RU2762719C2 - Composition modifying sweetness - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: composition modifying sweetness is proposed containing one or more high-intensity sweetener selected from steviolic glycosides, such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubuzoside and naringenin dihydrochalcone; and/or mogrosides, such as mogroside III, mogroside IIIx, mogroside IV, siamenoside, neomogroside and mogroside V; and a low-effective sweetener of 11-O-mogroside V. A ratio of one or more high-intensity sweetener and low-effective sweetener ranges from 2:1 to 12:1. The composition modifying sweetness is intended for use in a sweetened composition in the amount having sweetness less than sweetness equivalent to 1.5% by wt./vol. of sucrose, or below a sweetness recognition threshold.

EFFECT: invention provides a product with necessary properties.

33 cl, 8 dwg, 20 tbl, 12 ex

Description

FIELD OF TECHNOLOGY

The present invention generally relates to the use of one or more low potency sweetener (s) to improve one or more sweetness characteristics of one or more high potency sweetener (s). Thus, the present invention also relates to compositions comprising a mixture of at least one high intensity sweetener and at least one low potency sweetener. The present invention also relates to the use of a combination of at least one high intensity sweetener and at least one low potency sweetener as a sweetness modifier when used in combination with at least one other sweetener and / or as a sweetener. The present invention also relates to the use of one or more mogrosides as a sweetness enhancer in sweetened compositions and to said sweetened compositions. The present invention also relates to methods of making the sweeteners and compositions disclosed herein.

PRIOR ART

Sweetness in edible products, that is, products intended to be eaten by mouth, either for final swallowing or temporarily for spitting out, is often a desirable characteristic. Traditionally, sweetness is provided by the addition of one or more sweeteners, in particular low potency nutritional sweeteners such as sucrose (table sugar), fructose, glucose, xylose, arabinose, rhamnose, sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol, and also sugar syrups such as high fructose corn syrup and starch syrup. They impart significant sweetness without any unwanted aftertaste. However, in order to reduce the calorie content of an edible product, it is desirable to use lower amounts of these sweeteners. Therefore, it is desirable to provide alternative sweeteners that can reduce the caloric content of an edible while maintaining the same or similar sweet taste.

For this purpose, high intensity sweeteners (HIS) are used. High intensity sweeteners can be natural or artificial and can have a sweetness that can be several hundred times that of sucrose and therefore could theoretically replace much more sugar in the composition. Examples of high-intensity sweeteners include sucralose, saccharin, aspartame, acesulfame potassium (AceK), neotame, advantam, steviol glycosides including stevioside, rebaudioside A, rebaudioside D, or preparations of a mixture of stevioladic glycosides with rebaudioside / or stevioside components as a rebaudioside / or steviol. However, these substances usually have the disadvantage that they can impart undesirable off-flavors to edible products, usually a bitter, metallic or licorice flavor, or an undesirable long-lasting sweetness.

Therefore, it is desirable to provide an alternative and / or improved sweetness modifying composition and sweetened compositions to address one or more of these problems.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there is provided a sweetness-modifying composition comprising:

one or more high intensity sweetener (s) selected from the group consisting of steviol glycosides and / or mogrosides; and

one or more low intensity sweetener (s) selected from the group consisting of cellobiose, psicose, cyclamate and / or 11-O-mogroside V;

which increases the sweetness of the sweetened composition by more than the sweetness of the sweetness modifying composition itself; and / or

wherein the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) ranges from about 2: 1 to about 12: 1.

In accordance with a second aspect of the present invention, there is provided a sweetened composition comprising:

at least one sweetener present in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose; and

a sweetness modifying composition according to any aspect or embodiment of the present invention.

According to a third aspect of the present invention, there is provided the use of one or more low potency sweetener (s) selected from the group consisting of cellobiose, psicose, cyclamate and / or 11-O-mogroside V to improve one or more sweetness characteristics of a sweetened a composition containing one or more high intensity sweetener (s) selected from the group consisting of steviol glycosides and / or mogrosides, where the total concentration of one or more low potency sweetener (s) and one or more high potency sweetener (s) the sweetener (s) that are used have a sweetness less than a sweetness equivalent to 1.5% (w / v) sucrose.

In accordance with a fourth aspect of the present invention, there is provided a method of enhancing the sweetness of a sweetened composition, comprising providing a base composition comprising at least one sweetener present in an amount at or above its sweetness detection threshold and / or having a sweetness equal to or greater than a sweetness equivalent to about 1 , 5% (w / v) sucrose, and the addition of one or more high intensity sweetener (s) selected from the group consisting of steviol glycosides and / or mogrosides and one or more low potency sweetener (s) ) selected from the group consisting of cellobiose, psicose, cyclamate and / or 11-O-mogroside V, where the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) is from about 2: 1 to about 12: 1; and / or wherein one or more high intensity sweetener (s) are added in a total amount equal to or greater than about 15 ppm. and optionally equal to or less than about 50 ppm, and one or more low potency sweetener (s) are added in a total amount equal to or greater than about 2 ppm. and possibly equal to or less than about 12 ppm; and / or wherein the total concentration of one or more high intensity sweetener (s) of one or more low potency sweetener (s) that are added has a sweetness less than a sweetness equivalent to 1.5% (w / v) sucrose ...

In accordance with a fifth aspect of the present invention, there is provided a method of making a sweetness-modifying composition according to any aspect or embodiment of the present invention, comprising combining one or more high intensity sweetener (s) and one or more low potency sweetener (s).

In accordance with a sixth aspect of the present invention, there is provided a method of making a sweetened composition according to any aspect or embodiment of the present invention, comprising combining a base composition, one or more high intensity sweetener (s), one or more low intensity sweetener (s), and at least one another sweetener.

According to a seventh aspect of the present invention, there is provided a sweetened composition comprising at least one sweetener present in an amount having a sweetness equal to or greater than that equivalent to 1.5% (w / v) sucrose; and one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside.

In accordance with an eighth aspect of the present invention, there is provided the use of one or more of mogroside IV, siamenoside and neomogroside for enhancing the sweetness of a sweetened composition. Thus, in a further aspect, there is provided a method of enhancing the sweetness of a sweetened composition, comprising providing a base composition and adding at least one sweetener and one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside.

In accordance with a ninth aspect of the present invention, there is provided a method of making a sweetened composition according to any aspect or embodiment of the present invention, comprising combining a base composition, one or more sweetness enhancer (s) selected from mogroside IV, siamenoside and neomogroside, and at least one other sweetener ...

In accordance with a tenth aspect of the present invention, there is provided a sweetened composition comprising one or more mogroside (s). One or more mogroside (s) may, for example, be present as a sweetness enhancer and thus may be present in an amount having a sweetness less than that equivalent to 1.5% (w / v) sucrose. The sweetened composition will then further comprise at least one sweetener present in an amount having a sweetness equal to or greater than that equivalent to 1.5% (w / v) sucrose.

In accordance with an eleventh aspect of the present invention, there is provided the use of one or more mogroside (s) to enhance the sweetness of a sweetened composition. Thus, there is provided a method of enhancing the sweetness of a sweetened composition, comprising preparing a base composition and adding at least one sweetener and one or more mogroside (s).

In accordance with a twelfth aspect of the present invention, there is provided a method of making a sweetened composition according to any aspect or embodiment of the present invention, comprising combining a base composition, one or more mogroside (s), and at least one other sweetener.

In some embodiments of any aspect of the present invention, one or more high potency sweeteners may include or be mogroside V, and / or one or more low potency sweeteners may include or be 11-O-mogroside V.

In some embodiments of any aspect of the present invention, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) is from about 2: 1 to about 12: 1. In some embodiments of any aspect of the present invention, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) is from about 5: 1 to about 12: 1. In some embodiments of any aspect of the present invention, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) may be from about 6: 1 to about 10: 1.

In some embodiments of any aspect of the present invention, one or more high intensity sweetener (s) may be present in a total amount ranging from about 15 ppm. up to about 30 ppm, and / or one or more low potency sweetener (s) may be present in a total amount ranging from about 2 ppm. up to about 10 ppm In some embodiments of any aspect of the present invention, one or more high intensity sweetener (s) may be present in a total amount ranging from about 22 ppm. up to about 28 ppm, and / or one or more low potency sweetener (s) may be present in a total amount ranging from about 2 ppm. up to about 5 ppm

In some embodiments of the seventh to twelfth aspects of the present invention, one or more mogroside (s) or one or more sweetness enhancer (s) may be present in an amount ranging from about 15 ppm. up to about 50 ppm In some embodiments, one or more mogroside (s) or one or more sweetness enhancer (s) may be present in an amount ranging from about 15 ppm. up to about 35 ppm

In some embodiments of any aspect of the present invention, the combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) may have a sweetness less than that equivalent to about 1.5% (w / v) sucrose. In particular, the concentration of one or more high intensity sweetener (s) and one or more low intensity sweetener (s) in the sweetened composition may have a sweetness less than that equivalent to about 1.5% (w / v) sucrose.

In some embodiments of the seventh to twelfth aspects of the present invention, one or more mogroside (s) or one or more sweetness enhancer (s) may have a total sweetness less than a sweetness equivalent to about 1.5% (w / v) sucrose. In some embodiments, one or more sweetness enhancer (s) increases the sweetness of the sweetened composition by more than the total sweetness of the one or more sweetness enhancer (s).

In some embodiments of any aspect of the present invention, the combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) may increase the sweetness of the sweetened composition by more than the sweetness of the combination itself. In some embodiments of any aspect of the present invention, the combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) may increase the sweetness of the composition by an amount equal to or greater than the sweetness equivalent to about 1.25% ( wt./about.) sucrose.

In some embodiments of any aspect of the present invention, one or more low potency sweetener (s) attenuates the long-term sweet taste of a sweetened composition containing one or more high-intensity sweetener (s) as compared to the long-term sweet taste of the sweetened composition in the absence of one or lower potency sweetener (s).

In some embodiments of any aspect of the present invention, one or more low potency sweetener (s) reduces the bitter and / or astringent taste of a sweetened composition containing one or more high intensity sweetener (s) over the bitter and / or astringent taste. a sweetened composition in the complete absence of one or more low potency sweetener (s).

One or more (eg, all) of the sweeteners used can be natural or synthetic (artificial). One or more sweeteners can be, for example, produced by a biological method, or by an enzymatic method, or by a synthetic method.

Some embodiments of any aspect of the present invention can provide one or more of the following advantages:

increasing the sweetness in the composition;

enhancing sweetness in a composition containing at least one sweetener;

reducing the amount of nutritive sweetener required to achieve the desired sweetness;

improving one or more sweetness characteristics to make the sweet taste closer to sugar (sucrose);

a decrease in long-term sweetness (eg, a decrease in the length of time the sweet taste persists and / or a more rapid decrease in the intensity of the sweet taste);

weakening the bitter taste and / or astringent taste and / or licorice taste and / or metallic taste;

improving the manifestation of sweetness (for example, increasing the maximum intensity of the sweet taste and / or reducing the length of time over which the sweet taste is detected) (for example, reducing the long-term sweetness).

Details, examples and preferences provided in relation to any particular one or more of the indicated aspects of the present invention will be further described herein, and they are applicable to all aspects of the present invention. Any combination of embodiments, examples, and preferences described herein, in all their possible variations, are encompassed by the present invention, unless otherwise indicated or clearly contradicted by context.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a chromatogram of an extract from Luo Han Guo (Extract 2 in Table 1 below).

FIG. 2 shows the chemical structures of mogrosides 1-6. FIG. 3 shows LC / MS (liquid chromatography / mass spectrometry) analysis of commercial extracts from Luo Han Guo.

