阅读说明：本技术 萜烯糖苷衍生物及其用途 (Terpene glycoside derivatives and use thereof ) 是由 印丹婷 甘贤文 于 2019-08-21 设计创作，主要内容包括：本公开总体而言涉及萜烯糖苷,例如从甜叶菊(Stevia rebaudiana Bertoni)、甜叶悬钩子(Rubus suavissimus)或罗汉果(Siraitia grosvenorii)中提取的某些此类化合物。本公开还提供了此类化合物作为食品成分、调味料和甜味剂的用途,以及相关方法。本公开还提供了包含此类化合物的可摄入组合物,以及从某些植物来源有选择地提取此类化合物的方法。(The present disclosure relates generally to terpene glycosides, such as certain such compounds extracted from Stevia (Stevia rebaudiana Bertoni), Rubus suavissimus (Rubus suavissimus) or luo han guo (Siraitia grosvenorii). The present disclosure also provides for the use of such compounds as food ingredients, flavorings and sweeteners, and related methods. The disclosure also provides ingestible compositions comprising such compounds, and methods of selectively extracting such compounds from certain plant sources.)

1. A method of preparing a glucosylated terpene glycoside, the method comprising:

(a) providing an aqueous composition comprising an alpha-glucosyl sugar compound, a terpene glycoside and a transglucosidase; and

(b) reacting the alpha-glucosyl sugar compound with the terpene glycoside in the presence of the transglucosidase to form a glucosylated terpene glycoside having a terpene glycoside moiety and one or more alpha-glucosyl sugar moieties;

wherein the glucosylated terpene glycoside has an alpha-1, 6 glucosyl glycoside bond between the terpene glycoside moiety and one of the one or more alpha-glucosyl sugar moieties.

2. The method according to claim 1, wherein the reaction comprises incubating the aqueous composition.

3. The method according to claim 1 or 2, wherein the terpene glycoside is selected from the group consisting of: stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside M, dulcoside a, steviolbioside, rubusoside, terpene glycosides of Stevia (Stevia rebaudiana Bertoni) plants, terpene glycosides of Rubus suavissimus (Rubus suavissimus) plants, terpene glycosides of momordica grosvenori (Siraitia grosvenori) plants, and any combination thereof.

4. The method according to any one of claims 1 to 3, wherein the alpha-glucosyl sugar compound is selected from the group consisting of: maltose, maltotriose, maltotetraose, partial hydrolysates of starch, maltodextrin, glucose, sucrose, and any combination thereof.

5. The method according to any one of claims 1 to 4, wherein the transglucosidase is transglucosidase L.

6. The method according to any one of claims 1 to 5, wherein the glucosylated terpene glycoside is selected from the group consisting of: mono alpha-1, 6 glucosylated stevioside, mono alpha-1, 6 glucosylated rebaudioside a, mono alpha-1, 6 glucosylated rebaudioside B, mono alpha-1, 6 glucosylated rebaudioside C, mono alpha-1, 6 glucosylated rebaudioside D, mono alpha-1, 6 glucosylated rebaudioside E, mono alpha-1, 6 glucosylated rebaudioside F, mono alpha-1, 6 glucosylated rebaudioside M, mono alpha-1, 6 glucosylated dulcoside a, mono alpha-1, 6 glucosylated steviolbioside, mono alpha-1, 6 glucosylated rubusoside, and any combination thereof.

7. The method according to any one of claims 1 to 5, wherein the glucosylated terpene glycoside is selected from the group consisting of: a compound of formula I, a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, and a compound of formula IX.

8. The method according to claim 7, wherein the glucosylated terpene glycoside is selected from the group consisting of: a compound of formula I, a compound of formula II, a compound of formula VII, a compound of formula VIII, and a compound of formula IX.

9. The method according to claim 7, wherein the glucosylated terpene glycoside is selected from the group consisting of: a compound of formula III, a compound of formula IV, a compound of formula V, and a compound of formula VI.

10. Use of a compound to enhance the sweet taste of a flavoured product, wherein the compound is a glucosylated terpene glycoside formed by the method of any of claims 1 to 9.

11. Use according to claim 10, wherein the compound is present in the flavoured article at a concentration of 1ppm to 1000 ppm.

12. Use according to claim 10 or 11, wherein the flavoured article comprises a sweetener, such as a caloric sweetener, a low-caloric sweetener or a non-caloric sweetener.

13. The use according to claim 12, wherein the sweetener is selected from the group consisting of: sucrose, fructose, erythritol, xylitol, steviol glycosides, rebaudiosides, mogrosides, sucralose, acesulfame potassium, aspartame, and any combination thereof.

Technical Field

The present disclosure relates generally to terpene glycosides, such as certain such compounds extracted from Stevia (Stevia rebaudiana Bertoni), Rubus suavissimus (Rubus suavissimus) or luo han guo (Siraitia grosvenorii). The present disclosure also provides for the use of such compounds as food ingredients, flavorings and sweeteners, and related methods. The disclosure also provides ingestible compositions comprising such compounds, and methods of selectively extracting such compounds from certain plant sources.

Background

The taste system provides sensory information about the external chemical composition. Taste transduction is a more complex form of chemically triggered sensation in animals. Taste cues can be found throughout the animal kingdom, from simple metazoans to the most complex vertebrates. Mammals are believed to have five basic taste forms: sweet, bitter, sour, salty and umami.

Sweetness is the most commonly perceived taste when eating foods with high sugar content. Sweetness is generally perceived as a pleasant sensation by mammals unless there is an excess. Caloric (calorie) sweeteners, such as sucrose and fructose, are typical examples of sweetening substances. Despite the existence of various non-caloric and low-caloric alternatives, these caloric sweeteners remain the primary means by which edible products elicit a perception of sweetness upon consumption.

Metabolic and related diseases, such as obesity, diabetes and cardiovascular disease, are major public health problems worldwide. Their prevalence has increased at an alarming rate in almost every developed country. Caloric sweeteners are a key contributor to this trend because they have been included in various packaged food and beverage products, making them more palatable to the consumer. In many cases, non-caloric or low-caloric substitutes can be used in foods and beverages in place of sucrose or fructose. Even so, the sweetness imparted by these compounds is different from that of caloric sweeteners and many consumers fail to consider them as suitable substitutes. Moreover, such compounds may be difficult to incorporate into certain products. In some cases, they may be used as partial substitutes for caloric sweeteners, but their presence can result in unpleasant astringency being perceived by many consumers. Thus, the use of low-calorie sweeteners still faces certain challenges.

