be inactivated by heat treatment or other suitable means (step 5). The resulting reaction product may then be directly purified (step 9) to provide a glycosylated steviol glycoside composition suitable for use in consumables described elsewhere herein. Alternatively, as shown in step 8, the reaction product may be base-treated according to embodiment C of the invention prior to purification step 9. In a further variation (step 6), the reaction product containing glycosylated steviol glycosides may be contacted with an amylase or other inter-glucose bond cleavage enzyme under conditions effective to cause cleavage of at least certain inter-glucose bonds present in the glycosylated steviol glycoside, thereby altering the types and / or relative amounts of individual glycosylated steviol glycosides present in the reaction product. The amylase or other enzymes may then be inactivated (step 7) prior to further purification of the reaction products (step 9).

[0097] The processing steps described herein can be combined and carried out according to the following exemplary process flow: I). Base treatment of the starting steviol glycoside composition (optional if III or VII is performed). II). Treatment of dextrin or other glucose donors with amylase and CGTase (optional). III). Contacting the starting steviol glycoside composition (which may or may not be base-treated) with a CGTase and a glucose donor at a pH>7.5 (if either I or VII is performed, it may be performed at a pH≦7.5). IV). Inactivation of CGTase (e.g., by heat treatment). V). Contact of the glycosylated steviol glycoside composition with amylase or other glucose bond-cleaving enzyme (selectively). VI). Inactivation of amylase or other enzymes (e.g., by heating). VII). Base treatment of the glycosylated steviol glycoside composition (optional if I or III is performed). VIII). Purification of the glycosylated steviol glycoside composition (by activated carbon, ion exchange resin and / or hydrophobic resin treatment and / or other methods to remove impurities). Uses of glycosylated steviol glycoside compositions

[0098] The glycosylated steviol glycoside compositions produced by the methods of the invention can be used as ingredients or components of products intended for consumption, including, for example, foods, beverage products, cosmetics (personal care), and pharmaceuticals. The glycosylated steviol glycoside compositions are useful, for example, as the sole sweetener or flavor modifying ingredient or in combination with other sweeteners and / or flavor modifiers, as sweeteners and / or flavor modifiers or enhancers.

[0099] Suitable sweeteners that may be used in combination with the glycosylated steviol glycoside compositions produced according to the present invention include natural sweeteners, synthetic sweeteners, nutritive and non-nutritive sweeteners, as well as high-intensity and low-intensity sweeteners.Sweeteners include sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, lyxose, ribose, xylose, ribulose, xylulose, allose, galactose, glucose, gulose, idose, mannose, talose, fructose, allulose (psicose), sorbose, tagatose, mannoheptulose, sedoheptulose, octorose, fucose, rhamnose, arabinose, turanose, Sialose, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside I, Rebaudioside H, Rebaudioside L, Rebaudioside K, Rebaudioside J, Rebaudioside M (also called Rebaudioside X), Rebaudioside N, Rebaudioside O, Dulcoside A, Dulcoside B, Rubusoside, Stevia Extract, Stevioside, Mogroside IV, Mogroside V, Luo Han Guo Guo extract, siamenoside, monatin and its salts (monatin SS, RR, rS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, bayunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abru The glycoside may be selected from the group consisting of, but is not limited to, soside A, steviolbioside and cyclocaryoside I, sugar alcohols such as erythritol, sucralose, acesulfame potassium, acesulfamic acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and its salts, neotame, advantame, glucosylated steviol glycosides (GSG) produced by methods other than those described herein, and combinations thereof.

[0100] According to one embodiment of the present invention, the glycosylated steviol glycoside composition is combined with rebaudioside B in an amount effective to enhance the taste and flavor characteristics of the glycosylated steviol glycoside composition. The glycosylated steviol glycoside composition can be produced according to any of the procedures described herein, but can also be a glycosylated steviol glycoside composition obtained using any other method known in the art for subjecting a starting steviol glycoside composition to a glycosylation reaction. In certain embodiments, the glycosylated steviol glycoside composition can contain from 15% to about 40% by weight on a dry solids basis (e.g., from about 20% to about 35% by weight on a dry solids basis) of unreacted steviol glycoside, with the remainder being wholly or primarily (e.g., at least 80%, at least 85%, or at least 90% by weight on a dry solids basis) glycosylated steviol glycoside.

[0101] It has been found that combining rebaudioside B with a glycosylated steviol glycoside composition (e.g., a steviol glycoside composition obtained according to the present invention) can significantly improve the hedonic properties of the glycosylated steviol glycoside composition. Preferably, the glycosylated steviol glycoside composition is prepared according to embodiment A (the aspect of the present invention in which the starting steviol glycoside composition, the glucose donor and the cyclodextrin glycosyltransferase are contacted in an aqueous medium having a pH of greater than 7.5 to less than or equal to 10). In an advantageous embodiment of the present invention, a glycosylated steviol glycoside composition and a sweetener composition are provided that comprise, consist essentially of, or consist of the glycosylated steviol glycoside composition and at least 1%, at least 2%, or at least 3% rebaudioside B by weight based on the total dry weight of the glycosylated steviol glycoside composition. The glycosylated steviol glycoside composition can contain, on a dry weight basis, less than 2%, less than 1%, or less than 0.5%, or even 0% rebaudioside B. The sweetener composition, in various embodiments, can contain 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less rebaudioside B, based on the total dry weight of the glycosylated steviol glycoside composition and rebaudioside B. For example, the sweetener composition can contain 1-10%, 2-8%, 3-6%, or about 4% rebaudioside B, based on the total weight of the glycosylated steviol glycoside composition and rebaudioside B.