FIG. 4 shows Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy (HSQC-TOCSY) (hsqcgpmlph) of isomogroside VI with different mixing times (d9) based on heteronuclear one-quantum coherence. A: mixing time 10ms. B: mixing time 30ms. C: mixing time 60 ms. D: mixing time 100 ms. Due to the overlap of H-1 Glc II and H-6a Glc III, the Glc II correlation intensity determined by the HSQC-COZY method (correlation spectroscopy based on heteronuclear one-quantum coherence) was not analyzed here.

FIG. 5 shows a quantitative determination of the intensity of the HSQC-TOCSY (hsqcgpmlph) peaks of isomogroside VI glucopyranosyls with different mixing times (* at a mixing time of 100 ms, the C-3 and C-5 signals on HSQC-TOCSY turned out to be overlapping; therefore, the overall C-3 integration was used on the histogram and C-5);

FIG. 6 shows a strategy for identifying the sugar chain of mogroside experiments with different mixing times, the binding sequence can still be determined. ** Natural C-3 glycosylation of glucopyranosyl mogroside is absent at the moment. C-3 glycosylation on glucopyranosyl can cause a shift in the weak field from δ76 to δ81 and can be easily determined by conducting HSQC-TOCSY experiments). The sequence of steps in FIG. 6 can be outlined as follows:

In Stage 1, heteronuclear multilink correlation (HMBC) was used to determine the anomeric C-1 and H-1 sugar. They start with the connection between sugar and aglycone. Stage 2 used HSQC-TOCSY with a mixing time of 100 ms to determine the entire group from C-2 to C-6. HSQC-COZY or HSQC-TOCSY (d9 = 10 ms) was used to determine C-2. HSQC-TOCSY (d9 = 30 ms) was used to determine C-3. HSQC-TOCSY (d9 = 60 ms) was used to determine C-4. HSQC-TOCSY (d9 = 100 ms) was used to determine C-5 and C-6. In Stage 3, with a C-2 weak field shift from about δ75 to about δ81, a C-4 weak field shift from about δ71 to about δ81, or a C-6 weak field shift from about δ62 to about 69, a check is performed. HMBC for glycosylation at these positions.

With a shift in the weak field C-2 from δ75 to δ81, a shift in the weak field C-4 from δ71 to δ81, or a shift in the weak field C-6 from δ62 to 69, the HMBC is checked for glycosylation at these positions.

FIG. 7 shows the chemical structure of isomogroside VI, which has the chemical formula C ₆₆ H ₁₁₂ O ₃₄ and an exact mass of 1448.70. This chemical structure is designated as Formula I.

FIG. 8 shows the chemical structure of 11-epimogroside V, which has the chemical formula C ₆₀ H ₁₀₂ O ₂₉ and an exact mass of 1286.65. This chemical structure is designated as Formula II.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that a combination of one or more high intensity sweetener (s) (e.g. mogroside V) and one or more low potency sweetener (s) (e.g. 11-O-mogroside V) can act synergistically with at least one other sweetener (eg, sucrose) to provide a composition having a sweetness that is greater than the total sweetness of the individual sweeteners. The present invention is also based on the surprising finding that one or more low potency sweetener (s) can correct one or more negative sweetness characteristics of one or more high potency sweetener (s). For example, a combination of one or more high intensity sweetener (s) (e.g., mogroside V) and one or more low potency sweetener (s) (e.g., 11-O-mogroside V) can provide improved sweetness characteristics in a sweetened composition. (i.e., a composition containing at least one other sweetener such as sucrose in an amount above its sweetness detection threshold and / or in an amount equal to or greater than the sweetness equivalent to about 1.5% (w / v) sucrose) compared to using only one or more high intensity sweetener (s). Thus, the sweetness characteristics can be, for example, close to the sweetness characteristics of sucrose.

Thus, provided herein are various compositions comprising one or more high intensity sweetener (s) and one or more low potency sweetener (s) as disclosed herein, in particular sweetened compositions comprising at least one sweetener in an amount above its sweetness detection threshold and / or in an amount equal to or greater than the sweetness equivalent to about 1.5% (w / v) sucrose, and one or more high intensity sweeteners and one or more low potency (s) sweetener (s). Sweetened compositions can also be referred to as edible compositions. Also provided are various uses of one or more high intensity sweetener (s) and one or more low potency sweetener (s) disclosed herein, and methods for making the various compositions disclosed herein.

The present invention is also based on the surprising finding that mogroside such as mogroside IV, siamenoside and neomogroside can act as sweetness enhancers (i.e., can increase the sweetness of a sweetened composition by more than the sweetness of the sweetness enhancer itself).

Thus, various compositions are provided herein, in particular sweetened compositions containing one or more of mogroside IV, siamenoside and neomogroside.

Compositions

Provided herein are various compositions comprising at least one high intensity sweetener and at least one low potency sweetener. This document also provides compositions containing one or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside. In some embodiments, the compositions are edible compositions.

In some embodiments, there is provided a sweetness-modifying composition comprising, consisting essentially of or consisting of at least one high intensity sweetener selected from the group consisting of steviol glycosides and / or mogrosides, and at least one low potency sweetener selected from the group consisting of cellobiose, psicose, cyclamate and / or 11-O-mogroside V. In some embodiments, the sweetness modifying composition comprises, consists essentially of, or consists of one high intensity sweetener and one low potency sweetener. The sweetness-modifying composition can be, for example, a concentrate, which, for example, can be reconstituted in a sweetened (eg, edible) composition to provide an edible composition with the desired sweetness. The term "sweetened composition" refers to a composition containing at least one sweetener present in an amount above its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than that equivalent to about 1.5% (w / v. ) sucrose.

In some embodiments, a sweetened composition (eg, an edible composition) is provided comprising one or more mogroside (s), eg, one or more of mogroside IV, siamenoside, and neomogroside. In some embodiments, a sweetened composition (eg, an edible composition) is provided comprising at least one high intensity sweetener and at least one low potency sweetener. The combination of high intensity sweetener (s) and low potency sweetener (s) may be referred to as a sweetness modifying composition. One or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside, may also be referred to as a sweetness modifying composition. Thus, in some embodiments, there is provided a sweetened composition comprising at least one sweetener present in an amount above its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1.5% (w / v .) sucrose, and a sweetness-modifying composition comprising, consisting essentially of or consisting of at least one high intensity sweetener and at least one low potency sweetener. The sweetened composition can be, for example, an edible composition.

The term "enhancement" when used in relation to a particular sweetness-modifying composition refers to a synergistic sweetening effect when the sweetness-modifying composition is used in combination with at least one other sweetener. The sweetness modifying composition increases the sweetness of the sweetened composition by more than the sweetness of the sweetness modifying composition itself. In other words, the sweetness of a composition containing at least one sweetener and at least one sweetness modifying composition is greater than the total sweetness of all sweeteners in the composition. The sweetness modifying compositions described herein are used in sweetened (eg, edible) compositions in amounts that have undetectable sweetness or no detectable sweetness (below their sweetness detection threshold). Typically, a sweetness modifying composition with a sweetness below the sweetness equivalent to 1.5% (w / v) sucrose is considered by the Flavor & Extract Manufacturers Association (FEMA) as "not actually sweet ". Sweetness modifiers can also be referred to as sweetness enhancers.

A sweetened composition comprising a sweetness-modifying composition as disclosed herein and at least one sweetener in an amount above its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1.5% (wt ./vol.) sucrose may have a sweetness that is equal to or greater than a sweetness equivalent to about 1.0% (w / v) sucrose, greater than the sweetness of the sweetened composition in the absence of the sweetness modifying composition. For example, a sweetened composition may have a sweetness equal to or greater than a sweetness equivalent to about 1.1% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.15% (w / v) sucrose. or equal to or more sweetness equivalent to about 1.2% (w / v) sucrose, or equal to or more sweetness equivalent to about 1.25% (w / v) sucrose, greater than the sweetness of the sweetened composition in the absence of the composition, modifying sweetness. In other words, the sweetness modifying composition can increase the sweetness of the sweetened composition by an amount equal to or greater than a sweetness equivalent to about 1% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.1% (w / vol.) sucrose, or equal to or greater than the sweetness equivalent to about 1.15% (w / v) sucrose, or equal to or greater than the sweetness equivalent to about 1.2% (w / v) sucrose, or equal to or more sweetness equivalent to about 1.25% (w / v) sucrose. The comparative composition is identical to the specified composition except that it does not contain the specified sweetness modifying composition.

The term "sucrose equivalent sweetness" refers to the sweetness equivalence of a composition containing at least one non-sucrose sweetener to a sucrose reference solution. Typically, tasters are trained to detect the sweetness of sucrose reference solutions containing 1% to 15% sucrose (w / v). Other non-sucrose sweeteners can then be tested in a dilution series to determine the concentration of the non-sucrose sweetener at which it is as sweet (ie, isosweet) as a given sucrose reference. The term "sweet" refers to those compositions that have an equivalent sweetness. Typically, the sweetness of a given composition is measured relative to a sucrose solution (see "A Systematic Study of Concentration-Response Relationships of Sweeteners," GE DuBois, DE Walters, SS Schiffman, ZS Warwick, BJ Booth, SD Pecore, K. Gibes, BT Carr, and LM Brands, Sweeteners: Discovery, Molecular Design and Chemoreception, DE Walters, FT Orthoefer, and GE DuBois, Eds., American Chemical Society, Washington, DC (1991), pp 261-276).

The combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) (e.g., sweetness modifying composition) may, for example, have a sweetness less than a sweetness equivalent to about 1.5 wt%. / vol.) sucrose. For example, the combination of high intensity sweetener (s) and low potency sweetener (s) (e.g., sweetness modifying composition) may have a sweetness equal to or less than a sweetness equivalent to about 1.45% (w / v) sucrose , or equal to or less than about a sweetness equivalent to 1.4% (w / v) sucrose, or equal to or less than a sweetness equivalent to about 1.35% (w / v) sucrose, or equal to or less than about a sweetness, equivalent to 1.3% (w / v) sucrose. For example, the combination of high intensity sweetener (s) and low potency sweetener (s) (e.g., sweetness modifying composition) may have a sweetness equal to or greater than a sweetness equivalent to about 1% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.1% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.15% (w / v) sucrose, or equal to or greater than about a sweetness equivalent to 1 , 2% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.25% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.3% (w / v .) sucrose.

One or more mogroside (s), for example one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside, may, for example, have a sweetness less than a sweetness equivalent to about 1.5% (w / v) sucrose ... For example, one or more mogroside (s), for example one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside, may have a sweetness equal to or less than about a sweetness equivalent to 1.45% (w / v. ) sucrose, or equal or less sweetness equivalent to about 1.4% (w / v) sucrose, or equal or less sweetness equivalent to about 1.35% (w / v) sucrose, or equal or less sweetness equivalent to about 1.3% (w / v) sucrose. For example, one or more mogroside (s), for example one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside, may have a sweetness equal to or greater than that equivalent to about 1% (w / v) sucrose. , or equal to or greater than a sweetness equivalent to about 1.1% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.15% (w / v) sucrose, or equal to or greater than a sweetness equivalent about 1.2% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.25% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.3% (w / v) sucrose, / vol.) sucrose.

Each of the sweeteners and sweetness enhancers used in the compositions disclosed herein can be a natural or synthetic (artificial) sweetener. Examples of unnatural (i.e., synthetic) mogrosides are disclosed in WO 2017/075257, the contents of which are incorporated herein by reference. The term "natural sweetener" refers to sweeteners that are derived from natural sources, including mixtures that may have been enzymatically processed (e.g., glycosylated) to form non-naturally occurring compounds (this definition does not include purified compounds that have been enzymatically processed ). For example, a modified extract having a ratio of mogrol glycosides that is different (eg, expanded) from the naturally occurring ratio of mogrol glycosides may be classified as natural. For example, a mixture of glycosylated steviol glycosides and / or glycosylated mogrosides can be classified as natural. Each of the sweeteners used in the compositions disclosed herein can be edible. "Food" product refers to a product that is obtained under typical cooking conditions, such as, for example, using temperatures similar to those used in cooking methods. In some embodiments, the high intensity sweetener and low potency sweetener used in the compositions disclosed herein (eg, the sweetness-modifying composition disclosed herein) are both natural sweeteners. In some embodiments, all sweeteners used in the compositions disclosed herein are natural.