Terpene glycosides, such as steviol glycosides from Stevia (Stevia) (Stevia rebaudiana Bertoni) extract, rubusoside (rubusoside) from blackberry leaf (Rubus suavissimus) extract and mogroside from monk fruit (monk fruit) (silatia grosvenorii) extract are natural low calorie sweeteners. However, these products, like many other low calorie sugar substitutes, have undesirable taste attributes such as bitterness, residual aftertaste, or licorice flavor. Transglycosylation provides a means to mitigate some of these off-taste attributes. However, many of the currently disclosed glucosylated low calorie sweeteners continue to exhibit off-taste attributes, thereby preventing their widespread adoption. Thus, there is a continuing need to develop glucosylated products and transglucosylation processes that can more effectively mitigate the undesirable taste attributes.

Disclosure of Invention

The present disclosure provides transglucosylation methods and glucosylated natural low calorie sweeteners having an improved taste profile relative to other glucosylated derivatives of such compounds.

In a first form, the present disclosure provides a method of making a glucosylated terpene glycoside, the method comprising: (a) providing an aqueous composition comprising an alpha-glucosyl sugar compound, a terpene glycoside, and a transglucosidase; (b) reacting the alpha-glucosyl sugar compound with the terpene glycoside in the presence of the transglucosidase to form a glucosylated terpene glycoside having a terpene glycoside moiety and one or more alpha-glucosyl sugar moieties, wherein the glucosylated terpene glycoside has an alpha-1, 6 glucosidic linkage between the terpene glycoside moiety and one of the one or more alpha-glucosyl sugar moieties. In some embodiments, the reaction comprises incubating the aqueous composition. In some embodiments, the aqueous composition is an aqueous solution.

In a second form, the present disclosure provides a method of reducing the unpleasant taste of a terpene glycoside, the method comprising: (a) providing an aqueous composition comprising an alpha-glucosyl sugar compound, a terpene glycoside, and a transglucosidase; (b) reacting the alpha-glucosyl sugar compound with the terpene glycoside in the presence of the transglucosidase to form a glucosylated terpene glycoside having a terpene glycoside moiety and one or more alpha-glucosyl sugar moieties, wherein the glucosylated terpene glycoside has an alpha-1, 6 glucosidic linkage between the terpene glycoside moiety and one of the one or more alpha-glucosyl sugar moieties. In some embodiments, the reaction comprises incubating the aqueous composition. In some embodiments, the aqueous composition is an aqueous solution.

In some embodiments of the first or second forms, the terpene glycoside is selected from the group consisting of: stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside M, dulcoside a, steviolbioside, rubusoside, terpene glycosides of Stevia (Stevia rebaudiana Bertoni) plants, terpene glycosides of Rubus suavissimus (Rubus suavissimus) plants, terpene glycosides of momordica grosvenori (Siraitia grosvenori) plants, and any combination thereof.

In some embodiments of the first or second forms, the α -glucosyl sugar compound is selected from the group consisting of: maltose, maltotriose, maltotetraose, a partial hydrolysate of starch (batch), maltodextrin, glucose, sucrose, and any combination thereof.

In some embodiments of the first or second modality, the transglucosidase is transglucosidase L.

In some embodiments of the first or second forms, the glucosylated terpene glycoside is selected from the group consisting of: mono alpha-1, 6 glucosylated stevioside, mono alpha-1, 6 glucosylated rebaudioside a, mono alpha-1, 6 glucosylated rebaudioside B, mono alpha-1, 6 glucosylated rebaudioside C, mono alpha-1, 6 glucosylated rebaudioside D, mono alpha-1, 6 glucosylated rebaudioside E, mono alpha-1, 6 glucosylated rebaudioside F, mono alpha-1, 6 glucosylated rebaudioside G, mono alpha-1, 6 glucosylated rebaudioside M, mono alpha-1, 6 glucosylated dulcoside a, mono alpha-1, 6 glucosylated rebaudioside D, mono alpha-1, 6 glucosylated rebaudioside a, mono alpha-1, 6 glucosylated rebaudioside, and any combination thereof.

In a third form, the present disclosure provides a compound of formula I:

in a fourth form, the present disclosure provides a compound of formula II:

in a fifth form, the present disclosure provides a compound of formula III:

in a sixth form, the present disclosure provides a compound of formula IV:

in a seventh form, the present disclosure provides a compound of formula V:

in an eighth form, the present disclosure provides a compound of formula VI:

in a ninth form, the present disclosure provides a compound of formula VII:

in a tenth form, the present disclosure provides a compound of formula VIII:

in an eleventh form, the present disclosure provides a compound of formula IX:

in a twelfth form, the present disclosure provides a composition comprising at least one glucosylated terpene glycoside having a single α -1,6 glucosidic linkage selected from the group consisting of: mono alpha-1, 6 glucosylated stevioside, mono alpha-1, 6 glucosylated rebaudioside a, mono alpha-1, 6 glucosylated rebaudioside B, mono alpha-1, 6 glucosylated rebaudioside C, mono alpha-1, 6 glucosylated rebaudioside D, mono alpha-1, 6 glucosylated rebaudioside E, mono alpha-1, 6 glucosylated rebaudioside F, mono alpha-1, 6 glucosylated rebaudioside G, mono alpha-1, 6 glucosylated rebaudioside M, mono alpha-1, 6 glucosylated dulcoside a, mono alpha-1, 6 glucosylated rebaudioside D, and mono alpha-1, 6 glucosylated rubusoside. In some other embodiments thereof, the composition comprises one or more compounds of any one of the third to eleventh forms. In some other embodiments, the composition comprises one or more compounds prepared by the method of the first or second form. In some other embodiments, the composition comprises a compound of formula I, a compound of formula II, a compound of formula III, or a compound of formula VIII. In some other embodiments of any of the preceding embodiments, the glucosylated terpene glycosides in the composition impart, enhance, improve or modify the sweet taste of the flavored article. In some such embodiments, the terpene glycoside is present in the composition in an amount effective to impart, enhance, improve or modify the sweet taste of the flavored article. In some embodiments, the composition is a flavored article. In some embodiments, the composition is not a naturally occurring composition.

In a thirteenth form, the present disclosure provides the use of any compound of the third to eleventh forms or any compound prepared according to the first or second form for enhancing the sweetness of a composition, e.g., an ingestible composition. In some embodiments thereof, the composition comprises a sweetener, such as a non-caloric or caloric sweetener.

Other aspects and embodiments of the disclosure are set forth in the detailed description, figures, and claims.

Drawings

The following figures are provided to illustrate various embodiments of the compositions and methods disclosed herein. The drawings are provided for illustrative purposes only and are not intended to depict any preferred compositions or preferred methods, nor to be a source of any limitation on the scope of the claimed invention.

FIG. 1 shows an HPLC chromatogram of a product enzymatically produced by transglucosidase using rubusoside (Rubu) and corn maltodextrin with a Dextrose Equivalent (DE) of 18 as substrates.

FIG. 2 shows an HPLC chromatogram of a product enzymatically produced by transglucosidase using rebaudioside (Rubu) and maltose as substrates.