[0102] Advantageously, enhanced sweetener compositions can also be produced by combining glycosylated steviol glycoside compositions produced according to different aspects of the present invention.

[0103] For example, the sensory performance of a glycosylated steviol glycoside composition produced by contacting a starting steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium having a pH greater than 7.5 and less than or equal to 10, but without base treatment of the starting steviol glycoside composition before or after glycosylation ("GSG Composition Type A") can be compared to a glycosylated steviol glycoside composition produced by: a) contacting the starting steviol glycoside composition with a basic aqueous medium or a basic resin and treating the starting steviol glycoside composition with a base-treated steviol glycoside composition; The sweetener composition can be enhanced by combining with a glycosylated steviol glycoside composition ("GSG Composition Type B") produced by a) obtaining a glycoside composition and b) contacting the base-treated steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium (an aqueous medium having any suitable pH, e.g., about 5 to about 10; higher yields of glycosylated steviol composition can be obtained at a weakly basic pH, e.g., a pH of about 8 to about 9). The benefits of such blends are particularly evident at relatively high concentrations of sweetener composition (e.g., at least 1000 ppm total glycosylated steviol glycosides) in consumable products. The amount of GSG Composition Type B in the sweetener composition can be at least 5% and no more than 50% (or no more than 35% by weight), based on the total dry weight of GSG Composition Type A and GSG Composition Type B. Correspondingly, the amount of GSG Type A composition in the sweetener composition can be at least 50% by weight and no more than 95% by weight (or no more than 65% by weight), based on the total dry weight of GSG Type A composition and GSG Type B composition.

[0104] In another example, the sensory performance of a glycosylated steviol glycoside composition produced by contacting a starting steviol glycoside composition, a glucose donor, and a cyclodextrin glycosyltransferase in an aqueous medium having a pH greater than 7.5 and less than or equal to 10, but without base treatment of the starting steviol glycoside composition before or after glycosylation ("GSG Composition Type A") can be enhanced by combining such a composition with a glycosylated steviol glycoside composition produced by contacting the glycosylated steviol glycoside composition with a basic aqueous medium or a basic resin ("GSG Composition Type C"). The benefits of such blends are particularly evident at relatively high concentrations of the sweetener composition in consumable products (e.g., at least 1000 ppm total glycosylated steviol glycosides). The amount of GSG Composition Type C in the sweetener composition can be at least 5% and no more than 50% by weight, based on the total dry weight of GSG Composition Type A and GSG Composition Type C. Correspondingly, the amount of GSG Type A composition in the sweetener composition can be at least 50% by weight and up to 95% by weight, based on the total dry weight of GSG Type A composition and GSG Type C composition.

[0105] Further embodiments of the present invention provide a sweetener composition comprising, consisting essentially of, or consisting of: a) about 10 to about 25 (or about 15 to about 20) weight percent, on a dry solids basis, of a base-treated glycosylated steviol glycoside composition produced according to embodiment C of the present invention, wherein the glycosylated steviol glycoside composition is obtained by glycosylation of a starting steviol glycoside composition consisting of 25-30% stevioside, 55-65% rebaudioside A and other steviol glycosides (on a dry weight basis) to achieve a total steviol glycoside content of at least 95% or a starting steviol glycoside composition consisting of 55-65% rebaudioside A, 20-30% stevioside and a total of 3-8% rebaudioside C and dulcoside A and having a total steviol glycoside content of at least 90% (on a dry weight basis); b) about 65 to about 90 (or about 70 to about 85) weight percent, on a dry solids basis, of a glycosylated steviol glycoside composition that has not been base-treated according to embodiment C of the present invention, wherein the glycosylated steviol glycoside composition is obtained by glycosylation of a starting steviol glycoside composition consisting of 25-30% stevioside, 55-65% rebaudioside A and other steviol glycosides (on a dry weight basis) to reach a total steviol glycoside content of at least 95% or a starting steviol glycoside composition consisting of 55-65% rebaudioside A, 20-30% stevioside, and a total of 3-8% rebaudioside C and dulcoside A and having a total steviol glycoside content of at least 90% (on a dry weight basis); and c) 0 to about 10 (or 0 to about 5) weight percent total residual glucose donor and / or carbohydrate products derived from the glucose donor on a dry solids basis.

[0106] In another embodiment of the present invention, there is provided a sweetener composition comprising, consisting essentially of, or consisting of: a) about 10 to about 25 (or about 15 to about 20) weight % on a dry solids basis of a glycosylated steviol glycoside composition produced according to embodiment A of the present invention, wherein the glycosylated steviol glycoside composition is obtained by base-treating a starting steviol glycoside composition consisting of 25-30% stevioside, 55-65% rebaudioside A and other steviol glycosides (on a dry weight basis) and having a total steviol glycoside content of at least 95% or a starting steviol glycoside composition consisting of 55-65% rebaudioside A, 20-30% stevioside and a total of 3-8% rebaudioside C and dulcoside A and having a total steviol glycoside content of at least 90%, to obtain a base-treated starting steviol glycoside composition, which is then glycosylated; b) about 65 to about 90 (or about 70 to about 85) weight percent of a glycosylated steviol glycoside composition obtained by glycosylation of a starting steviol glycoside composition consisting of 25-30% stevioside, 55-65% rebaudioside A, and other steviol glycosides (on a dry solids basis) to reach a total steviol glycoside content of at least 95% or consisting of 55-65% rebaudioside A, 20-30% stevioside, and a total of 3-8% rebaudioside C and dulcoside A (on a dry solids basis) to obtain a glycosylated steviol glycoside composition having a total steviol glycoside content of at least 90%, wherein the starting steviol glycoside composition is not base-treated prior to glycosylation; and c) 0 to about 10 (or 0 to about 5) weight percent total residual glucose donor and / or carbohydrate products derived from the glucose donor on a dry solids basis.