The sweeteners disclosed herein can be used in pure or purified form and can be chemically synthesized, obtained by biotechnological methods (e.g., fermentation), or isolated from a natural source (e.g., a botanical source, including but not limited to fruits, sugarcane, sugar beet).

One or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside, may, for example, have a purity of at least 80 wt%. For example, one or more mogroside (s), such as one or more of mogroside IV, siamenoside, and neomogroside, may have a purity of at least about 85% by weight, or at least about 90% by weight, or at least about 95 % of the mass., or at least about 98% of the mass, or at least about 99% of the mass. For example, one or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside, can be up to 100 wt% pure, or up to 99 wt%.

The term "high intensity sweetener" refers to compounds having a sweetness that is at least 100 times that of sucrose. In some embodiments, the high intensity sweetener has a sweetness that is at least about 120, or at least about 140, or at least about 150, or at least about 160, or at least about 180, or at least about 200, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 280, or at least about 250 about 300, or at least about 320, or at least about 340, or at least about 350, or at least about 360, or at least about 380, or at least about 400, or at least about 420, or at least about 440, or at least about 450 times the sweetness of sucrose. The high intensity sweetener can, for example, have a sweetness that is up to 1000 times that of sucrose. Although the high intensity sweetener has a sweetness that is at least 100 times that of sucrose, under the circumstances of its use in a sweetness-modifying composition as described herein, it will be used in a sweetened composition in an amount that has no detectable sweetness or that not recognized as sweet (in an amount less than sweetness equivalent to 1.5% (w / v) sucrose, which is considered by FEMA to be "not really sweet").

The one or more high intensity sweetener (s) may, for example, be one or more steviol glycosides and / or one or more mogrosides. For example, one or more high intensity sweeteners can be a mixture of steviol glycosides and mogrosides. For example, one or more high intensity sweeteners can be one or more steviol glycosides. For example, one or more high intensity sweetener (s) can be one or more mogrosides. In some embodiments, mogrosides may perform better than steviol glycosides in terms of enhancing sweetness and reducing off-flavors (eg, reducing a long-lasting sweet aftertaste).

The high intensity sweetener may, for example, be one or more steviol glycoside (s). Examples of steviol glycosides include, for example, stevioside (CAS: 57817-89-7), rebaudioside A (CAS: 58543-16-1), rebaudioside B (CAS: 58543-17-2), rebaudioside C (CAS: 63550-99 -2), rebaudioside D (CAS: 63279-13-0), rebaudioside E (CAS: 63279-14-1), rebaudioside F (CAS: 438045-89-7), rebaudioside G (CAS: 127345-21-5 ), rebaudioside H, rebaudioside I (CAS: 1220616-34-1), rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M (CAS: 1220616-44-3), rebaudioside N (CAS: 1220616-46-5), rebaudioside O (CAS: 1220616-48-7), dulcoside A (CAS: 64432-06-0), dulcoside B (CAS: 63550-99-2), rubusoside (CAS: 64849-39-4) and naringin dihydrochalcone ( CAS: 18916-17-1).

The high intensity sweetener can, for example, be one or more mogroside (s). In some embodiments, the high intensity sweetener can be one or more of the mogrosides listed herein. In some embodiments, the high intensity sweetener can be one or more of mogroside IV, siamenoside, neomogroside, and mogroside V (including all isomers thereof). For example, the high intensity sweetener can be a mixture of mogroside IV, siamenoside, and mogroside V (including all of their isomers). One or more mogroside (s) can, for example, be obtained or obtainable from extracts from Luo Han Guo fruits.

The term "low potency sweetener" refers to compounds having a sweetness that is less than 100 times that of sucrose. In some embodiments, the low potency sweetener has a sweetness that is up to about 95 times, or up to about 90 times, or up to about 85 times the sweetness of sucrose.

The one or more low potency sweetener (s) are selected from one or more cellobiose, psicose, cyclamate and / or 11-O-mogroside V (CAS: 126105-11-1). For example, one or more low intensity sweetener (s) can be one or more of cellobiose, psicose, and 11-O-mogroside V.

In some embodiments, the one or more high intensity sweetener (s) include or are high intensity mogroside. In some embodiments, the one or more low potency sweetener (s) include or are low potency mogroside. In some embodiments, one or more high intensity sweetener includes or is a high intensity mogroside, and one or more low potency sweetener (s) includes or is a low potency mogroside.

In some embodiments, the one or more high intensity sweetener includes or is mogroside V. In some embodiments, the one or more low potency sweetener includes or is 11-O-mogroside V. In some embodiments, one or more the high intensity sweetener includes or is mogroside V, and the one or more low potency sweeteners includes or is 11-O-mogroside V.

Mogrosides are a group of triterpene glycosides and can be obtained from the fruit of Luo Han Guo {Siraitia grosvenorii), also known as monk fruit, or longevity fruit, or Swingle fruit. Mogrosides make up approximately 1% of fresh fruit. By extraction, an extract can be obtained in the form of a powder containing up to 80% mogrosides. Mogroside extract contains grosvenorin II, grosvenorin I, 11-O-mogroside II (I), 11-O-mogroside II (II), 11-O-mogroside II (III), mogroside II (I), mogroside II (II) , mogroside II (III), 11-dehydroxy-mogroside III, 11-O-mogroside III, mogroside III (I), mogroside III (II), mogroside IV (I) (siamenoside), mogroside IV (II), mogroside IV (III), mogroside IV (IV), deoxymogroside V (I), deoxymogroside V (II), 11-O-mogroside V (I), mogroside V isomer, mogroside V, isomogroside V, 7-O-mogroside V, 11 -O-mogroside VI, mogroside VI (I), mogroside VI (II), mogroside VI (III) (neomogroside) and mogroside VI (IV). The exact amount of mogroside V can vary depending on the ripeness of the fruit and / or the extraction process used.

Mogrosides include mogroside, which are naturally occurring, and mogroside, which are not found in nature. Examples of mogrosides include, for example, grosvenorin II, grosvenorin I, 11-O-mogroside II (I), 11-O-mogroside II (II), 11-O-mogroside II (III), mogroside II (I), mogroside II (II), mogroside II (III), 11-dehydroxy-mogroside III, 11-O-mogroside III, mogroside III (I), mogroside III (II), mogroside She, mogroside IIIx, mogroside IV (I) (siamenoside) , mogroside IV (II), mogroside IV (III), mogroside IV (IV), deoxymogroside V (I), deoxymogroside V (II), 11-O-mogroside V (I), mogroside V isomer, mogroside V, isomogroside V , 7-O-mogroside V, 11-O-mogroside VI, mogroside VI (I), mogroside VI (II), mogroside VI (III) (neomogroside) and mogroside VI (IV). Mogroside (s) can, for example, be obtained and obtainable from extracts from Luo Han Guo.

Mogroside V (CAS: 88901-36-4) is a glycoside of a cucurbitan derivative and has the chemical formula C ₆₀ H ₁₀₂ O ₂₉ and the chemical structure shown below. Mogroside V can be found in some plant extracts such as Luo Han Guo {Siraitia grosvenorii) fruit extracts. Pure mogroside V has been found to have a sweetness of at least 400 times that of sucrose.

Siamenoside (CAS: 126105-12-2) is cucurbitan found in the fruit of Siraitia grosvenorii, and has the following chemical structure:

Mogroside IV (CAS: 89590-95-4) is a triterpene heteroside found in the fruit of Siraitia grosvenorii and has the following chemical structure:

Neomogroside (CAS: 189307-15-1) is a cucurbitan glycoside also found in the fruit of Siraitia grosvenorii and has the following chemical structure:

11-O-Mogroside V (CAS: 126105-11-1) is a derivative of mogroside V and has the following chemical structure. It is also found in plant extracts such as the Luo Han Guo (Siraitia grosvenorii) fruit extracts. It was found that 11-O-mogroside V has a sweetness that is about 84 times that of sucrose.

The ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) is equal to or greater than about 2: 1. For example, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) may be equal to or greater than about 2.5: 1, or equal to or greater than about 3: 1, or equal to or greater than about 3.5: 1, or equal to or greater than about 4: 1, or equal to or greater than about 4.5: 1, or equal or greater than about 5: 1, or equal or greater than about 5.5: 1, or equal or greater than about 6: 1, or equal to or greater than about 6.5: 1, or equal to or greater than about 7: 1, or equal to or greater than about 7.5: 1, or equal or greater than about 8: 1. The ratio of high intensity sweetener to low potency sweetener is equal to or less than about 12: 1. For example, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) may be equal to or less than about 11.5: 1, or equal to or less than about 11: 1, or equal to or less than about 10.5: 1, or equal to or less than about 10: 1, or equal to or less than about 9.5: 1, or equal to or less than about 9: 1, or equal to or less than about 8.5: 1. For example, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) may range from about 5: 1 to about 11: 1, or from about 6: 1 to about 10 : 1, or from about 6.5: 1 to about 9.5: 1, or from about 7: 1 to about 9: 1, or from about 7.5: 1 to about 8.5: 1.

In some embodiments, the ratio of one or more high intensity sweetener (s) to one or more low potency sweetener (s) is from about 2: 1 to about 12: 1, or from about 4: 1 to about 12: 1, or from about 5: 1 to about 12: 1, or from about 6: 1 to about 10: 1, or from about 7: 1 to about 9: 1. The ratio can be a weight ratio or a volume ratio. This ratio only applies to the high intensity sweeteners and low potency sweeteners in the sweetness modifying composition (high intensity and low potency sweeteners that are used in the sweetened composition in an amount below the sweetness detection threshold or in an amount having a sweetness equivalent of less than 1.5% (w / v .) sucrose).

One or more high intensity sweetener (s) may be present in the composition in a total amount equal to or greater than about 15 ppm. For example, one or more high intensity sweetener (s) may be present in the composition in a total amount equal to or greater than about 16 ppm, or equal to or greater than about 17 ppm, or equal to or greater than about 18 ppm. or equal to or greater than about 19 ppm, or equal to or greater than about 20 ppm, or equal to or greater than about 21 ppm, or equal to or greater than about 22 ppm, or equal to or greater than about 23 ppm, or equal to or greater than about 24 ppm, or equal to or greater than about 25 ppm. For example, one or more high intensity sweetener (s) may be present in the composition in a total amount equal to or less than about 50 ppm, or equal to or less than about 48 ppm, or equal to or less than about 46 ppm. or equal to or less than about 45 ppm, or equal to or less than about 44 ppm, or equal to or less than about 42 ppm, or equal to or less than about 40 ppm, or equal to or less than about 38 ppm, or equal to or less than about 36 ppm, or equal to or less than about 35 ppm, or equal to or less than about 34 ppm, or equal to or less than about 32 ppm ., or equal to or less than about 30 ppm. For example, one or more high intensity sweetener (s) may be present in the composition in a total amount ranging from about 15 ppm. up to about 50 ppm, or from about 15 ppm. up to about 45 ppm, or from about 15 ppm up to about 40 ppm, or from about 15 ppm. up to about 35 ppm, or from about 15 ppm. up to about 30 ppm For example, one or more high intensity sweetener (s) may be present in the composition in a total amount ranging from about 15 ppm. up to about 30 ppm, or from about 20 ppm. up to about 30 ppm, or from about 22 ppm. to about 28 ppm, or from about 23 ppm. to about 27 ppm, or from about 24 ppm. up to about 26 ppm For example, one or more high intensity sweetener (s) may be present in the composition in a total amount of about 20 ppm. or about 25 ppm. The composition may, for example, be a sweetened composition containing at least one sweetener in an amount having a sweetness above its sweetness detection threshold and / or equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose.