FIG. 3 shows an HPLC chromatogram of a product enzymatically produced by transglucosidase with corn maltodextrin having rebaudioside A (RebA) and Dextrose Equivalent (DE) of 18 as substrate.

Figure 4 shows an HPLC chromatogram of the product enzymatically produced by transglucosidase with steviol glycoside (Layn, 60% RebA) and 90% pure composition of corn maltodextrin with Dextrose Equivalent (DE) of 18 as substrate.

FIG. 5 shows a mixture of a compound of formula I and a compound of formula II¹H NMR spectrum.

FIG. 6 shows a mixture of a compound of formula I and a compound of formula II¹³C NMR spectrum.

FIG. 7 shows a compound of formula III¹H NMR spectrum.

FIG. 8 shows a compound of formula III¹³C NMR spectrum.

FIG. 9 shows a compound of formula VIII¹H NMR spectrum.

FIG. 10 shows a compound of formula VIII¹³C NMR spectrum.

Detailed Description

In the following description, reference is made to specific embodiments which may be practiced and are shown by way of illustration. These embodiments are described in detail to enable those skilled in the art to practice the invention described herein, and it is to be understood that other embodiments may be utilized and that logical changes may be made without departing from the scope of the aspects set forth herein. The following description of example embodiments is, therefore, not to be taken in a limiting sense, and the scope of various aspects set forth herein is defined by the appended claims. The abstract is provided to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims

Method

In certain forms, the present disclosure provides a method of making a glucosylated terpene glycoside, the method comprising: (a) providing an aqueous composition comprising an alpha-glucosyl sugar compound, a terpene glycoside, and a transglucosidase; (b) reacting the alpha-glucosyl sugar compound with the terpene glycoside in the presence of the transglucosidase to form a glucosylated terpene glycoside having a terpene glycoside moiety and one or more alpha-glucosyl sugar moieties, wherein the glucosylated terpene glycoside has an alpha-1, 6 glucosidic linkage between the terpene glycoside moiety and one of the one or more alpha-glucosyl sugar moieties. In some such forms, the method is a method of reducing the unpleasant taste of terpene glycosides.

In some embodiments thereof, the present disclosure provides an enzymatic method for preparing a composition comprising, in glycosylated form: stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, dulcoside a, steviolbioside, rubusoside, terpene glycosides of Stevia (Stevia rebaudiana Bertoni) plants, terpene glycosides of Rubus suavissimus (Rubus suavissimus) plants, terpene glycosides of momordica grosvenori (Siraitia grosvenorii) plants, or any combination thereof.

In some embodiments, the starting material for the enzymatic process is an extract of a Stevia (Stevia rebaudiana Bertoni) plant, or an extract of a Rubus suavissimus (Rubus suavissimus) plant, or an extract of a luo han guo (Siraitia grosvenorii) plant. In some other embodiments, the plant extract contains one or more terpene glycosides.

For example, by way of non-limiting illustration, Stevia rebaudiana (Stevia rebaudiana Bertoni) produces a number of diterpene glycosides, including stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside M, dulcoside a, and steviolbioside. As another non-limiting example, rubusoside can be obtained from blackberry leaves (Rubus suavissimus) which contain essentially a single terpene glycoside, known as rubusoside. In some cases, small amounts of rubusoside may also be found in stevia leaves. In some cases, rubusoside is also found in extracts of the leaves of Stevia (Stevia rebaudiana Bertoni).

In some embodiments, the starting material for the enzymatic process is a terpene glycoside purified from an extract of the Stevia (Stevia rebaudiana Bertoni) plant, or an extract of the Rubus suavissimus (Rubus suavissimus) plant, or an extract of the luo han guo (Siraitia grosvenorii) plant.

In some embodiments, the terpene glycoside starting material is selected from the group consisting of: stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside M, dulcoside a, steviolbioside, rubusoside, and any combination thereof. In some other embodiments, the terpene glycoside starting material is selected from the group consisting of: stevioside, rebaudioside a, rebaudioside B, rebaudioside C, and rubusoside.

In some embodiments, the terpene glycoside starting material is a diterpene glycoside disclosed in U.S. patent 8,257,948. In some other embodiments, the terpene glycoside starting material is a terpene glycoside disclosed in PCT publication No. WO 2017/089444. In some other embodiments, the terpene glycoside starting material is a terpene glycoside disclosed in PCT publication No. WO 2013/019050. In some other embodiments, the terpene glycoside starting material is a terpene glycoside disclosed in european patent application publication No. EP 3003058.

As used herein, the term "glycoside" refers to an organic compound to which one or more saccharide units are covalently bound at one or more sites of the chemical structure.

As mentioned above, the reaction is carried out in an aqueous composition, e.g. an aqueous solution. In some such embodiments, the aqueous composition comprises deionized water. Alternatively, in some other embodiments, the aqueous composition comprises an aqueous solution of sodium acetate. In some other such embodiments, the concentration of sodium acetate in the aqueous solution is 0.2M.

The aqueous composition may have any suitable pH depending on the terpene glycoside, the sugars reacted and the nature of the enzyme used. In some embodiments, the pH of the aqueous composition is from 4.0 to 7.0. In some other embodiments, the pH of the aqueous composition is from 4.0 to 6.0. In some other embodiments, the pH of the aqueous composition is from 4.0 to 5.0. In some other embodiments, the pH of the aqueous composition is from 5.0 to 7.0. In some other embodiments, the pH of the aqueous composition is from 6.0 to 7.0. In some embodiments, the pH of the aqueous composition is 4.0 or 4.1 or 4.2 or 4.3 or 4.4 or 4.5 or 4.6 or 4.7 or 4.8 or 4.9 or 5.0 or 5.1 or 5.2 or 5.3 or 5.4 or 5.5 or 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5 or 6.6 or 6.7 or 6.8 or 6.9 or 7.0. In some embodiments, the pH of the aqueous composition is about 5.0.

The terpene glycoside may be added at any suitable concentration as the reaction proceeds. In some embodiments, the terpene glycoside is present in the aqueous composition at a concentration of 0.005g/mL to 0.5 g/mL. In some other embodiments, the terpene glycoside is present in the aqueous composition at a concentration of 0.05g/mL to 0.25 g/mL. In some other embodiments, the terpene glycoside is added to the aqueous solution in an amount of 0.1 to 0.2 g/ml.

As noted above, the methods disclosed herein employ alpha-glucosyl sugar compounds. As used herein, the term "α -glucosyl sugar compound" refers to a sugar containing at least one α -glucosyl residue. In some non-limiting embodiments, the alpha-glucosyl sugar compound is selected from the group consisting of: maltose, maltotriose, maltotetraose, partial hydrolysates of starch, maltodextrins, glucose, sucrose, and combinations thereof. In some embodiments, the alpha-glucosyl sugar compound is maltodextrin. In some embodiments, the alpha-glucosyl sugar compound is maltose. In some embodiments, the alpha-glucosyl sugar compound is selected from the alpha-glucosyl sugar compounds disclosed in british patent publication No. 2027432.