[0107] One aspect of the invention provides a food product comprising a glycosylated steviol glycoside composition produced according to the invention or a combination of such glycosylated steviol glycoside compositions, typically in combination with at least one additional food ingredient. Optionally, one or more individual steviol glycosides, including, inter alia, rebaudioside B, can be used in combination with such glycosylated steviol glycoside composition(s). For example, the food product can include, in addition to such glycosylated steviol glycoside compositions, 1-10%, 2-8%, 3-6% or about 4% rebaudioside B by weight based on the total dry weight of rebaudioside B and the glycosylated steviol glycoside composition. The amount of glycosylated steviol glycoside composition in the food product can be varied as needed to achieve a particular desired result, such as, for example, increased sweetness and / or improved flavor. For example, a food product can include the glycosylated steviol glycoside composition in a subsweetening or sweetening amount. The concentration of the glycosylated steviol glycoside composition in the food product can be from 5 to 2000 ppm on a dry weight basis. In other embodiments, the food product contains the glycosylated steviol glycoside composition in an amount effective to impart to the food product an SEV of at least 7, at least 8, at least 9, or at least 10, or at least 11, but generally no more than 20 or 15, wherein the food product can further include one or more other sweeteners that also contribute to the SEV. The glycosylated steviol glycoside composition can be added to a food product that also contains one or more other sweeteners in an amount effective to increase the SEV of the food product to at least 7, at least 8, at least 9, at least 10, or at least 11, thereby providing the food product with a predetermined desired sweetness. Thus, the food product may have an SEV of 1-20, 5-15 or 7-13 in various embodiments of the invention.

[0108] Non-limiting examples of food products include confectionery products (including, but not limited to, jelly candies, hard candies, and gums), dessert products such as yogurt (including, but not limited to, full fat, reduced fat, and low fat dairy yogurt, as well as non-dairy yogurt and lactose free yogurt, and all frozen equivalents thereof), frozen desserts (including, but not limited to, frozen dairy desserts such as ice cream - including regular ice cream, soft serve ice cream, and other types of ice cream - and non-dairy desserts), sweet bakery products (including, but not limited to, biscuits, cakes, rolls, pies, pastries, cookies), pre-made sweet bakery mixes for making sweet bakery products, pie fillings (including, but not limited to, fruit pie fillings, and nut pie fillings such as pecan pie filling), cereal products such as sweetened breakfast cereals (including, but not limited to, extruded breakfast cereals, flaked breakfast cereals, and puffed breakfast cereals), cereal coating compositions, bread products (leavened and non-leavened breads, yeast breads, and soda breads). The sweeteners may be any type of food product, including, but not limited to, unleavened breads such as bread, breads containing any flour type, breads containing any non-wheat flour (e.g., potato, rice and rye flour, etc.), gluten-free breads, pre-made bread mixes for bread making, frozen dairy products, meats, dairy products, condiments, snack bars (including, but not limited to, cereal, nut, seed and / or fruit bars), soups, dressings, mixes, prepared meals, baby foods, diet preparations, syrups, food coatings, dried fruits, sauces, gravies, spreads (jams / jellies, butter and other spreadable preserves, conserves), and the like. Although not mentioned herein, other types of food products, particularly reduced sugar or low sugar products, that typically contain one or more nutritive sweeteners, are also considered to be within the scope of the present invention. The food product may be an animal feed product, such as a pet food. The food product of the present invention may include the sweetener composition as a coating or icing formed on the surface of the food product.Such coatings not only enhance the flavor of the food product, but also improve the shelf life of the food product.

[0109] The glycosylated steviol glycoside compositions of the present invention are also useful in medical foods (foods specifically formulated and intended for the dietary management of diseases with unique nutritional needs that cannot be met by conventional diet alone).

[0110] Another aspect of the present invention provides a beverage product comprising a glycosylated steviol glycoside composition according to the present invention or a combination of glycosylated steviol glycoside compositions according to the present invention, optionally modified with one or more individual steviol glycosides such as rebaudioside B. For example, the beverage product may contain, in addition to such glycosylated steviol glycoside compositions, 1-10%, 2-8%, 3-6% or about 4% rebaudioside B by weight based on the total dry weight of rebaudioside B and the glycosylated steviol glycoside composition. The amount of glycosylated steviol glycoside composition in the beverage product may be varied as necessary to achieve a particular desired result, such as, for example, increased sweetness and / or improved flavor. For example, the beverage product may contain a sub-sweetening or sweetening amount of the glycosylated steviol glycoside composition. The concentration of the glycosylated steviol glycoside composition in the beverage product (on a dry solids basis) may be 5-2000 ppm. In one embodiment, the beverage product comprises 800-1800 ppm (dry) glycosylated steviol glycoside composition. In other embodiments, the beverage product contains the glycosylated steviol glycoside composition in an amount effective to impart to the beverage product an SEV of at least 7, at least 8, at least 9, or at least 10, or at least 11, but typically no more than 20 or 15, where the beverage product may further contain one or more other sweeteners that also contribute to the SEV. The glycosylated steviol glycoside composition may be added to a beverage product that also contains one or more other sweeteners in an amount effective to increase the SEV of the beverage product to at least 7, at least 8, at least 9, at least 10, or at least 11, thereby providing the beverage product with a predetermined desired sweetness. For example, non-diet soft drinks typically contain 12 grams of sucrose per 100 mL of water, i.e., 12% sucrose. Thus, a comparable sweetened diet soft drink can be prepared using a glycosylated steviol glycoside composition in an amount sufficient to achieve a SEV of 12 (along with one or more other sweeteners that may be present).Thus, the beverage product may have an SEV of 1-20, 5-15, or 7-13 in various embodiments of the invention.