One or more low potency sweetener (s) may be present in the composition in a total amount equal to or greater than about 2 ppm. For example, one or more low potency sweetener (s) may be present in the composition in a total amount equal to or greater than about 3 ppm. For example, one or more low potency sweetener (s) may be present in the composition in a total amount equal to or less than about 12 ppm, or equal to or less than about 11 ppm, or equal to or less than about 10 ppm. or equal to or less than about 9 ppm, or equal to or less than about 8 ppm, or equal to or less than about 7 ppm, or equal to or less than about 6 ppm, or equal to or less than about 5 ppm For example, one or more low potency sweetener (s) may be present in the composition in a total amount ranging from about 2 ppm. up to about 12 ppm, or from about 2 ppm to about 10 ppm, or from about 2 ppm up to about 5 ppm, for example in a total amount of about 3 ppm. The composition may, for example, contain at least one sweetener other than a combination of a high intensity sweetener and a low potency sweetener (eg, a sweetness modifying composition) as disclosed herein. These concentration ranges may, for example, be particularly suitable for liquid compositions such as beverages, or for compositions that do not contain proteins or fats. In compositions having a base such as milk and yogurt, or in other compositions that contain proteins or fats, higher concentrations of one or more high intensity sweetener (s) and one or more low potency sweetener (s) may be used ( her). For example, concentrations that are about 1.5 times higher than those used for liquid compositions or compositions that do not contain any proteins or fats can be used. For example, concentrations that are about 1.5 to 3 times higher than those used for liquid compositions or compositions that do not contain any proteins or fats can be used.

Therefore, for example, one or more high intensity sweetener (s) may be present in the composition (for example, in a composition having a base such as milk and yogurt, or in other compositions that contain proteins and / or fats) in a total amount ranging from about 20 ppm to about 75 ppm, for example from about 22 ppm. to about 74 ppm, or from about 24 ppm. to about 72 ppm, or from about 25 ppm to about 70 ppm, or from about 26 ppm. to about 68 ppm, or from about 28 ppm. up to about 66 ppm, or from about 30 ppm up to about 65 ppm, or from about 30 ppm up to about 60 ppm, or from about 30 ppm up to about 55 ppm, or from about 30 ppm up to about 50 ppm, or from about 30 ppm. up to about 45 ppm

Therefore, for example, one or more low potency sweetener (s) may be present in the composition (for example, in a composition having a base such as milk and yogurt, or in other compositions that contain proteins and / or fats) in a total amount ranging from about 3 ppm up to about 20 ppm, or from about 4 ppm. to about 18 ppm, or from about 4 ppm. to about 16 ppm, or from about 5 ppm. up to about 15 ppm, or from about 6 ppm. up to about 15 ppm

In some embodiments, the sweetened composition comprises at least one sweetener in an amount having a sweetness above its sweetness detection threshold and / or equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose, and a sweetness modifying composition, consisting of 15 ppm up to about 50 ppm one or more high intensity sweetener (s) as described herein, and from 2 ppm up to 12 ppm one or more low potency sweetener (s) as described herein. In some embodiments, the edible composition comprises at least one sweetener and a sweetness modifying composition of 15 ppm or more. up to about 30 ppm one or more high intensity sweetener (s) as described herein, and from 2 ppm up to 10 ppm one or more low potency sweetener (s) as described herein. In some embodiments, the edible composition comprises at least one sweetener and a sweetness modifying composition from 20 ppm. up to about 30 ppm one or more high intensity sweetener (s) as described herein, and from 2 ppm up to 10 ppm one or more low potency sweetener (s) as described herein. In some embodiments, the edible composition comprises at least one sweetener and a sweetness modifying composition from 22 ppm. up to about 28 ppm one or more high intensity sweetener (s) as described herein, and from 2 ppm up to 5 ppm one or more low potency sweetener (s) as described herein. In some embodiments, the high intensity sweetener is mogroside V. In some embodiments, the low potency sweetener is 11-O-mogroside V.

One or more mogroside (s), such as one or more of mogroside IV, siamenoside and neomogroside, may, for example, be present in the sweetened composition in a total amount equal to or greater than about 15 ppm. For example, one or more mogroside (s), such as one or more of mogroside IV, siamenoside and neomogroside, may, for example, be present in the sweetened composition in a total amount equal to or greater than about 16 ppm, or equal to or greater than about 17 ppm, or equal to or greater than about 18 ppm, or equal to or greater than about 19 ppm, or equal to or greater than about 20 ppm, or equal to or greater than about 21 ppm, or equal to or greater than about 22 ppm, or equal to or greater than about 23 ppm, or equal to or greater than about 24 ppm, or equal to or greater than about 25 ppm. For example, one or more mogroside (s), such as one or more of mogroside IV, siamenoside, and neomogroside, may, for example, be present in the sweetened composition in a total amount equal to or less than about 50 ppm, such as equal to or less than about 45 ppm, such as equal to or less than about 40 ppm, such as equal to or less than about 35 ppm. For example, one or more mogroside (s), such as one or more of mogroside IV, siamenoside, and neomogroside, may be present in the sweetened composition in a total amount ranging from about 15 ppm. up to about 50 ppm, or from about 15 ppm. up to about 45 ppm, or from about 15 ppm up to about 40 ppm, or from about 15 ppm. up to about 35 ppm, or from about 20 ppm. up to about 35 ppm, or from about 20 ppm. up to about 30 ppm

The term "ppm" refers to parts per million by weight, for example, by weight of a compound such as mogroside V (in milligrams), per kilogram of product containing such a compound (i.e., mg / kg), or by weight of a compound (for example, orally consumed / edible product from the present disclosure), such as mogroside V (in milligrams), per liter of product containing such a compound (i.e., mg / L), or by volume, for example, by volume of a compound such as mogroside V (in milliliters), per liter of product containing such a compound (i.e. ml / l).

The sweetness-modifying compositions described herein may, for example, contain high-intensity and low-intensity sweeteners at higher concentrations, and then they can be reconstituted in the sweetened composition to obtain the concentrations specified herein.

The sweetened composition contains at least one sweetener in an amount equal to or greater than its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than that equivalent to about 1.5% (w / v) sucrose. The term "sweetness detection threshold" refers to the lowest known concentration of a compound that is perceived to be sweet by the human palate. A sweetness equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose is considered by FEMA to be "proper sweet".

The at least one sweetener can be nutritive or non-nutritive. Nutritional sweeteners increase the calorie content of foods that contain them, while non-nutritive sweeteners are very low in calories or have no calories at all. Aspartame, the only nutritious high-intensity sweetener approved for use, has more than 2% calories in sugar equivalent, as opposed to non-nutritive sweeteners, which have less than 2% calories in sugar equivalent.

The at least one sweetener may, for example, be selected from one or more of sucrose, fructose, glucose, xylose, arabinose, rhamnose, tagatose, allulose, trehalose, isomaltulose, acesulfame potassium (ActK), aspartame, steviol glycoside ( ov), sucralose, high fructose corn syrup, starch syrup, saccharin, sucralose, neotame, advantam, Luo Han Guo extract, neohesperidin, dihydrochalcone, naringin dihydrochalcone, neohesperidin dihydrochalcone, starchydiosydosin, stupidosium sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol. Examples of sweeteners that can be used in sweetened compositions are disclosed, for example, in WO 2016/038617, the contents of which are incorporated herein by reference.

The at least one sweetener may, for example, be selected from one or more of sucrose, high fructose corn syrup, acesulfame potassium (AceK), aspartame, steviol glycoside (s) and / or sucralose.

How to sweeten consumable products using sufficient amounts of sweeteners is well known in the art. Depending on the usable product, the amount of sweetener can be reduced by adding a sweetness modifying composition as disclosed herein. For example, a reduction from about 1 ° to about 4 ° Brix or more can be achieved.

At least one other sweetener, present in an amount equal to or greater than its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than that equivalent to about 1.5% (w / v) sucrose, may be , for example, is used in a sweetened composition in an amount equal to or greater than about 0.01% (w / v). For example, at least one other sweetener can be used in the sweetened composition in an amount equal to or greater than about 0.1% (w / v), or equal to or greater than about 0.5% (w / v), or equal to or greater than about 1% (w / v), or equal to or greater than about 2% (w / v). For example, at least one other sweetener can be used in the edible composition in an amount equal to or less than about 20% (w / v), or equal to or less than about 15% (w / v), or equal to or less about 10% (w / v), or equal to or less than about 8% (w / v), or equal to or less than about 6% (w / v), or equal to or less than about 5% (w / v) ./about.).

At least one other sweetener, present in an amount equal to or greater than its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than that equivalent to about 1.5% (w / v) sucrose, may be used in the sweetened compositions disclosed herein (eg, in an edible composition), in amounts that are sweet with from about 2% (w / v) to about 15% (w / v) sucrose.

In some embodiments, a sweetness-modifying composition is provided of mogroside V and 11-O-mogroside V in a ratio ranging from about 2: 1 to about 12: 1, such as from about 6: 1 to about 10: 1. This sweetness modifying composition can, for example, be used as a sweetness enhancer or modifier in an edible composition. The edible composition may, for example, contain at least one other sweetener such as sucrose. Mogroside V can, for example, be used in an edible composition in an amount ranging from about 15 ppm. up to about 30 ppm, or from about 20 ppm up to about 30 ppm (for example, about 20 ppm or about 25 ppm). 11-O-mogroside V can be used in an edible composition in an amount ranging from about 2 ppm. up to about 12 ppm or from about 2 ppm. up to about 10 ppm (for example, about 8.5 ppm or about 3 ppm). At least one other sweetener may, for example, be present in the edible composition in an amount that is sweet with from about 2% (w / v) to about 15% (w / v) sucrose.

The compositions can be in any suitable form, for example in solid form (eg, powder, granules, tablets), or in the form of a solution (eg, aqueous solution), or in the form of an emulsion, or in the form of a suspension. For example, the compositions may further comprise a diluent or bulking agent such as dietary fiber.

Edible compositions that are disclosed herein include, for example, the following:

- Wet / liquid soups regardless of concentration or container, including frozen soups. For the purposes of this definition, soup (s) means food made from meat, poultry, fish, vegetables, grains, fruits and other ingredients, cooked as a liquid that may contain visible pieces of some or all of the ingredients. The soup can be clear (like a broth), thick (like a chowder), smooth, mashed or chunky, ready to eat, semi-thickened or thickened, and can be served hot or cold as a first course, or as a main course, or in as a snack between meals (drunk as a drink). Soup can be used as an ingredient in other food ingredients and can range from broths (consommé) to sauces (creamy and cheese-based).

- Dehydrated and culinary food products, including culinary auxiliary products such as powders, granules, pastes, concentrated liquid products, including concentrated broth, broth and broth-like products in pressed cubes, tablets or powder or granular form, which are sold by individually as a finished product or as an ingredient in a product, sauces and recipes (regardless of technology).

- Meal solution products, such as: dehydrated and freeze-dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated convenience soups, dehydrated or room temperature pre-cooked meals, flour single-serving packaged foods and foods, including pasta, potato and rice dishes.

- Food supplements such as condiments, marinades, salad dressings, salad toppings, sauces, breads, batter mixes, stable spreads, barbecue sauces, liquid potting mixes, concentrates, sauces or sauce mixes, including topping for lettuce, sold as a finished product or as an ingredient in a product, whether dehydrated, liquid, or frozen.

- Beverages, including drink mixes and concentrates, including, but not limited to, alcoholic and non-alcoholic, ready-to-drink and dry powdered drinks, carbonated and non-carbonated beverages such as soda, fruit or vegetable juices, alcoholic and non-alcoholic beverages.

- Confectionery, such as cakes, cookies, tarts, candy, gum, gelatins, ice cream, sorbets, puddings, jams, jellies, salad dressings and other condiments, cereals and other breakfast foods, canned fruits and fruit sauces, etc. ...