It is contemplated that the alpha-glucosyl sugar compounds for use herein can have any suitable value for their dextrose equivalents. In some embodiments, the alpha-glucosyl sugar compound has a dextrose equivalent of 10 to 25, or 12 to 20. In some embodiments, the alpha-glucosyl sugar compound has a dextrose equivalent of about 18.

In the methods contemplated herein, the alpha-glucosyl sugar compound can have any suitable concentration in the aqueous composition. In some embodiments, the concentration of the alpha-glucosyl sugar compound in the aqueous composition is from 10 weight% (w/w) to 40 weight%, or from 20 weight% to 30 weight%. In some embodiments, the concentration of the alpha-glucosyl sugar compound in the aqueous composition is from 0.005g/mL to 0.5 g/mL. In some embodiments, the concentration of the alpha-glucosyl sugar compound in the aqueous composition is about 0.2 g/mL.

In the methods contemplated herein, the terpene glycoside and the α -glucosyl saccharide may be present in the aqueous composition in any suitable ratio relative to each other. In some embodiments, the ratio (w/w) of terpene glycoside to alpha-glucosyl sugar compound in the aqueous composition is from 100:1 to 1:100, or from 10:1 to 1: 10. In some embodiments, the ratio (w/w) of terpene glycoside to alpha-glucosyl sugar compound in the aqueous composition is about 1: 1.

In the methods disclosed herein, transglucosidase undergoes a transglycosylation reaction, thereby producing a glucosylated terpene glycoside having a single α -1,6 glucosidic bond. Alternatively, in some embodiments thereof, the transglucosidase enzyme undergoes a transglycosylation reaction, thereby producing a glucosylated terpene glycoside having one or more glucose residues covalently attached to the terpene glycoside via an alpha-1, 6 glucosidic linkage. In some embodiments, the number of glucose residues added to the terpene glycoside can be controlled by parameters such as reaction duration, reaction temperature, concentration of terpene glycoside, concentration of alpha-glucosyl sugar compound, and the like.

In some embodiments, the transglucosidase enzyme undergoes a transglycosylation reaction using maltose or maltodextrin as a substrate, thereby producing a glucosylated terpene glycoside in which a single maltose residue is added to the terpene glycoside via an α -1,6 glucosidic linkage, or two glucose units are added to the terpene glycoside via an α -1,6 glucosidic linkage.

Transglucosidase may be provided in any suitable manner. In some embodiments, the transglucosidase is in the form of a cell-free medium, a concentrated cell-free medium, a spray-dried or freeze-dried cell-free medium, or a high purity protein. In some embodiments, free and immobilized enzyme preparations are used. In some embodiments, the transglucosidase is transglucosidase L. In certain embodiments thereof, the activity of transglucosidase may be determined according to the procedure described in Hale w.s., Rawlins L.C (1951) amidase of Bacillus macerans.cellular chem.28, 49-58.

In the methods contemplated by the present disclosure, the transglucosidase enzyme is present in the aqueous composition at any suitable concentration. In some embodiments, the transglucosidase enzyme is present in the mixture in an amount in the range of 0.2 to 0.4 units per gram of the alpha-glucosyl sugar compound.

In the methods contemplated herein, the enzyme may be present in any suitable ratio relative to the terpene glycoside. In some embodiments, the ratio of the amount of enzyme in weight% to the amount of terpene glycoside in weight% is from 1:1000 to 1: 1. In some other embodiments, the ratio of the amount of enzyme in weight% to the amount of terpene glycoside in weight% is from 1:100 to 1: 2. In some embodiments, the ratio of the amount of enzyme in weight% to the amount of terpene glycoside in weight% is from 1:10 to 1: 5.

In the methods contemplated herein, the reaction may be carried out in any suitable manner. In some embodiments, the aqueous composition is incubated for a time and at a temperature sufficient to produce the glucosylated terpene glycoside. In some such embodiments, the temperature is from 30 ℃ to 90 ℃, or from 70 ℃ to 90 ℃. In some embodiments, the temperature is about 60 ℃.

The reaction may be allowed to proceed for any suitable length of time. In some embodiments, the duration is 24 hours or more, such as 24 to 48 hours. Alternatively, in some other embodiments, the duration is 24 hours or less, such as 4 to 24 hours. In some embodiments, the duration is 24 hours, or 23 hours, or 22 hours, or 21 hours, or 20 hours, or 19 hours, or 18 hours, or 17 hours, or 16 hours, or 15 hours, or 14 hours, or 13 hours, or 12 hours, or 11 hours, or 10 hours, or 9 hours, or 8 hours, or 7 hours, or 6 hours, or 5 hours, or 4 hours, or 3 hours, or 2 hours, or 1 hour. In some embodiments, the duration is about 24 hours.

In some embodiments, after the reacting step, the mixture containing glucosylated terpene glycosides is further processed. Such further processing may comprise, for example, an inactivation step or a purification step, wherein the glucosylated terpene glycosides are isolated or purified. Non-limiting examples of purification steps include, for example, enrichment, isolation or purification of the glucosylated terpene glycoside, or removal of solids from the reaction mixture. For example, solids may be removed from the reaction mixture by techniques such as filtration, centrifugation, or other techniques known to those skilled in the art. In another example, the carbohydrates may be removed from the mixture using an adsorbent resin, precipitation, or other techniques known to those skilled in the art.

In some embodiments, the further treatment comprises inactivating the transglucosidase. In one non-limiting example, transglucosidase is inactivated by heating. In some embodiments, the transglucosidase is inactivated by heating the reaction mixture to a temperature sufficient to inactivate the transglucosidase. In some embodiments, the temperature is at least 100 ℃.

U.S. patent No. 8,257,948 discloses some examples of purification steps that may be used to isolate or purify glucosylated terpene glycosides in some forms of the present disclosure. PCT publication No. WO2017/089444 discloses further examples of purification steps that may be used in some forms of the present disclosure to isolate or purify glucosylated terpene glycosides. PCT publication No. WO 2013/019050 discloses further examples of purification steps that may be used in some forms of the present disclosure to isolate or purify glucosylated terpene glycosides. European patent application publication No. 3003058 discloses other examples of purification steps that may be used in some forms of the present disclosure to isolate or purify glucosylated terpene glycosides. U.S. patent No. 8,257,948 discloses some examples of inactivation steps that may be used in some forms of the present disclosure. PCT publication No. WO2017/089444 discloses other examples of inactivation steps that may be used in some forms of the present disclosure.