[0111] Non-limiting examples of beverage products include carbonated beverages (including, but not limited to, carbonated soft drinks), non-carbonated beverages (including, but not limited to, flavored water and non-carbonated soft drinks such as sweet tea or coffee drinks), fruit-flavored beverages, fruit juices, tea, milk, coffee, and especially reduced sugar or low sugar products. Frozen beverage products (sometimes known as slushies) are also expressly contemplated. Although not mentioned herein, other types of beverage products, particularly reduced sugar or low sugar products, that typically contain one or more nutritive sweeteners are also considered to be within the scope of the present invention.

[0112] A further aspect of the present invention provides a table sweetener comprising at least one glycosylated steviol glycoside composition according to the present invention. The table sweetener of the present invention can optionally comprise one or more additional ingredients selected from the group consisting of bulking agents (such as maltodextrin, polydextrose, gums such as xanthan gum or guar gum, soluble corn fiber (SCF), starch and polyols), natural and / or artificial flavors, flavor enhancers, natural and / or artificial colors, fiber, acidulants, vitamins, antioxidants, preservatives, starch hydrolysates, etc. In one embodiment, the table sweetener comprises both at least one glycosylated steviol composition according to the present invention and at least one individual steviol glycoside, such as rebaudioside B.

[0113] According to one embodiment, the table sweetener is a dry table sweetener. For example, the sweetener can be in the form of tablets, granules or powder. Liquid table sweeteners are also contemplated, typically in the form of an aqueous solution of the components.

[0114] According to one embodiment, the table sweetener can further comprise one or more nutritive sweeteners. The nutritive sweeteners can be selected from the group consisting of sucrose, glucose, glucose syrup, isoglucose, fructose, glucose-fructose syrup, maltose, lactose, corn syrup, high fructose corn syrup, invert sugar, molasses, honey and agave. In a preferred embodiment, the nutritive sweetener is sucrose. When the table product comprises a nutritive sweetener, the nutritive sweetener can be present in an amount of up to about 30% by weight based on the total weight of the table sweetener. For example, the nutritive sweetener can be present in an amount of about 26% by weight based on the total weight of the table sweetener. According to one embodiment, the table sweetener can further comprise one or more auxiliary sweeteners selected from the group consisting of high-intensity sweeteners and sugar alcohols. Various synthetic high-intensity sweeteners can also be used as one or more auxiliary sweeteners. Specific examples include sucralose, aspartame, and acesulfame potassium (Ace K). Various sugar alcohols can also be used as one or more auxiliary sweeteners in the present invention. Specific examples include maltitol, xylitol, and erythritol.

[0115] The table sweetener according to the invention can be used to sweeten typical beverages, especially hot beverages such as tea and coffee.

[0116] Another aspect of the present invention provides a bulking agent comprising a glycosylated steviol glycoside composition according to the present invention.

[0117] A further aspect of the present invention provides a coating agent comprising a glycosylated steviol glycoside composition according to the present invention.

[0118] Another aspect of the present invention provides a pharmaceutical product comprising a glycosylated steviol glycoside composition according to the present invention and at least one other pharmaceutical ingredient, such as an active ingredient or excipient. Exemplary pharmaceutical products include cough syrups, chewable tablets, lozenges, vitamins, and the like.

[0119] Another aspect of the present invention provides a nutritional product or a sports product comprising a glycosylated steviol glycoside composition according to the present invention.

[0120] Another aspect of the present invention provides a cosmetic (personal care) comprising a glycosylated steviol glycoside composition according to the present invention and at least one cosmetic ingredient. Exemplary cosmetic products include dentifrices, mouthwashes, and the like.

[0121] It will be understood that the amount of glycosylated steviol glycoside composition obtained according to the present invention present in a food, beverage product, pharmaceutical, nutritional product, sports nutrition or cosmetic product will depend on the sweetness, taste, flavor and other sensory attributes of the glycosylated steviol glycoside composition and the type(s) and amount(s) of other sweetener(s) and / or flavor modifier(s) present in the product and other factors such as the desired sweetness or other flavor characteristics of the product as well as the desired caloric content of the product. The glycosylated steviol glycoside composition can be used as a flavor enhancer at concentrations in the product below its sweetness detection limit (the minimum concentration at which the glycosylated steviol glycoside imparts a perceptible sweetness to the product in the absence of any other sweeteners) and can be used as a sweetener at concentrations in the product at or above its sweetness detection limit.

[0122] An alternative aspect of the present invention provides the use of the sweetener composition of the present invention as a bulking agent or as a coating agent in food, beverage products, pharmaceuticals, nutritional products, sports nutrition or cosmetics.

[0123] The glycosylated steviol glycoside compositions produced according to the present invention can be formulated in any ingestible form, such as a syrup, powder form, tablet form, granules, solution or any other suitable form, including beverage products and food products. EXAMPLES

[0124] Example 1

[0125] In this example, a highly purified combination of steviol glycosides (containing rebaudioside A as the major component) was used as the starting material and was first glycosylated. The resulting glycosylated steviol glycoside composition was then treated with base. material

[0126] 1) Maltodextrin (dextrose equivalent = 1). 2) Cyclodextrin glucotransferase (CGTase). 3) Maltogenic amylase 4) SG95 steviol glycoside blend supplied by GLG Life Tech Corporation (a highly purified combination of nine sweet steviol glycosides found in stevia leaves, with Reb A accounting for approximately 60% by weight of the blend and stevioside accounting for approximately 30% by weight of the blend). 5) Granular activated carbon, recycled. 6) Strong base anionic resin and strong acid cation resin mixed in a ratio of 60:40 strong base anionic resin to strong acid cation resin by weight. 7) Macroporous adsorbent resins (bead-shaped nonionic aliphatic acrylic polymers with a macroreticulated structure and high surface area). reaction