- Dairy products such as milk, cheese, yogurt.

- Pharmaceutical compositions, which can be, for example, in the form of a syrup, emulsion, suspension, solution or other liquid form.

- Dental compositions, including, for example, mouth fresheners, throat rinses, toothpastes, dental polishes, dental elixirs, mouth sprays, and dental floss.

- Food gel compositions.

The compositions disclosed herein may further comprise a base composition. For example, the edible compositions disclosed herein may further comprise an edible base composition. This applies to all ingredients required for the composition, except for the combination of a high intensity sweetener and a low potency sweetener (eg, a sweetness modifying composition). The base composition may, for example, be a sweetened base composition containing at least one other sweetener present in an amount equal to or greater than its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1, 5% (w / v) sucrose. They will naturally vary in both nature and proportion, depending on the nature and use of the composition, but they are also generally known in the art and can be used in proportions known in the art. The composition of such a base composition for various purposes is therefore within the skill of the art.

Ingredients in the base composition may include, but are not limited to, anti-caking agents, anti-foaming agents, antioxidants, binders, colorants, diluents, disintegrants, emulsifiers, encapsulating agents or formulations, enzymes, fats, flavor enhancers, flavoring agents, gums, binding agents, polysaccharides, preservatives, proteins, solubilizers, solvents, stabilizers, sugar derivatives, surfactants, sweeteners, vitamins, waxes and the like. Solvents that can be used are known to those skilled in the art and include, for example, ethanol, ethylene glycol, propylene glycol, glycerin, and triacetin. Encapsulating agents and gums include maltodextrin, gum arabic, alginates, gelatin, modified starch, and polysaccharides.

Examples of additives, excipients, carriers, diluents or solvents for flavoring or aromatic compounds can be found, for example, in "Perfume and Flavor Materials of Natural Origin", S. Arctander, Ed., Elizabeth, N.J., 1960; in "Perfume and Flavor Chemicals", S. Arctander, Ed., Vol. I & II, Allured Publishing Corporation, Carol Stream, USA, 1994; in Flavourings, E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998, and in the CTFA Cosmetic Ingredient Handbook, J.M. Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988.

The ratio of the combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) (e.g. sweetness modifying composition) or one or more sweetness enhancer (s) selected from mogroside IV, siamenoside and neomogroside will depend on the nature of the composition and the degree and characteristics of sweetness that are desired. A specialist can easily determine the quantitative content in each case as a result of simple non-inventive experimentation. The amounts and ratios disclosed herein are illustrative only, and the specialist in flavoring will be able to achieve specific effects by working outside this range, and this should be considered as an indication only.

The pH of the compositions disclosed herein can be any pH that does not adversely affect the taste of the sweetener mixture. For example, the pH can range from about 1.8 to about 8, or from about 2 to about 5. One skilled in the art is able to identify the appropriate concentration of each sweetener for use depending on the pH of the composition.

The use of one or more low potency sweetener (s) with one or more high intensity sweetener (s) may, for example, improve one or more sweetness characteristics in the sweetened composition as compared to using only one or more high intensity sweetener (s). (her). Thus, the sweetened compositions disclosed herein have, for example, one or more improved sweetness characteristics over sweetened compositions in the absence of one or more low potency sweetener (s). The use of one or more sweetness enhancer (s) selected from mogroside IV, siamenoside, and neomogroside may, for example, improve one or more sweetness characteristics of the sweetened composition over using another sweetness enhancer, such as an extract from Luo Han Guo, instead of one or more from mogroside IV, siamenoside and neomogroside.

Sweetened compositions disclosed herein may, for example, have one or more sweetness characteristics that are more similar to sucrose as compared to sweetened compositions in the absence of one or more low potency sweetener (s) or compared to sweetened compositions containing another sweetness enhancer.

Sweetened compositions disclosed herein may, for example, have a reduced long-term sweet taste when compared to sweetened compositions in the absence of one or more inferior sweetener (s) or compared to sweetened compositions containing another sweetness enhancer.

The sweetened compositions disclosed herein may, for example, have a reduced long-term bitter taste and / or astringency and / or metallic taste and / or licorice taste compared to sweetened compositions in the absence of one or more low potency sweetener (s) (s) or compared to sweetened compositions containing another sweetness enhancer.

Sweetened compositions disclosed herein may, for example, have an increased sweetness manifestation compared to sweetened compositions in the absence of one or more inferior sweetener (s) or compared to sweetened compositions containing another sweetness enhancer.

The comparative sweetened composition is identical except that it does not contain any one or more of the low potency sweetener (s), or is identical except that it contains a different sweetness enhancer in place of one or more mogroside (s). for example one or more of mogroside IV, siamenoside and neomogroside.

Applications

It is proposed to use a combination of one or more high intensity sweetener (s) and one or more low potency sweetener (s) to enhance sweetness of a composition containing at least one other sweetener present in an amount equal to or greater than its sweetness detection threshold and / or in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose. The combination of one or more high intensity sweetener (s) and one or more low intensity sweetener (s) has a sweetness less than that equivalent to 1.5% (w / v) sucrose. One or more high intensity sweetener (s), one or more low potency sweetener (s), and at least one other sweetener may be in accordance with any embodiment disclosed herein.

There is provided the use of one or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside, to enhance the sweetness of a composition containing at least one other sweetener present in an amount equal to or greater than its sweetness detection threshold, and / or in an amount having a sweetness equal to or greater than a sweetness equivalent to about 1.5% (w / v) sucrose.

Thus, there is provided a method for enhancing the sweetness of a sweetened composition, comprising obtaining a base composition containing at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and / or in an amount having a sweetness equal to or greater than the sweetness equivalent to about 1, 5% (w / v) sucrose, and the addition of at least one low potency sweetener, at least one high intensity sweetener; or the addition of one or more mogroside (s), for example one or more of mogroside IV, siamenoside and neomogroside. Each component of the final composition can be added in any order to obtain the desired final composition. The method may, for example, include mixing the components.

One or more high intensity sweetener and / or a combination of one or more high intensity sweeteners and one or more low potency sweeteners (e.g., sweetness-modifying composition) and / or one or more mogroside (s), e.g., one or more of mogroside IV, siamenoside, and neomogroside may, for example, increase the sweetness of the sweetened composition by an amount equal to or greater than the sweetness equivalent to about 1.0% (w / v) sucrose. For example, high intensity sweetener (s) and / or a combination of high intensity sweetener (s) and low potency sweetener (s) and / or one or more mogroside (s), such as one or more of mogroside IV, siamenoside and neomogroside, can increase the sweetness of the sweetened composition by an amount equal to or greater than the sweetness equivalent to about 1.1% (w / v) sucrose, or equal to or greater than the sweetness equivalent to about 1.15% (w / v. ) sucrose, or equal to or greater than a sweetness equivalent to about 1.2% (w / v) sucrose, or equal to or greater than a sweetness equivalent to about 1.25% (w / v) sucrose. The composition may, for example, be a composition containing at least one other sweetener.

Also provided is the use of one or more low potency sweetener (s) to improve one or more sweetness characteristics of a sweetened composition containing one or more high intensity sweetener (s). The one or more high intensity sweetener (s) and one or more low potency sweetener (s) are used in amounts having a sweetness less than that equivalent to about 1.5% (w / v) sucrose.

Thus, there is provided a method for improving one or more sweetness characteristics of a sweetened composition containing one or more high intensity sweetener (s) in an amount having a sweetness less than the sweetness equivalent to 1.5% (w / v) sucrose, comprising obtaining a composition containing one or more high intensity sweetener (s) and the addition of one or more low potency sweetener (s). Each component of the final composition can be added in any order to obtain the desired final composition. The method may, for example, include mixing the components.

Improving one or more sweetness characteristics of a sweetened composition containing a high intensity sweetener may, for example, provide sweetness characteristics that are more similar to those of sucrose.

Sweetness characteristics may include a flavor profile (flavor profile), which refers to the intensity of flavor and aroma and the perceptual attributes of a given compound. Examples of sweetness perception attributes are sweetness intensity, bitterness, black liquorice flavor, etc.

Sweetness characteristics may include a time profile, which refers to changes in the perception of sweetness over time. Each sweetener exhibits a characteristic time of appearance (AT) and time of disappearance (ET). The most highly effective sweeteners, as opposed to carbohydrate sweeteners, exhibit prolonged ET (long-term). Typically, the found sweetness, equivalent to sucrose, rises sharply to the maximum level of response, then decreases over time. The longer the decay lasts, the longer the discovered sweetness of the compound lasts.

In some embodiments, one or more low potency sweetener (s) can be used to reduce the long-lasting sweet taste of a sweetened composition containing one or more high intensity sweetener (s). In other words, the low potency sweetener can be used to shorten the sweet taste disappearance time (ET) of a sweetened composition containing one or more high intensity sweetener (s). This refers to an undesirable long-lasting sweetness taste in the mouth following initial swallowing or spitting out of the composition. Long-term sweetness can, for example, refer to the length of time that the sweet taste persists after its initial detection, to how quickly the intensity of the sweet taste decreases or gradually disappears after its initial detection, and to the intensity of the sweet taste after its initial detection. The one or more low potency sweetener (s) may, for example, shorten the length of time that sweet taste persists after initial detection and / or increase the rate at which the sweet taste decreases after initial detection, and / or reduce the intensity of the sweet taste after its initial detection.

In some embodiments, one or more low potency sweetener (s) can be used to reduce the bitter taste and / or astringent taste and / or metallic taste and / or licorice taste of a sweetened composition containing one or more high intensity (s) sweetener (s). The term "licorice" refers to the sweet taste of a compound.

In some embodiments, one or more low potency sweetener (s) can be used to enhance the sweetness of a sweetened composition containing one or more high intensity sweetener (s). The onset of sweetness refers to the length of time required for the initial detection of sweetness and the intensity with which the sweetness is initially detected. The one or more low potency sweetener (s) may, for example, decrease the amount of time required to initially detect sweetness and / or increase the intensity with which sweetness is initially detected.

The degree of sweetness and other characteristics of sweetness described in this document can be evaluated by a tasting panel of qualified experts, for example, as described in the examples below.

Manufacturing methods

Additionally, methods of making the compositions disclosed herein are provided. The compositions can be in accordance with any embodiment disclosed herein.

The methods can include combining each component of the desired composition in the desired ratios and optionally mixing the components together. Components can be combined and mixed in any suitable order.

One skilled in the art will be able to identify a suitable method of making the composition (eg, the appropriate order in which to combine or mix the components) depending on the nature of the composition and the degree and characteristics of sweetness that are desired. The methods may, for example, include preparing a desired base composition and adding sweeteners thereto.

Each of the sweeteners disclosed herein can be made synthetically, or biologically (eg, enzymatic), or by fermentation, or can be isolated from a natural source such as a plant or fruit.

The method may, for example, include contacting at least one mogrol precursor substrate with a mogroside pathway enzyme. The enzyme may, for example, be present in a cell lysate, or may be present in a host cell (eg, a recombinant host cell). The enzyme may, for example, be a UGT (UDP (uridine diphosphate) -glucuronosyltransferase) enzyme.

For example, a mogroside compound can be made biosynthetically as disclosed in WO 2013/076577 or WO 2014/086842, the contents of which are incorporated herein by reference.

For example, mogroside V can be made biosynthetically disclosed in Itkin et al., "The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorif," PNAS, 7 November 2016, E7619-E7628, and WO 2016/038617, the contents of which are incorporated herein by reference.

For example, a mogroside compound can be made by modifying (eg, redistributing a glycosidic moiety) another mogroside compound. For example, a mogroside compound can be made by redistributing the glycosidic moiety of another mogroside compound using an acid or enzymes, as disclosed in WO 2014/150127, the contents of which are incorporated herein by reference.