Glucosylated terpene glycosides

In some embodiments, the glucosylated terpene glycoside is selected from the group consisting of: mono alpha-1, 6 glucosylated stevioside, mono alpha-1, 6 glucosylated rebaudioside A, mono alpha-1, 6 glucosylated rebaudioside B, mono alpha-1, 6 glucosylated rebaudioside C, mono alpha-1, 6 glucosylated rebaudioside D, mono alpha-1, 6 glucosylated rebaudioside E, mono alpha-1, 6 glucosylated rebaudioside F, mono alpha-1, 6 glucosylated rebaudioside G, mono alpha-1, 6 glucosylated rebaudioside M, mono alpha-1, 6 glucosylated dulcoside A, mono alpha-1, 6 glucosylated bisglucoside, mono alpha-1, 6 glucosylated rubusoside, and any combination thereof

In some embodiments, the glucosylated terpene glycoside is selected from the group consisting of: di-alpha-1, 6 glucosylated stevioside, di-alpha-1, 6 glucosylated rebaudioside a, di-alpha-1, 6 glucosylated rebaudioside B, di-alpha-1, 6 glucosylated rebaudioside C, di-alpha-1, 6 glucosylated rebaudioside D, di-alpha-1, 6 glucosylated rebaudioside E, di-alpha-1, 6 glucosylated rebaudioside F, di-alpha-1, 6 glucosylated rebaudioside G, di-alpha-1, 6 glucosylated rebaudioside M, di-alpha-1, 6 glucosylated dulcoside a, di-alpha-1, 6 glucosylated bisglucoside, di-alpha-1, 6 glucosylated rubusoside, and any combination thereof.

In some embodiments, the glucosylated terpene glycoside is selected from the group consisting of: mono alpha-1, 6 maltosylated stevioside, mono alpha-1, 6 maltosylated rebaudioside a, mono alpha-1, 6 maltosylated rebaudioside B, mono alpha-1, 6 maltosylated rebaudioside C, mono alpha-1, 6 maltosylated rebaudioside D, mono alpha-1, 6 maltoglycosylated rebaudioside E, mono alpha-1, 6 maltoglycosylated rebaudioside F, mono alpha-1, 6 maltoglycosylated rebaudioside G, mono alpha-1, 6 maltoglycosylated rebaudioside M, mono alpha-1, 6 maltoglycosylated dulcoside a, mono alpha-1, 6 maltoglycosylated rebaudioside bioside, mono alpha-1, 6 maltoglycosylated rubusoside, and any combination thereof.

In some forms, the present disclosure provides compounds of formula I:

in some forms, the present disclosure provides a compound of formula II:

in some forms, the present disclosure provides a compound of formula III:

in some forms, the present disclosure provides a compound of formula IV:

in some forms, the present disclosure provides a compound of formula V:

in some forms, the present disclosure provides a compound of formula VI:

in some forms, the present disclosure provides a compound of formula VII:

in some forms, the present disclosure provides a compound of formula VIII:

in some forms, the present disclosure provides a compound of formula IX:

in at least one form disclosed herein, the present disclosure provides a composition comprising at least one glucosylated terpene glycoside selected from the group consisting of: mono alpha-1, 6-glycosylated stevioside, mono alpha-1, 6-glycosylated rebaudioside A, mono alpha-1, 6-glycosylated rebaudioside B, mono alpha-1, 6-glycosylated rebaudioside C, mono alpha-1, 6-glycosylated rebaudioside D, mono alpha-1, 6-glycosylated rebaudioside E, mono alpha-1, 6-glycosylated rebaudioside F, mono alpha-1, 6-glycosylated rebaudioside G, mono alpha-1, 6-glycosylated rebaudioside M, mono alpha-1, 6-glycosylated dulcoside A, mono alpha-1, 6-glycosylated rebaudioside D, di alpha-1, 6-glycosylated rebaudioside A, di alpha-1, 6-glycosylated rebaudioside B, di-alpha-1, 6-glucosylated rebaudioside C, di-alpha-1, 6-glucosylated rebaudioside D, di-alpha-1, 6-glucosylated rebaudioside E, di-alpha-1, 6-glucosylated rebaudioside F, di-alpha-1, 6-glucosylated rebaudioside G, di-alpha-1, 6-glucosylated rebaudioside M, di-alpha-1, 6-glucosylated dulcoside A, di-alpha-1, 6-glucosylated steviolbioside, di-alpha-1, 6-glucosylated rubusoside, mono-alpha-1, 6-maltosylated stevioside, mono-alpha-1, 6-maltosylated rebaudioside A, mono-alpha-1, 6-maltosylated rebaudioside B, mono-alpha-1, 6-maltosylated rebaudioside C, mono-alpha-1, 6-maltosylated rebaudioside D, mono-alpha-1, 6 maltosylrebaudioside E, mono alpha-1, 6 maltosylrebaudioside F, mono alpha-1, 6 maltosylrebaudioside G, mono alpha-1, 6 maltosylrebaudioside M, mono alpha-1, 6 maltosyldulcoside A, mono alpha-1, 6 maltosylsteviolbioside, mono alpha-1, 6 maltosylrubusoside.

In at least another form, the present disclosure provides a composition comprising at least one glucosylated terpene glycoside selected from the group consisting of: a compound of formula I, a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, and a compound of formula IX. In a further embodiment thereof, the present disclosure provides a composition comprising a compound of formula I, a compound of formula II, a compound of formula III, and a compound of formula VIII.

Sweetener and/or sweetness enhancer

The glucosylated terpene glycosides described herein can be used as sweetness enhancers, flavor enhancers or sweeteners in a variety of flavored articles. In some embodiments thereof, the present disclosure provides the use of such glucosylated terpene glycosides for imparting, enhancing, improving or modifying the sweet taste of a flavored article.

In some embodiments, the concentration effective to impart, enhance, improve or modify the sweet taste of the flavored article ranges from 1ppm to 1000ppm, or from 1ppm to 500ppm, or from 1ppm to 200ppm, or from 1ppm to 100ppm, or from 1ppm to 75ppm, or from 1ppm to 50 ppm. In some embodiments, the concentration effective to impart, enhance, improve or modify the sweet taste of the flavored article is about 40 ppm. In some forms, the amount effective to impart, enhance, improve or modify the sweet taste of the flavored article is less than 40 ppm. In some forms, the amount effective to impart, enhance, improve or modify the sweet taste of the flavored article is greater than 40 ppm. In some forms, the amount effective to impart, enhance, improve or modify the sweet taste of the flavored article is from 0 to 1000 ppm.

In some embodiments, the composition comprises a glucosylated terpene glycoside (according to any of the preceding embodiments) and a food base. Non-limiting examples of suitable foods, such as foods or beverages, are also provided herein. For the purposes of this disclosure, "food base" refers to an edible product, such as a food or beverage. Thus, the flavored articles provided herein comprise a functional formula, and optionally an additional benefit agent, corresponding to a desired edible product, such as a savory cube, and a flavoring-effective amount of at least one glucosylated terpene glycoside as described herein.