[0127] The procedure was carried out in a 500 mL effector tank. Three samples of each reaction (see Table 1) were prepared simultaneously using the following procedure: 1) Suspend 20 g of SG95 steviol glycosides in 340 mL of warm water in a 1000 mL beaker. 2) Add 40 g of maltodextrin (DE=1) to the above suspension and blend to hydrate the material. 3) Adjust the pH to 8.2 using sodium hydroxide. 4) Add 2mL of CGTase enzyme to the above solution. 5) Incubate at 50°C for 24 hours with agitation at 200 rpm. 6) The mixture was transferred to a flask and heated in boiling water (approximately 95°C) for 30 minutes to denature the CGTase enzyme, then allowed to cool to room temperature. 7) Pour the mixture back into the fermenter beaker and rinse the flask with DI water, ensuring complete transfer of material from the flask to the beaker and bringing the total volume of the mixture to approximately 500ml. 8) Add 5mL (1%) of maltogenic amylase to the above solution. 9) Incubate at 30°C for 24 hours with agitation at 150 rpm. 10) The reaction mixture was heated in boiling water (about 95°C) for 30 minutes to denature the maltogenic amylase, and then allowed to cool to room temperature. 11) Filter the solution through a Buchner funnel to remove any unreacted solids / dextrin. 12) Wash the activated carbon with DI water to remove particulates. Pack a Pack 1 #15 x 300 mm jacketed column with granular activated carbon. Preheat the carbon column to 50°C and subsequently wash with 1.5 L of DI water to remove particulates. 13) Prepare a mixed bed resin by washing 29.95 g of strong base anionic resin and 20.00 g of strong acid cation resin with DI water. Pack the mixed bed resin into a Pack 1 #15 x 300 mm jacketed column. Preheat the column to 25°C and subsequently wash with 1.5 L of DI water. 14) Pass the reaction solution through a carbon column and a mixed bed resin column at a flow rate of 1.5 mL / min. Wash the columns with water. 15) Rinse the macroporous adsorption column with 1500 g of degassed DI water. 16) The reaction solution is filtered through a macroporous adsorption column. 17) Rinse the macroporous adsorption column with 1500 g of degassed DI water. 18) Wash the macroporous adsorption column with 1500 g of a 50% ethanol solution (758.68 g degassed DI water and 750.63 g 190 proof ethanol). 19) Collect the ethanol washed solution and evaporate the ethanol from the solution. 20) Transfer the solution (740.11 g) to a 2 L glass beaker. Measure the pH (4.24) and adjust to a final pH of 10.03 with sodium hydroxide. Heat the solution to 50° C. and stir at 350 rpm for 24 h. 21) Cool the solution and adjust the pH to 8.20-5.00 using 7% w / w hydrochloric acid. 22) Place the solution in the freeze dryer. 23) Collect the dried material (24.17 g total) and submit for analytical testing. [Table 1] result

[0128] The primary analytical technique used for such samples was LC-MS. Mass spectrometry (MS) reports the molecular weight of the peak from chromatography (LC) divided by the charge, which was generally 1. Steviol glycosides are expected to have the molecular weights shown in the table below. The most common sugar residues added to the steviol core are glucose, rhamnose, xylose, 6-deoxyglucose and fructose. From a mass standpoint, glucose = fructose and rhamnose = 6-deoxyglucose. Another phenomenon in mass spectrometry is that adducts can be formed during ionization. These reaction products add mass to the expected mass-to-charge ratio. In the case of this experiment, m / z 687, shown in Figure 2, corresponds to rubusoside and the formic acid adduct. Such an m / z peak is also seen in the steviol glycoside standard containing rubusoside. FIG. 2 shows that the majority of the reaction products correspond to mono-, di-, and tri-glycosylated steviol glycosides, with the highest peaks at 965 and 803 being rebaudioside A and stevioside, respectively. [Table 2]

[0129] As seen in Table 2 above, stevioside has an m / z of 803 and rebaudioside A has an m / z of 965. In Figures 2 and 3, these correspond to the large peaks eluting at retention times of 9-12 minutes. [Table 3] Discussion

[0130] The major products expected from this procedure were glycosylated rebaudioside B and glycosylated steviolbioside, since rebaudioside B and steviolbioside are the major products of base treatment of the starting material. These products would be expected to appear on the LC-MS after 10 minutes. Generally, higher degrees of glycosylation correlate with lower retention times, so the products with the large peaks before 7 minutes would be expected to contain significant amounts of steviol glycosides with larger glycosides. Although some such products are produced, they appear to be less prevalent than in more typically produced samples (Figure 4). Comparing Figure 3 to Figure 4, the most notable difference between the figures is the suppression of the 1127 m / z and 1451 m / z peaks at approximately 7.5 minutes. Such peaks likely correspond to di- and tri-glycosylated rebaudioside A and tri-glycosylated stevioside. Both procedures appear to produce lower abundances of components with higher degrees of glycosylation compared to commercial glycosylated steviol glycoside products (Figure 5). Example 2

[0131] This example demonstrates transglycosylation of a steviol glycoside composition following initial base treatment to produce a glycosylated steviol glycoside composition. Base Treatment