The method may, for example, include extracting one or more sweetening compounds from a natural source such as a plant or fruit. This may, for example, be followed by a purification step to produce a high intensity sweetener, a low intensity sweetener, or a mixture of sweeteners (eg, a mixture of high intensity sweeteners such as a mixture of mogrosides). The extract may, for example, have a relatively high content of mogroside V and / or 11-O-mogroside V (e.g., at least about 30 wt% or at least about 40 wt%). This method may, for example, include fractionation, such as flash chromatography. One or more mogroside compounds (eg mogroside V) can be obtained from the fruit of Luo Han Guo (Siraitia grosvenorii).

When using the fermentation method to make the desired product (for example, the desired mogroside product), the target product can be extracted from the aqueous fermentation reaction medium using a suitable solvent (for example, heptane), followed by fractional distillation. The chemical composition of each fraction can be quantified by GC / MS (gas chromatography / mass spectrometry). The fractions can be mixed to provide the desired mogroside compounds (eg, mogroside V and 11-O-mogroside V) for use in flavoring or other applications.

Verification of the acceptability of the final blended product can be made by direct comparison with a reference mogroside flavor (eg, an existing commercial natural flavor obtained from a commercial supplier).

EXAMPLES

Example 1 Methods

Luo Han Guo fruit extracts obtained from Blue California (Tomas, Rancho Santa Margarita, California) (extract 4), Azile LCC (Rolling Hills Est, California) (extracts 1 and 2) and Chr. Olesen Group (Gentofte, Denmark) (extract 3) were analyzed to identify the compounds present in the extract.

Sample solutions of the extracts were prepared by dissolving 16.52 μg of the sample in 25.0 ml of solvent (acetonitrile / water 20/80 v / v). From this solution, 100 μl was transferred to an HPLC (high performance liquid chromatography) vial and 900 μl solvent (66.1 ppm solution) was added. From the sample solution, 10 μl was transferred to an HPLC vial and 990 μl solvent (6.61 ppm solution) was added. Both solutions are 66.1 ppm. and 6.61 ppm. was injected twice on LC / MS (liquid chromatography / mass spectrometry).

Calibration (standard) solutions of mogroside V were prepared by dissolving 9.22 mg of mogroside V (98.5% mogroside V, obtained from AAPIN Chemicals Ltd., Oxfordshire, UK) in 10.0 ml of a solvent (acetonitrile / water 20/80 about. / about.). This stock solution was stored in a freezer and used to prepare solutions of mogroside V at various concentrations (0.11 ppm, 0.34 ppm, 1.02 ppm, 3.07 ppm, and 9.22 ppm). These solutions were also injected twice on LC / MS.

2 μl of each solution was injected onto an Acquity C18 BEN 1.7 μm 150 x 2.1 mm column (Waters, Milford, Massachusetts, United States) at 40 ° C. Compounds were eluted using a mixture of acetonitrile and 0.1% formic acid in water, starting at 20% acetonitrile sludge and going up to 50% acetonitrile in 14 minutes. The gradient was returned to its initial values after 1 minute and stabilized for 5 minutes. The flow was set at 400 μL throughout the run.

The eluted compounds were detected using liquid chromatography / mass spectrometry (LC / MS). The mass spectrometer operated in the ERI (electrospray ionization) mode with the registration of negative ions with a measurement of 150-2000 amu. (atomic mass unit) with a resolution of 70,000. The gas flow rates were: envelope 60, auxiliary 20 and purge 3. The capillary temperature and the auxiliary gas heater temperature were set at 380 ° C and 400 ° C, respectively.

The% of each component in the extract was calculated using the following equation and calibrated against the curve of the calibration solutions of mogroside V (reference) at various concentrations described above.

ratio =% component

area = area of the component in the sample (average area of 2 shots)

V = volume of sample solvent in liters

d = sample dilution (from sample solution to vial)

slope = slope from mogroside V calibration curve at b (cutoff) = 0

SW = sample weight in mg

results

FIG. 1 is a chromatogram of an extract from Luo Han Guo (extract 2 in Table 1 below).

Table 1 shows the composition of four different Luo Han Guo extracts. Mogroside V is the mogroside having the highest concentration in all four extracts (approximately 45 wt% in extract 1).

Example 2 Methods

An extract from Luo Han Guo fruits obtained from Azile LCC (Rolling Hills Est, California) (extract 1 from Example 1 above), containing about 68% by weight of the agrosides, was fractionated by reverse phase (C-18) flash chromatography.

Compounds were eluted using a mixture of methanol (MeOH) in water, starting at 30% MeOH followed by a linear gradient of 30-80% MeOH, then finally the column was washed with 80% MeOH. Solvents were introduced at a flow rate of 30 ml / min throughout the separation procedure. The eluted compounds were visualized with a UV detector set at 210 nm and a coronal light scatter detector. The% of each component in the extract was calculated using the equation described in Example 1 above.

The collected fractions were pooled according to Table 2 below and then freeze-dried to powders. Powders corresponding to the different pooled fractions shown in Table 2 below were dissolved at different concentrations in addition to 5% sucrose. Three panelists (qualified tasters) compared the taste of these samples to 5% sucrose controls. In this way, the sweetness enhancing effect was determined for each fraction or pool of fractions, demonstrated in 5% sucrose.

results

The results are shown in the table below. The entire extract was collected in 22 fractions. Fractions 1-10 do not contain mogroside V.

Fractions 1-10 and 19-22 have an off-flavor that is characteristic of Luo Han Guo fruits, there is no manifestation of increased sweetness.

The best effect of enhancing sweetness was observed in fractions 12-17, which contain mainly mogroside. When the fractions are nearly pure mogroside V, a long, unpleasant fermentation note is more noticeable, for example in fractions 14 and 15. Thus, pure mogroside V has its inherent long-lasting off-flavor.

Fraction 12 was the purest sweet, but to a lesser extent initially due to the small percentage of mogroside V. Fraction 13 has better sweetness quality, but slightly higher astringent flavor. 11-O-mogroside V and mogroside V are the two main mogroside in these two fractions. , but in a different ratio (in the F12 fraction mogroside V: 11-O-mogroside V is 4: 9, and in the F13 fraction mogroside V: 11-O-mogroside V is 13: 3).

Mogroside V was very sweet, 425 times sweeter than sucrose, while 11-O-mogroside V was rated 84 times sweeter than sucrose.

Example 3

The ways

Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est, California) (extract 1 from Example 1 above) containing about 68% of the mass. mogrosides, were fractionated, and the composition of each fraction was determined by the chromatographic method described above in Example 1.

Each fraction was combined with 5% sucrose solution, and three panelists (qualified tasters) compared the taste of these samples with 5% sucrose controls.

results

Table 3 shows the chemical composition of fractions 11 -20 of the extract.

Table 4 presents the results of tasting fractions 12-15. The results are similar to those obtained in Example 2.

Example 4

Mogroside V and 11-O-mogroside V were isolated from extracts from Luo Han Guo using an Agilent 1100 preparative HPLC system with a Phenomenex Luna CI8 (2) column (5 μm, 210 × 21.4 mm) and combined to form solutions having different concentration.

These solutions were combined with a solution containing 5% (w / v) sucrose and 0.03% (w / v) citric acid to form test samples, and a panel of five experts (qualified tasters) assessed the sweet taste.

The results are shown in Table 5 below.

Surprisingly, it has been found that mixing 11-O-mogroside V with mogroside V improves the sweetness quality over mogroside V alone. 11-O-mogroside V added to mogroside V helped to reduce long-term sweetness (reduce late sweetness) and reduce astringent aftertaste and bitter aftertaste compared to mogroside V alone. Thus, 11-O-mogroside V makes sweetness more similar to that of sugar than mogroside V alone (i.e. it helps provide a temporal profile that is closer to sugar) ... This makes it possible to use higher concentrations of mogroside V to obtain higher sweetness and at the same time overcome the disadvantages associated with the use of higher concentrations of this sweetener (eg, long-lasting sweetness, bitter aftertaste and astringent aftertaste). This was surprising given that mogroside V is the most effective mogroside sweetener and 11-O-mogroside V is much less effective.

Example 5

The Sweet Taste Panel evaluated the sweetness of the sweetener blend solutions ("Blend 1") versus sucrose solutions over a range of concentrations to determine the sweetness equivalent to sucrose. Mixture 1 was a combination of fractions 11-18 from Example 2 and contained 8.16% of the mass. 11-O-mogroside V and 61.6% of the mass. mogroside V.

The results are shown in Table 6 below.

The data demonstrates that Blend 1 has a sweetness lower than the sweetness equivalent to 1% sucrose (as determined by seven panelists), which FEMA considers "not actually sweet". Therefore, Blend 1 is suitable for use as a sweetness modifier or blends at the indicated concentrations because it has no detectable sweetness at these levels.

Mixture 1 at a concentration having an isosweet threshold close to 1% (35 ppm) was selected and added to 5% (w / v) sucrose solutions. This solution was then evaluated against 5, 6, 6.5 and 7% (w / v) sucrose solutions. The same was repeated for 45 ppm. extract from Luo Han Guo. The average score was determined for each solution. The results are shown in Table 7 below.

Surprisingly, it was found that Blend 1 and the Luo Han Guo extract act as sweetness enhancers because the increase in sweetness of the 5% (w / v) sucrose solution to which they were added was greater than the sweetness of the sweetener alone.

The flavor of Blend 1 at various concentrations was tested by a panel of three experts (trained tasters) in solutions containing 5% sucrose and 0.03% citric acid. The flavor was compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC (Rolling Hills Est, California) (extract 1 from Example 1 above) and containing about 68 wt% mogrosides) (combined with the same solution containing 5% sucrose and 0.03% citric acid). The results are presented in Table 8.

Overall, Blend 1 provides better quality sweet taste (less baggage, sweeter) than Luo Han Guo extract.

Example 6

Members of the sweet taste tasting panel evaluated the sweetness of solutions of various sweeteners (mogroside V, mogroside IV, siamenoside, neomogroside, 11-O-mogroside V) relative to sucrose solutions in a concentration range to determine the sweetness equivalent to sucrose. Sweeteners were prepared using an Agilent 1100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 μm, 210 x 21.4 mm). The results are shown in Tables 9-13.

The data demonstrate that mogroside V (25 ppm), mogroside IV (30 ppm), siamenoside (25 ppm), mogroside V (25 ppm) in combination with 11-O-mogroside V (3 ppm) and neomogroside (30 ppm) all have a sweetness below the sweetness equivalent to 1.5% sucrose (as determined by seven panelists), which FEMA considers "not actually sweet". Therefore, these compounds and mixtures are suitable for use as sweetness modifiers at the indicated concentrations since they have no detectable sweetness at these levels.

The tested sweeteners were selected at concentrations with an isosweet threshold close to 1% and added to 5% (w / v) sucrose solutions. These solutions were then evaluated against 5, 6, 6.5 and 7% (w / v) sucrose solutions. The average score was determined for each solution. The results are shown in Table 14 below.

Surprisingly, it was found that mogroside V, siamenoside, neomogroside and mogroside V act as sweetness enhancers because the increase in sweetness of the 5% (w / v) sucrose solution to which they were added was greater than the sweetness of the sweetener alone.

The taste of these sweeteners was tested by a panel of three experts (trained tasters) in solutions containing 5% sucrose and 0.03% citric acid. The flavor was compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC (Rolling Hills Est, California) (extract 1 from Example 1 above) and containing about 68 wt% mogrosides). Mogroside IV, siamenoside and neomogroside all proved to be better than the Luo Han Guo extract in terms of sweetness quality when added to a solution containing 5% sucrose and 0.03% citric acid. These 3 compounds provide a sugar-like taste with a less long-lasting sweet taste. Siamenoside has been described as having "a sweeter body, sweeter, rounder with a slightly larger initial and rounder lasting sweet taste." Mogroside IV has been described as having "good sweetness and similar sweetness to mogroside V". Neomogroside has been described as having a "sweet but slightly bitter aftertaste." The results for mogroside V are presented in Table 15.