The composition may comprise any suitable sweetener, or combination of sweeteners. In some embodiments, the sweetener is a common carbohydrate sweetener, such as sucrose, fructose, glucose, and sweetener compositions comprising natural sugars, such as corn syrup (including high fructose corn syrup) or other syrups or sweetener concentrates from natural fruit and vegetable sources. In some embodiments, the sweetener is sucrose, fructose, or a combination thereof. In some embodiments, the sweetener is sucrose. In some other embodiments, the sweetener is selected from the group consisting of rare natural sugars, including D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose, D-turanose, and D-albicans disaccharide. In some embodiments, the sweetener is selected from semi-synthetic "sugar alcohol" sweeteners, such as erythritol, isomalt (isomalt), lactitol, mannitol, sorbitol, xylitol, maltodextrin, and the like. In some embodiments, the sweetener is selected from artificial sweeteners, such as aspartame, saccharin, acesulfame potassium, cyclamate (cyclamate), sucralose, and alitame. In some embodiments, the sweetener is selected from the group consisting of: cyclamic acid, mogroside, tagatose, maltose, galactose, mannose, sucrose, fructose, lactose, neotame and other aspartame derivatives, glucose, D-tryptophan, glycine, maltitol, lactitol, isomalt, Hydrogenated Glucose Syrup (HGS), Hydrogenated Starch Hydrolysate (HSH), stevioside, rebaudioside a and other sweet steviol glycosides, carrelame and other guanidinium sweeteners. In some embodiments, the sweetener is a combination of two or more sweeteners set forth in this paragraph. In some embodiments, the sweetener may be a combination of two, three, four or five sweeteners disclosed herein. In some embodiments, the sweetener may be a sugar. In some embodiments, the sweetener may be a combination of one or more sugars with other natural and artificial sweeteners. In some embodiments, the sweetener is a sugar. In some embodiments, the sugar is cane sugar (cane sugar). In some embodiments, the sugar is beet sugar. In some embodiments, the sugar may be sucrose, fructose, glucose, or a combination thereof. In some embodiments, the sugar may be sucrose. In some embodiments, the sugar may be a combination of fructose and glucose.

Sweeteners may also include, for example, sweetener compositions comprising one or more natural or synthetic carbohydrates, such as corn syrup, high fructose corn syrup, high maltose corn syrup, glucose syrup, sucralose syrup, Hydrogenated Glucose Syrup (HGS), Hydrogenated Starch Hydrolysate (HSH), or other syrups or sweetener concentrates from natural fruit and vegetable sources, or semi-synthetic "sugar alcohol" sweeteners, such as polyols. In some embodiments, non-limiting examples of polyols include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerol), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, isomaltulose (isomaltulose), maltodextrin, and the like, as well as non-taste-reducing sugar alcohols or any other carbohydrates or combinations thereof that can be reduced.

The sweetener may be a natural or synthetic sweetener including, but not limited to, agave inulin, agave nectar, agave syrup, japan liqueur (amazake), brazzein (brazzein), brown rice syrup, coconut crystal, coconut sugar, coconut syrup, jujube sugar, fructan (also known as inulin fiber, fructo-oligosaccharide or fructo-oligosaccharide), green Stevia powder, Stevia rebaudiana (Stevia rebaudiana), rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside N, rebaudioside O, rebaudioside M and other steviol glycosides, stevioside, glycoside extract, honey, Jerusalem artichoke (Jerusalem artichoke) syrup, licorice root, luo han guo (fruit, powder or extract), mayonnaise (lucuma) (fruit, powders or extracts) maple sap (including sap extracted from, for example, maple sugar (Acer saccharum), black maple (Acer nigrum), Acer rubrum (Acer rubrum), silver maple (Acer saccharum), Norway maple (Acer platanoides), Acer negundo, Acer major, Acer grandiflorum (Acer grandifoliatum), Acer grandiflorum (Acer grandidentatum), Acer glabrum (Acer glaberum), Acer nigrum (Acer glaberber), Acer mono), maple syrup, maple sugar, walnut sap (including, for example, sap extracted from white walnut (Juglans cinerea), black walnut (Juglans nigra), walnut (Juglans regia), birch sap (Juglans nigra), birch (Betula), birch paper (Betula), Betula platyphylla (Betula), Betula (Betula) extract, Betula (Betula) and Betula (Betula) extract, Ironwood sap (e.g., sap extracted from ironwood americana (Ostrya virginiana)), unrefined sucrose (mascobado), molasses (molasses) (e.g., blackstrap molasses (blackstrap molasses)), molasses sugar, monatin, monellin (monellin), cane sugar (cane sugar) (also known as natural sugar, unrefined glycoside sucrose or sucrose (sucrose)), palm sugar, crude mexican sugar (panocha), crude mexican cane (pilonacilo), brown sugar brick (rapadura), raw sugar, rice syrup, sorghum syrup, cassava syrup (also known as tapioca syrup), thaumatin (thaumatin), yacon (yacon root), malt syrup, barley malt meal, beet sugar, cane sugar (cane sugar), crystallized fruit syrup, crystallized fruit juice crystals, caramel, carbitol, castor bean syrup, hydrogenated water-based juices, hydrolyzed starch, invert sugar, anethole, arabinogalactan, concentrated grape juice (arope), syrup, P-4000, acesulfame potassium (also known as acesulfame potassium or ace-K), alitame (also known as alitame), edmuntin, aspartame, bantam (baiyunoside), neotame, benzamide derivatives, bernadame, the alternative to aspartame (conderel), guanidine sweeteners (carrelame) and other guanidino sweeteners, vegetable fibres, corn sugar, conjugated sugars, curculin, cyclamate (cyclamates), cyclocarioside i (cyclocaryoside i), demerara (demerara), dextran, dextrin, saccharified malt (diastatic malt), glycine (dulcin), cyclamine (sucrin), ethoxyphenylurea (valzin), dulcoside a, dulcoside B, xylolysin (idonin), maltodextrin (maltodextrin ), estragole, ethyl maltol, hydroxyphenylglycine (glucin), gluconic acid, gluconolactone, glucosamine, glucuronic acid, glycerol, glycine, glycocalylin, glycyrrhizin, chrysose, yellow sugar, golden syrup, granulated sugar, gynostemma pentaphyllum, hernandulcin (southern andulicin), isomerized liquid sugar, jallab juice, chicory root dietary fiber, kynurenine derivatives (including N '-formyl-kynurenine, N' -acetyl-kynurenine, 6-chloro-kynurenine), galactitol, rituxin (lites), Jianmeiogen sugarcane golden sugar (licane), licarin (lycasin), N- (4-cyanophenyl) -N- (2, 3-methylenedioxybenzyl) guanidinoacetic acid (lugduname), guanidine, Falleran syrup (falerum), mabinlin I, mabinlin II, maltol, crystalline maltitol (maltorb), maltodextrin, maltotriol (maltotriol), mannosamine, miraculin (micuclin), maltose (mizuram), mogrosides (including, for example, mogroside IV, mogroside V and neomogroside), sapindoside (mukurozioside), nanomose (nano sugar), naringin dihydrochalcone, neohesperidin dihydrochalcone, crude sugar (nib sugar), black oligosaccharide, nougat (norbu), almond syrup, fossil keel (osladin), parkez (pekmez), talofuran (pennantadin), brazilin I (periandrin I), perillaldehyde, perillaseed (perillartine), petyllum, phenylalanine, pseudoqinolicin I physosiphoside I, beta-carotene I (peyroside A), pterocaryoside A (dihydropterocaryoside A), pterocaryoside (dihydropterocaryoside A), refined syrup, friction syrup (rub syrup), rubusoside (rubusoside), selliguenin A (selliguein A), suger sugar (shugr), siamenoside I, siraitia grosvenorii (siraitia grosvenorii), soybean oligosaccharide, goon sugar (Splenda), SRI oxide V, stevioside, steviolbioside, stevioside, strongins (strigins) 1, 2 and 4, saccharonic acid (sucronic acid), succinonate, sugar, sodium p-nitrophenyl ureidopropionates (suosan), phlorizin (phloridzin), super aspartame, tetrasaccharides, threitol, molasses (treacle), trilobatin (trilobatin), tryptophan and derivatives (6-trifluoromethyl-tryptophan, 6-chloro-D-tryptophan), vanillyl sugar, heptanol, birch syrup, aspartame-acesulfame, elsugrin (assugrin), and combinations or blends of any two or more thereof.