[0132] 1) 39.99 g of steviol glycoside mixture (SG95, GLG Life Tech Corporation) was dissolved in 360.00 g of 1.25 N sodium hydroxide solution. The pH of the resulting mixture was 13.6. 2) The above mixture was heated to 50° C. and stirred at 360 rpm for 24 hours. 3) The mixture was then cooled in an ice bath. 4) Once cooled, the mixture was then adjusted to pH 5 (initial pH 13.60, final pH 4.99) with 7% w / w hydrochloric acid. 5) The resulting material was centrifuged three times (4500 rpm for 30 min at 25° C. in a 50 mL tube) and dried. The precipitate was analyzed for steviol glycoside content and used in the transglycosylation step. Glycosyl transfer

[0133] 1) 20 g of the base-treated SG95 steviol glycoside mixture was suspended in 340 mL of warm water in a 1000 mL beaker. 2) 40 g of maltodextrin (DE=1) was added to the above suspension and blended to hydrate the material. 3) The pH was adjusted to 8.2 and the mixture was held at 50°C. 4) 2mL of CGTase enzyme was added to the above solution. 5) The mixture was incubated at 50°C for 24 hours with stirring at 200 rpm. 6) The reaction mixture was then transferred to a flask and heated in near boiling water (about 95° C.) for 30 minutes; the sample was then allowed to cool to room temperature. 7) The mixture was again transferred back into the fermentor and the flask was washed with DI water to ensure complete transfer of material from the flask to the beaker. The total volume of the mixture was approximately 500 ml. 8) 5mL (1%) of maltogenic amylase was added to the above solution. 9) The mixture was incubated at 30°C for 24 hours with stirring at 150 rpm. 10) The reaction mixture was then transferred to a flask and heated in near boiling water (about 95°C) for 30 minutes, and then the sample was allowed to cool to room temperature. 11) The mixture was then filtered through filter paper to remove poorly dispersed dextrin. 12) The granular activated carbon was then washed with DI water and packed into a 15 x 300 mm jacketed column. The carbon column was then preheated to 50°C and washed with 1.5 L of DI water to remove particulates. 13) The filtrate from step 11 was then passed through a carbon column at a flow rate of 1.5 mL / min. The column was then washed with water. 14) 24.13 g of strong base anionic resin was washed with DI water along with 16.10 g of strong acid cation resin and packed into a 15 x 300 mm jacketed column. The mixed bed column was then heated to 25°C and washed with 1.5 L of DI water. 15) The filtrate from step 13 was then passed through a mixed bed column at a flow rate of 1.5 mL / min. The column was then washed with water. 16) The macroporous adsorption column was washed with 1500 g of degassed DI water. 17) The filtrate from step 15 was then passed through a macroporous adsorption column and then washed with 1500 g of degassed DI water. 18) The macroporous adsorption column was then washed with 1500 g of a 50% ethanol solution (760.69 g of degassed DI water and 729.07 g of 190 proof ethanol). 19) The ethanol fraction was collected and dried via rotovap and freeze dryer. 20) The dried material (20.07 g) was then submitted for analytical testing for heavy metals, residual solvents and glycoside analysis. result

[0134] LC-MS analysis of the base-treated SG95 steviol glycoside mixture before enzyme treatment shows peaks corresponding to rebaudioside B and steviolbioside (FIG. 6).

[0135] The LC-MS chromatogram of the enzyme-treated material is shown in Figure 7. All assigned masses correspond to sugar (glucose) addition to the steviol core.

[0136] The steviol glycosides are expected to have the molecular weights shown in Table 4 below (the "X glc" designation refers to the number of glucose units added to the steviol core): [Table 4]

[0137] The large 641 and 803 peaks eluting after 17 min are rebaudioside B and steviolbioside. The later peaks with m / z of 787 and 773 correspond to dulcoside B and deglycosylated rebaudioside F: these would be the expected products of base treatment of rebaudioside C and rebaudioside F. The rhamnose and xylose residues on such products may inhibit their glycosylation.

[0138] Base treatment of the SG95 steviol glycoside mixture appears to produce steviolbioside and rebaudioside B as expected. Under glycosylation conditions using elevated (basic) pH and a 2:1 glucose donor to steviol glycoside ratio (w / w), most of the rebaudioside B (and possibly steviolbioside as well) was expected to remain unreacted. The small peaks at 6-11 min in Figure 7 were somewhat unexpected when compared to previous results obtained with rebaudioside B where glycosylation was performed at a slightly acidic pH and a 1:1 (w / w) glucose donor:steviol glycoside ratio (Figure 8). For example, the chromatogram in Figure 7 shows four peaks with m / z of 803: these could only be stevioside, mono-glycosylated steviolbioside, and rebaudioside B. This suggests that steviolbioside may be glycosylated at multiple positions or fragmentation peaks are recorded in the chromatograms. However, the relative complexity of Figure 7 compared to Figure 8 strongly suggests that steviolbioside glycosylation is complex compared to rebaudioside B.

[0139] The structural difference between steviolbioside and rebaudioside B is that rebaudioside B has 1-2, 1-3 linked glucose moieties, while steviolbioside only has 1-2 linked moieties. While it is possible that the two glucose residues of steviolbioside can be glycosylated together due to reduced steric hindrance, this possibility is strongly hindered for rebaudioside B. Example 3

[0140] The taste performance of various steviol glycoside and glycosylated steviol glycoside compositions was evaluated. The compositions evaluated are described as follows: Reb A = rebaudioside A, 97% purity. Reb AG = glycosylated rebaudioside A (glycosylated at pH 5.5–6.0, dextrin:steviol glycoside weight ratio = 1:1). Reb BG = glycosylated rebaudioside B (glycosylated at pH 5.5–6.0, dextrin:steviol glycoside weight ratio = 1:1). SG95 BG=glycosylated SG95 steviol glycoside mixture (source: GLG Life Tech Corporation, containing about 60% rebaudioside A and about 30% stevioside by weight), base-treated prior to glycosylation (glycosylated at about pH 8.5, dextrin:steviol glycoside weight ratio=2:1). SG95 GB=glycosylated SG95 steviol glycoside mixture (source: GLG Life Tech Corporation, containing about 60% rebaudioside A and about 30% stevioside by weight), base-treated after glycosylation (glycosylated at about pH 8.5, dextrin:steviol glycoside weight ratio=2:1). Stevioside G = glycosylated stevioside (glycosylated at pH 5.5-6.0, dextrin:steviol glycoside weight ratio = 1:1). Steviten = A commercial glycosylated steviol glycoside product marketed under the trade name "Steviten" by Daepyung Co., Ltd.