In general, panelists agreed that 80% Mogroside V did not have the same full rounded sweet profile as the Luo Han Guo extract. 80% mogroside V is more acidic in taste when applied to a sugar / acid / water solution.

Example 7

The ways

Mogroside V, siamenoside, mogroside IV, and neomogroside were prepared using an Agilent 1100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 μm, 210 × 21.4 mm).

Mogroside V, siamenoside, mogroside IV and neomogroside were added to a solution containing 5% sucrose and 0.03% citric acid at a concentration of 25 ppm. (mogroside V), 25 ppm (siamenoside), 30 ppm (mogroside IV) and 30 ppm. (neomogrozid) respectively.

These test solutions were tasted by a panel of seven experts. For various aspects of sweet taste (initially sweet, sweet from start to finish, long-lasting sweet, astringent, volatile off-flavor), each panel member evaluated the test solutions versus the stock solution (solution containing 5% sucrose and 0.03% lemon acid).

A score of 0 means the palatability was the same, 1 means slightly higher, 2 means higher, 3 means much higher, -1 means slightly lower, -2 means lower, and -3 means much lower. The average score for each test solution was calculated for each taste aspect. The results are shown in Table 16 below.

Example 8

As shown above, siamenoside, neomogroside and mogroside IV all had similar or better sweet taste quality in addition to a solution containing 5% sucrose and 0.03% citric acid compared to mogroside V. Therefore, the taste of 11-O-mogroside V with each of these mogrosides was scored as shown in Table 17.

Example 9

The taste of mogroside V with 11-O-mogroside V and without 11-O-mogroside in various milk or yoghurt bases was evaluated as shown in Table 18. The sweet threshold for mogroside V in milk and yogurt was also evaluated.

The milk base (2% fat) contains 2% fat milk and 5% sucrose. Low-fat yogurt base contains low-fat yogurt without additives and 5% sucrose. Unfatted yogurt base contains unfatted unflavored yogurt and 5% sucrose. Higher dosage levels are used for dairy and yoghurt compositions due to fat, protein and other ingredients. The Luo Han Guo extract was used at 75 ppm. for these applications.

Example 10

Identification of New Minor Cucurbitan Glycosides from Siraitia grosvenorii

Introduction

Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang is a perennial herbaceous vine of the Cucurbitaceae family (cucurbitaceae) endemic to southern China and northern Thailand. The fruit of S. grosvenorii, commonly known as 'luo han guo', has been used in traditional medicine in China for centuries to treat respiratory infections, bronchitis, gastritis, constipation, etc. Modern pharmacological studies have confirmed that extracts from S. grosvenorii or their components have various types of bioactivity, such as antibacterial, anti-inflammatory, antidiabetic, anticancer and immunostimulating [1]. In China, Luo Han Guo is used as a sweetener in foods and beverages. It is now one of the best known natural high-intensity sweeteners in the world. Since the cucurbitan glycoside mogroside V was discovered as the sweet component of S. grosvenorii, more than 40 cucurbitan triterpenoids from S. grosvenorii have been reported to date [1-4]. Researchers in the food and flavoring industries are actively discovering and adding newer compounds to the mogroside pool to find new mogrosides with better sweet characteristics [5-7]. New molecules under well-known categories of natural sweeteners with better sweet characteristics are in demand in the food and flavoring industries. The commercialization of rebaubioside M (also known as rebaubioside X) is a good example. Although it is a minor natural product from stevia (less than 0.1%), discovered in 2010, rebaubioside M rapidly progressed to the stage of commercialization due to cost reduction due to the development of technologies in plant biology, molecular biology and enzymatic engineering [8, 9] ... Rebaudioside M received a non-objection letter regarding its Generally Recognized Safe (GRAS) status from the US FDA in 2013, 2014 and 2017 (GRN No. 473, 512 and 667) [10-12 ].

The inventors carried out research to find the mogrosides with the best characteristics or their combinations using a commercial extract from Luo Han Guo [13]. Herein, the inventors report two new minor cucurbitan glycosides from S. grosvenorii and highlight their new oligosaccharide identification strategy based on HSQC-TOCSY experiments with different mixing times.

Materials and methods

General experimental techniques

Optical rotations were measured on a Rudolph Autopol IV polarimeter. NMR spectra were recorded on a Bruker DRX Avance 300 or 500 spectrometers. Chemical shifts are reported in δ (ppm) relative to the residual solvent peak. Low pressure chromatography was performed on a Biotage Flash System SP1. Preparative HPLC was performed on an Agilent 1100 preparative HPLC system with a Phenomenex Lunar C18 (2) column (5 μm, 210 × 21.4 mm) or TSKgel Amide-80 (5 μm, 300 × 21.5 mm) (Tosoh Bioscience LLC). Analytical HPLC was performed on an Agilent 1100 analytical HPLC system equipped with an ESA Corona CAD (charged aerosol detector) detector. LC / MS was performed using a Waters Q-Tof micro-mass spectrometer coupled to a Waters 2795 separation module.

Plant material

Luo Han Guo extract (trade name Swingle, ~ 60% mogrosides) was purchased from Blue California Co., Ltd. Equipment

Chromatographic Conditions: Chromatography was performed on a Waters Acquity H UPLC. Separation was carried out at 25 ° C. using a 1.0 x 100 mm Acquity UPLC HSS T3 column (Waters) with a particle size of 1.8 mm, equipped with a 0.2 mm pre-column filter. Solvent A was water and solvent B was acetonitrile; both solvents contained 0.1% formic acid. The injected volume was 10 μl. The chromatographic flow rate was 200 μl / min. The sample was eluted from the LC column using the following linear gradient (curve number 6): 0-40 min: 90% A-30% A; 40-45 min: 30-10% A; 45-50 min: 10% A; 50-51 min 10% -90% A; 51-55 min 90% A for rebalancing.

Mass spectrometry

The U-HPLC (ultra high performance liquid chromatography) system was coupled to a hybrid quadrupole orthogonal time-of-flight (TOF) mass spectrometer (SYNAPT G2 HDMS, Waters MS Technologies, Manchester, UK). The mass spectrometer operated in the electrospray ionization mode with the registration of positive ions (ERI). Cone voltage 40, capillary voltage 0.7 kV, source temperature 40 ° C, desolvation temperature 450 ° C, solvation gas flow rate 800 l / h, and cone gas flow rate 50 l / h were optimized. Leucine-enkephalin was used as a fixed mass [M + H] ^- at m / z 556.2771. The sodium formate solution was used for external calibration of the instrument.

Cleaning

3 g of the Luo Han Guo extract was dissolved in 15 ml of water and loaded onto a pre-equilibrated C-18 Snap cartridge (KP-C18-HS, 120 g, column volume 132 ml). The gradient system (A: water; B: methanol) was as follows: 30% 2 CV (column volume), 30% -80% 10 CV, 80% -100% 2 CV, 100% 2 CV. The flow rate was 30 ml / min. Fractions were collected at 27 ml per tube. Four applications were fractionated for a total of 12 g of Luo Han Guo. All fractions were analyzed by analytical HPLC to localize fractions with the target mogroside (isocratic mobile phase: 24% acetonitrile in water; column: Luna C18 5 μm 4.6 x 150 mm); fractions 36-38 with isomogroside VI and 11-epimogroside V were combined to evaporate the solvents. After additional purification by preparative HPLC of fractions 36-38, isomogroside VI (1.22 mg) and 11-epimogroside V (2.17 mg) (24% acetonitrile in water, 10 ml / min, retention time 13.1 min and 14 , 3 min, respectively). 11-Oxo-mogroside V (4) and neomogroside (3) were found mainly in flash fractions 39-40 with 11-oxo-mogroside V as the main component. On reverse phase C-18 preparative HPLC, neomogroside appeared as a terminal arm of 11-oxo-mogroside (24% acetonitrile in water, 10 ml / min, retention times 17.0 min and 18.0 min, respectively). After collecting the front of the peak, 105 mg of compound 11-oxo-mogroside V was obtained (4). Additional purification of the shoulder neomogroside (3, 15 mg) was carried out by preparative HPLC on TSKgel Amide-80 (65% acetonitrile in water, 20 ml / min, retention time 15.5 min).

Isomogroside VI (1): White amorphous powder; [α] ²⁰ _D -8.2 (c 0.12, MeOH); for ¹ H NMR and ¹³ C spectroscopy data, see Tables 1; -ERI-HRMS (high resolution mass spectrometry with electrospray ionization): m / z 1449.7075 [M + H] ^- (calculated for C ₆₆ H ₁₁₃ O ₃₄ 1447.7113, Δ2.6 ppm).

Epimogroside V (2): White amorphous powder; [α] ²⁰ _D +4.5 (c 0.13, MeOH); for ¹ H NMR and ¹³ C spectroscopy data, see Tables 1; ERI-HRMS: m / z 1287.6558 [M + H] ^- (calculated for C ₆₀ H ₁₀₃ O ₂₉ 1287.6585, Δ2.1 ppm).

Acid hydrolysis and determination of the absolute configuration of sugars

Compound 1 (1.2 mg) or 2 (1.8 mg) was incubated in 1 ml of 1M HCl at 80 ° C for 3 h. After hydrolysis, the solution was extracted with EtOAc (1 ml × 3). The remaining aqueous solutions were evaporated by blowing nitrogen gas and freeze-dried. The absolute configuration of the sugar in the residue was determined by GC / MS analysis of its O-silylated derivative and comparison with the derivatives of D-glucose and L-glucose as standards. Briefly, sugar residues, D-glucose (2 mg) or L-glucose (2 mg), were dissolved in pyridine (0.5 ml). To this solution was added 0.1 M L-cysteine methyl ester hydrochloride (Aldrich, Milwaukee, WI) in pyridine (0.5 ml). The mixture was kept at 60 ° C for 2 hours and dried by blowing nitrogen gas. To the residue was added 1-trimethylsilylimidazole (Fluka, Buchs, Switzerland) (0.5 ml) and incubated at 60 ° C for 1 hour. The mixture was separated by adding n-hexane and water (1.0 ml each). The n-Hexane extract was analyzed by GC / MS under the following conditions: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent); column temperature 180-230 ° С with a step of 5 ° С / min; injection temperature 250 ° С; carrier, He gas; split ratio 20: 1. O-silylated D-glucose and L-glucose derivatives showed retention times of 16.02 and 16.39 minutes, respectively. By comparing retention times and co-chromatography, acid hydrolysis sugar residues 1 and 2 were identified as D-glucose.

Reduction of 11-oxo-mogroside V using NaBH ₄

25 mg of 11-oxo-mogroside V (4) was dissolved in 50% dioxane, 20 mg of NaBH _{4 was} added, and the mixture was heated at 50 ° C for 3 days. The reaction mixture was periodically analyzed by HPLC to monitor the progress of the reaction. After the reaction, the mixture was acidified with acetic acid and concentrated to dryness by passing nitrogen gas. The residue was redissolved in water and passed through a pre-equilibrated C-18 SPE column. The methanol eluents from the SPE column were concentrated. The residue was then separated by semi-preparative HPLC. The two recovered products had the same retention time and the same molecular weight as the isolated mogroside V and 11-epimogroside V as determined by LC / MS and analytical HPLC co-chromatography. 1-D and 2-D NMR data also confirmed that the structures of these two reduced products were those of mogroside V and 11-epimogroside V.