In other embodiments, the sweetener may be a chemically or enzymatically modified natural high potency sweetener. The modified natural high-potency sweetener comprises glycosylated natural high-potency sweetener, such as glucosyl, galactosyl or fructosyl derivatives containing 1-50 glucoside residues. Glycosylated natural high-potency sweeteners can be prepared by enzymatic transglycosylation reactions catalyzed by various enzymes having transglycosylation activity. In some embodiments, the modified sweetener may be substituted or unsubstituted.

Additional sweeteners also include combinations of any two or more of any of the above sweeteners. In some embodiments, the sweetener may comprise a combination of two, three, four, or five sweeteners disclosed herein. In some embodiments, the sweetener may be a sugar. In some embodiments, the sweetener may be a combination of one or more sugars with other natural and artificial sweeteners. In some embodiments, the sweetener is a caloric sweetener, such as sucrose, fructose, xylitol, erythritol, or a combination thereof. In some embodiments, the ingestible composition is free of (or in some embodiments is substantially free of) stevia-derived sweeteners, such as steviol glycosides, glucosylated steviol glycosides or rebaudiosides. For example, in some embodiments, the ingestible composition is free of stevia-derived sweeteners or comprises stevia-derived sweeteners at a concentration of no more than 1000ppm, or no more than 500ppm, or no more than 200ppm, or no more than 100ppm, or no more than 50ppm, or no more than 20ppm, or no more than 10ppm, or no more than 5ppm, or no more than 3ppm, or no more than 1 ppm.

The ingestible compositions disclosed herein may be formulated into any kind of food product. The compositions and methods provided herein can be used in food or beverage products. When the food product is a granular or powdered food, the dry particles can be easily added thereto by dry blending. Typical food products are selected from the group consisting of instant soups or sauces, breakfast cereals, milk powders, baby foods, powdered beverages, powdered chocolate beverages, spreads, powdered cereal beverages, chewing gums, effervescent tablets, cereal bars and chocolate bars. The powdered food or beverage may be intended for consumption after reconstitution of the product with water, milk and/or fruit juice or another aqueous liquid.

The food product may be selected from the group consisting of a condiment, a baked good, a powdered food, a bread filling, and a fluid dairy product. Condiments include, but are not limited to, ketchup, mayonnaise, salad dressing, worthler sauce, fruit dressing, chocolate sauce, tomato sauce, chili sauce, and mustard.

Baked goods include, but are not limited to, cakes, biscuits, pastries, breads, donuts, and the like.

Bread fillings include, but are not limited to, low or neutral pH fillings, high, medium or low solids fillings, fruit or milk (pudding or mousse type) based fillings, hot or cold complementary fillings and non-fat to full fat fillings.

Fluid dairy products include, but are not limited to, non-frozen, partially frozen, and frozen fluid dairy products such as milk, ice cream, sorbets, and yogurt. Beverage products include, but are not limited to, carbonated soft drinks including cola, lemon-lime, root beer, citrus maxima ("cooling"), fruit flavored and cream sodas; powdered soft drinks, as well as liquid concentrates such as fountain syrups and fruit juices concentrates (cordials); coffee and coffee-based beverages, coffee substitutes and cereal beverages; tea, including dry blended products and ready-to-drink tea (herbal and tea based); fruit and vegetable juices and fruit juice flavored beverages as well as fruit juice beverages, nectars, concentrates, punch and various fruit drinks ("ads"); carbonated and non-foaming sweet and flavored waters; sports/energy/health drinks; alcoholic beverages plus non-alcoholic and other low-alcoholic products including beer and malt beverages, cider and wine (non-foaming, fortified wine and wine fruit drinks (wine cooler)); other beverages in hot processed (infusion, pasteurization, ultra high temperature, electric heating or commercial aseptic sterilization) and hot-fill packages; and cold-filled products made by filtration or other freshness-preserving techniques.

The nature and type of the food or beverage ingredients do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of his general knowledge and according to the nature of the product.

The proportion of the at least one glucosylated terpene glycoside having a single α -1,6 glucosidic linkage described herein that can be incorporated into the various aforementioned articles or compositions can vary over a wide range of values. These values, when the compounds according to the invention are mixed with flavouring co-ingredients, solvents or additives commonly used in the art, depend on the nature of the article to be flavoured and on the desired organoleptic effect as well as on the nature of the co-ingredients in a given base.

In the case of flavoring compositions, typical concentrations are from about 0.0001 wt.% to 1 wt.%, or even higher levels of the at least one glucosylated terpene glycoside described herein, based on the weight of the consumer product into which it is incorporated. When at least one of the glucosylated terpene glycosides having a single α -1,6 glucosidic linkage as described herein is incorporated into a flavored article, concentrations below these, e.g., in amounts of 0.001 wt.% to 0.5 wt.%, percent relative to the weight of the article, can be used.

The present invention is best illustrated, but is not limited to the following examples.

Examples

Example 1: generation of mono alpha-1, 6-glucosylated stevioside compounds (Compounds I and II) by methods according to some of the modalities set forth herein using rebaudioside and maltodextrin as starting materials

Rubusoside (2g) and corn maltodextrin with Dextrose Equivalent (DE) of 18 (2g) were dissolved in 10mL NaOAc-HOAc (pH 6.0, 0.2M, 10mL) buffer or deionized water at room temperature. Subsequently, 100. mu.l of transglucosidase L (Amano) was added to the mixture. The enzyme-containing mixture is then heated to 60 ℃ and incubated at 60 ℃ for 24 hours to allow the transglucosidation reaction to proceed, thereby producing a glucosylated terpene glycoside having a single α -1, 6-glucosidic linkage. The reaction was stopped by inactivating the transglucosidase by incubating the reaction mixture at 100 ℃ for 30 minutes.