[0141] The starting steviol glycoside composition and dextrin were contacted with CGTase at 50° C. for 24 hours to produce Reb AG, Reb BG, SG95 BG, SG95 GB and stevioside G compositions. After the heat kill (enzyme inactivation) step, each resulting composition was treated with amylase at 30° C. for 24 hours to chain-shorten. After the second enzyme killing step, the compositions were purified by ion exchange resin, carbon filtration and adsorption resin. All compositions were then dried using lyophilization.

[0142] Each stevia product was tested at two levels during the day of testing, and testing was performed at both 500 ppm and 1000 ppm utilization rates. Due to known carryover effects, products were provided at increasing concentrations to allow clear readings for both data points. Solutions were provided in 2-ounce soufflé cups with 3-digit codes attached at room temperature. Panelists were allowed to use RO water and unsalted crackers to cleanse their palate before and during testing. Each daily study included 30 individuals, but sometimes as few as 28 panelists were tested due to exclusion criteria (i.e., nursing women, pregnant women, those taking medication, etc. who started the study but dropped out during the course).

[0143] The study was designed as a ranking study using a standard anchored scale of sucrose solutions in neutral pH water. In this study, panelists were provided with a series of reference samples labeled and identified by their SEV equivalents. The range exceeded the expected sweetness of the test samples, and panelists ranked the sweetness of the test samples in excess of the expected sweetness using a 15-pt maximum.

[0144] Preliminary screening of the test solutions suggested that most individuals would perceive these solutions as below 10 SEV. Because it is not possible to effectively predict the sensitivity of all individuals prior to testing, more extensive criteria were used. Specifically, the SEV criteria were as follows (Table 5): [Table 5]

[0145] Panelists were first asked to familiarize themselves with the strength of each standard solution and its corresponding SEV. They then waited 30 seconds and cleansed their palate with water and crackers. Panelists were then asked to taste the test samples (randomly identified by a 3-digit code). Panelists were then instructed to drag the marker that matched the sweetness of their test sample compared to the criteria listed above on a line scale, which ranged from 0 to 15. A box to the right of the line scale showed the panelists the value to drag to ensure that they were not confused regarding the sweetness equivalent they were grading. Once this was completed, questions were asked regarding the preference of the test sample, the similarity of the test sample to sugar taste, the intensity of any off-flavors, and the substance of any off-flavors, using the format shown in Figure 9.

[0146] The values ​​used for the preference scale were a standard 7-pt liking scale ranging from 1 to 7, the similarity scale was a standard 5-pt intensity scale ranging from 1 to 5, and the off-flavor scale was a zero-pt anchored 6-pt intensity scale ranging from 0 to 5. There were no restrictions on the descriptions of the off-flavors so that panelists could label potential off-notes with any name they wished.

[0147] The resulting taste performance data is summarized in the following table (Table 6). SEV is the sucrose equivalent value (% sucrose solution). The phrase "sucrose equivalent value" or "SEV" is the amount of non-sucrose sweetener required to provide the sweetness of a given percentage of sucrose in the same food, beverage or solution. % inversion is the percentage of panelists who rated the 500 ppm sample as sweeter than the 1000 ppm sample. This indicates being near the plateau of the intensity curve, indicating a bitter or other unpleasant taste that masks the sweetness at high concentrations. A low % inversion is optimal, along with a high SEV and high preference value. A low difference from the sugar score, a low SEV standard deviation and a low off-flavor score are also preferred. [Table 6]

[0148] The taste data shows that all glycosylated samples are superior to rebaudioside A in all measures except SEV. At 1000 ppm, some samples can be considered equivalent to rebaudioside A for sweetness while having a smaller standard deviation in the grading. This implies that the general consumer population has a more consistent experience with these products as ingredients in consumable products such as foods and beverages compared to rebaudioside A, thereby providing an advantage to the manufacture of such products. Among the compositions tested, SG95G-B is particularly potent at 500 ppm. It is therefore not surprising that the composition has the lowest sweetness threshold among the compositions tested. This attribute would be beneficial when using such compositions as flavor enhancers at low levels. The potency advantage of the composition is most evident when compared to Steviten. As seen in SG95G-B, since the glycosylated SG95 type product is mainly composed of rebaudioside A, stevioside and their glycosylated products, it is not surprising that its taste performance may be between that of glycosylated rebaudioside A and glycosylated stevioside. However, surprisingly, SG95G-B was found to be sweeter than either of the above mentioned products. Similarly, SG95B-G is mainly composed of rebaudioside B, steviolbioside and glycosylated products. Considering that steviolbioside has been reported to be unsweet and has poor taste quality compared to rebaudioside A, rebaudioside B or stevioside, it was surprising that the above product (SG95B-G) behaved very similarly to glycosylated rebaudioside B. Example 4

[0149] In this example, the following glycosylated steviol glycoside samples were tested: SG95G=glycosylated SG95 steviol glycoside mixture (supplied by Sweet Green Fields Corporation, containing about 60% rebaudioside A and about 25% stevioside by weight), glycosylated at about pH 8.5, dextrin:steviol glycoside weight ratio=2:1. SG95G with 4% Reb B = 96% SG95G as above with 4% rebaudioside B (dry blended). SG95G with 15% SG95B-G = 85% SG95G with 15% SG95B-G (as described in Example 3).