The resulting reaction mixture was analyzed by UPLC-UV and a mixture comprising glucosylated terpene glycosides having a single α -1,6 glucosidic bond was identified (see fig. 1). The identified mixture was purified by preparative LC. The compounds in the mixture are identified as compounds of formula I and compounds of formula II. FIGS. 5 and 6 show mixtures of two compounds, respectively¹H and¹³c NMR spectrum.

Example 2: generation of mono alpha-1, 6-glucosylated stevioside compounds (Compounds I and II) by methods according to some of the modalities set forth herein using rebaudioside and maltose as starting materials

Rubusoside (2g) and maltose (2g) were dissolved in 10mL NaOAc-HOAc (pH 6.0, 0.2M, 10mL) buffer or deionized water at room temperature. Subsequently, 100. mu.L of transglucosidase L (Amano) was added to the mixture. The enzyme-containing mixture is then heated to 60 ℃ and incubated at 60 ℃ for 24 hours to allow the transglucosidation reaction to proceed, thereby producing a glucosylated terpene glycoside having a single α -1, 6-glucosidic linkage. The reaction was stopped by inactivating the transglucosidase by incubating the reaction mixture at 100 ℃ for 30 minutes.

The resulting reaction mixture was analyzed by UPLC-UV and a mixture comprising glucosylated terpene glycosides having a single α -1,6 glucosidic bond was identified (see fig. 2). The identified mixture was purified by preparative LC. The compounds in the mixture are identified as compounds of formula I and compounds of formula II.

Example 3: generation of mono alpha-1, 6-glucosylated rebaudioside A (Compound III) by methods according to some of the modalities set forth herein using rebaudioside as starting material

Rebaudioside a (2g) and corn maltodextrin with a Dextrose Equivalent (DE) of 18 (2g) were dissolved in 10ml of deionized water at room temperature. Subsequently, 100. mu.l of transglucosidase L (Amano) was added to the mixture. The enzyme-containing mixture is then heated to 60 ℃ and incubated at 60 ℃ for 24 hours to allow the transglucosidation reaction to proceed, thereby producing a glucosylated terpene glycoside having a single α -1, 6-glucosidic linkage. The reaction was stopped by inactivating the transglucosidase by incubating the reaction mixture at 100 ℃ for 30 minutes.

The resulting reaction mixture was analyzed by UPLC-UV and glucosylated terpene glycosides having a single α -1,6 glucosidic bond were identified (see fig. 3). The identified mixture was purified by preparative LC. Glucosylated terpene glycosides having a single alpha-1, 6 glucosidic linkage are identified as compounds of formula III and compounds of formula VIII. FIGS. 7 and 8 show the compounds of formula III¹H and¹³c NMR spectrum.

Example 4: generation of mono alpha-1, 6-glucosylated rebaudioside A (Compound III) by methods according to some of the modalities set forth herein using rebaudioside as starting material

A composition of steviol glycoside with a Dextrose Equivalent (DE) of 18 (90% w/w) (1g) and corn maltodextrin (1g) was dissolved in 5ml of deionized water at room temperature. Subsequently, 100. mu.l of transglucosidase L (Amano) was added to the mixture. The enzyme-containing mixture is then heated to 60 ℃ and incubated at 60 ℃ for 24 hours to allow the transglucosidation reaction to proceed, thereby producing a glucosylated terpene glycoside having a single α -1, 6-glucosidic linkage. The reaction was stopped by inactivating the transglucosidase by incubating the reaction mixture at 100 ℃ for 30 minutes.

The resulting reaction mixture was analyzed by UPLC-UV and glucosylated terpene glycosides having a single alpha-1, 6 glucosidic bond were identified (see fig. 4). The identified mixture was purified by preparative LC. Glucosylated terpene glycosides having a single alpha-1, 6 glucosidic linkage are identified as compounds of formula III and compounds of formula VIII. FIGS. 9 and 10 show the compounds of formula VIII¹H and¹³c NMR spectrum.

Example 5: organoleptic properties of compositions comprising mono alpha-1, 6-glucosylated stevioside, a compound of formula I and a compound of formula II

A composition comprising compounds I and II was produced according to the method described in example 1. The composition was dissolved in (i) water or (ii) a 2% w/w sucrose solution, wherein the final concentration of the composition in the solution was 40 ppm. Control solutions of the corresponding (i) 1.5% or (ii) 2% w/w sucrose were also generated. A panel of 6 experts will use a three point matching method (3-Alternative formed Choice,3-AFC) or sweetness intensity rating method to assess the difference between the test composition solution and the sucrose solution. All samples will be tested in a random order in a blind test.

The sweetness intensity of a solution comprising 40ppm of a composition comprising compounds I and II is equal to a 1.5% sucrose solution based on the 3-AFC method. This indicates that the sweetness intensity of a solution containing 40ppm of the composition comprising compounds I and II in an aqueous solution is low, almost at the sweetness threshold. 40ppm of a composition comprising a compound of formula I and a compound of formula II was added to a 2% sucrose solution, increasing the sweetness intensity from 3 to 5 based on the sweetness intensity scale (scale range: 0-10). This indicates that a solution containing 40ppm of a composition comprising compounds I and II increases the sweetness of sucrose from a marginal sweetness to a moderate sweetness. Thus, a solution containing 40ppm of a composition comprising compounds I and II is an effective sweetness enhancer for sucrose.

Example 6: organoleptic properties of compositions comprising at least one mono alpha-1, 6-glucosylated terpene glycoside provided herein

According to the method described in the preceding examples, a composition comprising at least one glucosylated terpene glucoside was generated. The compounds are dissolved in water (alternatively, the compounds may also be combined as a 2% (w/w) or 4% (w/w) sucrose solution or a 7% (w/w) invert sugar plus 0.15% citric acid (w/w) solution), wherein the final concentration of the composition in the solution may range from 1 to 1000 ppm. A panel of taste experts evaluated these solutions.

When a sample of the purified compound was tasted at a concentration of 500ppm in a water base, a mixture comprising a compound of formula I and a compound of formula II was perceived: moderately sweet, about 100 to 200 times sweeter than sucrose.

When a sample of the purified compound was tasted at a concentration of 500ppm in the water base, the compound of formula III was perceived: intense sweetness, about 200 to 300 times sweeter than sucrose.

When a sample of the purified compound was tasted at a concentration of 500ppm in the water base, the compound of formula VIII was perceived: intense sweetness, about 200 to 300 times sweeter than sucrose.