[0150] SG95G with 20% GB = glycosylated SG95 steviol glycoside mixture (supplied by Sweet Green Fields Corporation, containing about 60% rebaudioside A and about 25% stevioside by weight), glycosylated at about pH 8.5, dextrin:steviol glycoside weight ratio = 2:1, where 20% of the material is base treated after glycosylation.

[0151] The starting steviol glycoside composition and dextrin were contacted with CGTase at 50° C. for up to 24 hours to produce the SG95G sample. The product was then chain-shortened with amylase at 30° C. for 24 hours. After an enzyme killing step (heating at 95° C. for 30 minutes), the product was purified by ion exchange resin, carbon filtration and adsorption resin. The sample was then spray dried.

[0152] The starting steviol glycoside composition and dextrin were contacted with CGTase at 80°C for up to 18 hours to produce SG95G samples with 20% GB. The product was then chain-shortened with amylase at 50°C for 2 hours. After an enzyme killing step (heating at 95°C for 30 minutes), residual carbohydrates were removed. 20% of the solution was then base treated at 80°C. After neutralization, the material was recombined with the original solution and treated with ion exchange resin and carbon filtration. The sample was then dried using lyophilization.

[0153] Samples were evaluated at additional concentrations including 1500 ppm and tested as described in Example 3. The results obtained are summarized in Table 7. [Table 7]

[0154] As can be seen in Table 7, SG95G (while extremely sweet) has higher off-flavors and lower preference scores than the other products tested in this study. 4% rebaudioside B was found to improve the preference and off-flavor scores of SG95G. The inclusion of base-treated glycosylated stevia (base-treated either before or after glycosylation) was found to further improve the taste performance of the sweetener by reducing off-flavors and / or increasing preference. Additionally, a higher sweetness level is likely to be achieved by reducing bitter off-flavors compared to the 1500 ppm solution of SG95G.

Claims

1. 1. A composition comprising steviol glycosides and glycosylated steviol glycosides, comprising: The composition contains 85% or more in total of steviol glycosides and glycosylated steviol glycosides, based on the dry weight of the composition; and glycosylated rebaudioside B, glycosylated steviolbioside, steviolbioside and rebaudioside B are each present and collectively comprise 5-50% by dry weight of the composition; composition.

2. 2. The composition of claim 1, wherein glycosylated rebaudioside B, glycosylated steviolbioside, steviolbioside and rebaudioside B collectively comprise 10-25% by dry weight of the composition.

3. 2. The composition of claim 1, wherein glycosylated rebaudioside B and glycosylated steviolbioside collectively comprise 5-15% by dry weight of the composition.

4. The composition according to any one of claims 1 to 3, wherein the total amount of steviol glycosides and glycosylated steviol glycosides accounts for 90% or more by dry weight of the composition.

5. The composition according to any one of claims 1 to 3, wherein the total amount of steviol glycosides and glycosylated steviol glycosides accounts for 95% or more by dry weight of the composition.

6. The composition of any one of claims 1 to 3, wherein the composition comprises 15% to 40% by weight of non-glycosylated steviol glycosides and 60% to 85% by weight of glycosylated steviol glycosides, based on the dry weight of the total of non-glycosylated steviol glycosides and glycosylated steviol glycosides, and wherein the total weight of non-glycosylated steviol glycosides and glycosylated steviol glycosides is 90% or more, based on the dry weight of the total weight of the composition.

7. The composition of any one of claims 1 to 3, wherein the composition comprises 20% to 35% by weight of non-glycosylated steviol glycosides and 65% to 80% by weight of glycosylated steviol glycosides, based on the dry weight of the total of non-glycosylated steviol glycosides and glycosylated steviol glycosides, and wherein the total weight of non-glycosylated steviol glycosides and glycosylated steviol glycosides is 95% or more, based on the dry weight of the total weight of the composition.

8. below: i) a composition according to any one of claims 1 to 7, and ii) one or more additional food, beverage product, cosmetic or pharmaceutical ingredients; including food, beverage products, cosmetics or pharmaceuticals.

9. below: i) a composition according to claim 1, and ii) one or more additional food, beverage product, cosmetic or pharmaceutical ingredients; 16. A method for producing a food, beverage, cosmetic or pharmaceutical product, comprising combining

10. 10. The method of claim 9, wherein the one or more additional food, beverage, cosmetic, or pharmaceutical ingredients, in addition to the composition of claim 1, comprise 2-8% rebaudioside B by weight, based on the total dry weight of rebaudioside B and the composition of claim 1.

11. below: a) the composition of claim 1, and b) Rebaudioside B, 1. A sweetener composition prepared by combining a) and b), wherein the sweetener composition comprises 2 to 8% rebaudioside B by weight based on the dry weight of the sum of a) and b).

12. 12. The sweetener composition of claim 11, wherein the sweetener composition comprises 3 to 6% rebaudioside B by dry weight of the sum of a) and b).

13. below: i) a sweetener composition according to claim 11 or 12, and ii) one or more additional food, beverage product, cosmetic or pharmaceutical ingredients; including food, beverage products, cosmetics or pharmaceuticals.

14. below: i) a sweetener composition according to claim 11 or 12, and ii) one or more additional food, beverage product, cosmetic or pharmaceutical ingredients; 16. A method for producing a food, beverage, cosmetic or pharmaceutical product, comprising combining

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