Results and discussion

Isolation and identification of isomogroside VI (1) and 11-epimogroside V (2)

In an LC / MS study of commercial extracts from Luo Han Guo with 60% mogrosides, several mogrosides with six or five sugar groups in the extracts attracted the attention of the inventors (Figures 2 and 3). Since there were few reports on the sweet properties of the isomers of mogroside V and mogroside VI, the inventors decided to purify and identify these isomers for their evaluation. The concentrations of mogrosides 1, 2, 3 and 4 in the extracts were estimated to be 0.8%, 0.5%, 0.6%, 4.9%, respectively, according to the Corona universal detector. After fractionation on a flash chromatography system and subsequent purification by preparative HPLC, four target mogroside 1-4 were purified and determined to be isomogroside VI (1), 11-epimogroside V (2), neomogroside (3), 11 -oxo-mogroside V (4).

The molecular formula of mogroside 1 was determined as C ₆₆ H ₁₁₂ O ₃₄ from its ERI-HRMS spectral data ([M-H] ^- , m / z 1447.6957; calculated for C ₆₆ H ₁₁₁ O ₃₄ 1447.6957). The NMR spectral data of mogroside 1 confirmed the structure of the saponin triterpenoid hexasaccharide: 30 out of 66 carbon atoms belonged to the triterpenoid aglycone, and 36 out of 66 belonged to six hexose groups.

The ¹³ C and ¹ H NMR spectra of mogroside 1 showed signals from seven singlets of tertiary methyls, a doublet of secondary methyl and olefinic methine (Table 19), which confirmed the typical aglycone mogrol (24R) -cucurbit-5-ene-3β, 11α, 24,25- tetraola. Mogrol aglycone of mogroside 1 was additionally confirmed by comprehensive analysis of its ¹ H, ¹³ C and 2D (COZY, TOCSY, HSQC and NOESY (Nuclear Overhauser Effect Spectroscopy)) NMR data, as well as comparison with NMR data of mogroside V as a standard.

GC / MS analysis of water-soluble products of acid hydrolysis 1 showed that D-glucose was the only monosaccharide in structure 1. The HSQC spectra clearly displayed anomeric cross-peaks of six glucosides: Glc-I (δ _С 106.8 and δ _H 4.73), Glc-II (δ _С 105.1 and δ _H 5.10), Glc-III (δ _С 103.7 and δ _H 4.85), Glc-IV (δ _С 104.6 and δ _H 4.78), Glc-V (δ _С 104.7 and δ _H 5.43), Glc -VI (δ _C 104.8 and δ _H 5.03). From the constants of interaction of their anomeric protons ³ J _{H1, H2,} it was determined that the stereochemistry of all six glucopyranosyls is β-configuration. From the HSQC-TOCSY experiment (hsqcgpmlph) with a mixing time of 100 ms, the glucopyranosyl carbon signals can be divided into six groups (FIG. 4). The identification of the oligosaccharide began with glucopyranosyl linked at C-3 of cucurbitan aglycone. It was determined that Glc-I is bound to C-3 aglycone according to HMBC (spectroscopy based on heteronuclear multiquantum coherence) correlation of its anomeric proton (δ _H 4.73, d, J = 7.9 Hz) with C-3 aglycone (δ _C 87.6) and NOESY correlations of H-1 Glc-I and H-3 aglycone. ^{The 13} C-signals Glc-I (δ _C 75.1, 77.8, 71.4, 77.2, 70.1), as determined by the HSQC-TOCSY method, lacked a typical carbon C-6 signal in the region of δ _C 62. The shift of the C-6 Glc-I signal ( δ _С 70.1) towards the weak field indicates glycosylation in this position. Comparing the HSQC-TOCSY spectra (hsqcgpmlph) with increasing mixing times of 10, 30, 60 and 100 ms, magnetization transfer can be observed gradually spreading from C-2 to C-6 (Fig. 4). As shown in FIG. 4, HSQC-TOCSY at 10 ms mixing time displayed the H-1 and C-2 correlation of glucopyranosyl. At a mixing time of 30 ms, in addition to the correlation of H-1 and C-2, a correlation of H-1 and C-3 appeared. At 60 ms, the carbon chain, as shown by the HSQC-TOCSY correlation, extends to C-4. Full HSQC-TOCSY correlation of H-1 with C-2-C-6 can be observed at 100 ms. Therefore, the C-2-C-6 signals can be assigned unambiguously. The relationship between Glc-II and Glc-I was established as a result of the HMBC correlation of anomeric H-1 Glc-II (δ _H 5.10, d, J = 7.8 Hz) with Glc-I С-6 (δ _С 70.1). ¹³ С-signals of Glc-II (δ _С 75.0, 77.8, 71.5, 78.0, 62.5) confirmed the absence of glycosylation on Glc-II. As a result, the sugar chain on the C-3 of the aglycone was represented as 3-O- (β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosyl.

HMBC correlation of the anomeric proton (δ _H 4.85, d, J = 7.5 Hz) with the carbon signal of the aglycone (δ _С 92.3) showed the connection of Н-1 Glc-III with С-24 aglycone. ^{The 13} C pattern Glc-III (δ _C 81.6, 78.3, 71.4, 76.4, 70.0) confirmed the shifts in C-2 and C-6 glycosylation. HSQC-TOCSY analysis with mixing times of 10, 30, 60 and 100 ms resulted in the assignment and confirmation of the C-2 and C-6 weak field shifts. Glc-IV was identified as associated with С-6 Clc-III, as follows from the NMSC correlation of its Н-1 (δ _H 4.78, d, J = 7.5 Hz) with С-6 Glc-III (δ _С 70.0). Glc-IV was a regular terminal glucopyranosyl without any substitution (δ _C 74.5, 77.7, 71.2, 78.2, 62.4). The connection between Glc-V and С-2 Glc-III was established by means of the HMBC correlation of anomeric Н-1 Glc-V (δ _H 5.43, d, J = 7.8 Hz) with С-2 Glc-III (δ _С 81.6). The relative shift towards the weak field H-1 Glc-V (δ _H 5.43) is consistent with previous reports with a similar structure. ¹³ С chemical shift С-4 usually at δ _С 70-71 was absent in the set of Glc-V signals (δ _С 104.7, 75.4, 76.4, 82.0, 76.5, 62.6), which confirms glycosylation at С-4. Based on the observation of the transfer from C-2 to C-6 in the HSQC-TOCSY experiment with mixing times of 10, 30, 60 and 100 ms, δ _C 82.0 was clearly assigned to C-4 Glc-V (Fig. 4). HMBC cross-peak between С-4 Glc-V (δ _С 82.0) and Н-1 Glc-VI (δ _H 5.03, d, J = 7.7 Hz) additionally confirmed that Glc-VI is bound to Glc-V at this position ... Glc-VI was a terminal glucopyranosyl without an additional sugar branch. Based on the above evidence, the structure of isomogroside (1) was determined as 3-O-β-B-glucopyranosyl (1 → 6) -β-D-glucopyranosyl-mogrol-24-O-β-D-glucopyranosyl- (1 → 6- [β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranosyl- (1 → 2)] - β-D-glucopyranosyl.

Compound 2 was assigned the molecular formula C ₆₀ H ₁₀₂ O ₂₉ based on its ERI-HRMS data ([M-H] ^- m / z 1285.6429). The NMR data of oligosaccharide part 2 was comparable to that of mogroside V. Detailed 2-D NMR experiments including HSQC, HMBC, NOESY, COZY, and HSQC-TOCSY confirmed that 2 has the same sugar moieties as mogroside V. Then attention was turned on NMR data of aglycone. HMWS correlation between С-11 and Н ₃ -19 revealed a significant shift towards the strong field С-11 (δ _C 72.5) compared to mogroside V (δ _C 77.8). Additional assignment of aglycone data by 2-D NMR experiments showed that the main changes in the ¹³ C chemical shift occurred at C-8, C-10 and C-12 when compared with mogroside V data (Table 19). This suggested β-OH instead of α-OH on C-11. β-OH stereo structure 2 was additionally established by NOE (nuclear Overhauser effect) correlation between H-8 and H ₃ -18, 19; H-10 and H ₃ -28, H ₃ -30; H-11 and H ₃ -30; H-17 and H ₃ -30. Previously, there were reports of one natural 11-β-OH cucurbitan and one semi-synthetic 11-β-OH cucurbitan.

[14, 15]. ^{The 13} C NMR data of the aglycone of compound 2 were in good agreement with the data of the semisynthetic 11-β-OH cucurbitan glycoside, which were recorded in pyridine-d ₆ [14]. ^{The 13} C NMR data of natural 11-β-OH cucurbitan published by Matsuda et al were obtained in methanol-d ₄ and were completely different in chemical shifts at C-11, C-8, C-10 and C-12 [15]. To further confirm the 11-β-OH structure of compound 2, the semisynthesis of compound 2 was carried out by chemical reduction of 11-oxo-mogroside V (4) to 11-β-OH and 11-α-OH isomers of mogroside V. According to LC / MS analyzes , HPLC co-chromatography and NMR determined that the semisynthetic 11-epimogroside V is identical to the isolated 11-epimogroside V. Therefore, the structure of 11-epimogroside V (2) was determined as 3-O-β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosyl-11β-OH-mogrol-24-O-β-D-glucopyranosyl- (1 → 2) - [β-D-glucopyranosyl- (1 → 6)] - β-D-glucopyranosyl. To the best of the present inventors' knowledge, this is the first report of a naturally occurring mogroside with an 11-β hydroxyl group.

HSQC-TOCSY with different mixing times for identification of the oligosaccharide chain

Gheysen et al conducted TOCSY experiments with different mixing times and concluded that 100 ms is the optimal spin-blocking time to discriminate between D-glucose, D-galactose and D-mannose [16]. Due to these results, the inventors of the present invention noticed that the spin blocking time can significantly affect the magnetization transfer efficiency of D-glucose H-1. The correlation between H-1 and H-2 through H-6 gradually extended to H-6 as the spin blocking time increased. Based on their research, the present inventors hypothesized that by increasing the HSQC-TOCSY spin blocking time, they would be able to see that the correlations of H-1 glucose with C-2-C-6 gradually spread from C-2 to C-6. as the magnetization transfer chain propagates. HSQC-TOCSY with increasing spin blocking time should provide carbon sequence information that can be very useful for oligosaccharide identification and assignment. FIG. 4 shows the HSQC-TOCSY (hsqcgpmlph) of isomogroside VI with mixing times of 10, 30, 60 and 100 ms. The cross peaks in FIG. 4 were quantified from their integrals and their comparison is shown in FIG. 5. The intensity of the peaks (presented as integrals) can serve as an indicator of their distance from H-1 in some cases. For example, all C-3 peaks were significantly weaker than the C-2 peaks in experiments with a mixing time of 30 ms; however, at 60 ms, the C-3 peaks become larger than the C-2 peaks. To ensure accurate identification, the carbon sequence should be determined by looking at all HSQC-TOCSY spectra with different mixing times, but only by peak intensities at the same mixing time.

Traditionally, NMR identification and assignment of the sugar chain of saponins begins with the sugar bound to the aglycone. Anomeric H-1 and C-1 signals with good resolution can be identified by HMBC or NOESY. Then, using COZY correlations and checking the ³ J (H, H) coupling constants, the proton signals of the monosaccharide can be assigned. Since a large interaction constant (> 7 Hz) usually indicates two adjacent axial CH bonds, and a small interaction constant (<4 Hz) indicates an axial-equatorial or equatorial-equatorial CH bond, it is possible to determine the type of monosaccharide. NOE correlations are useful for confirming the stereochemistry of axial-axial, axial-equatorial, or equatorial-equatorial relationships. The carbon signals of the saccharide (C-2-C-6) are assigned according to HSQC or HMQC. The chemical shifts of the signals of carbon atoms, which are determined by HSQC / HMQC, are very important information for confirming the type of monosaccharide, since the pattern of chemical shifts C-1-C-6 for another type of monosaccharide is characteristic and consistent. By detecting changes in chemical shifts of carbon atoms, the position of glycosylation on the sugar chain can be identified and further confirmed by HMBC correlation. In summary, the traditional pathway for identifying saponin sugar is: HMBC → C-1, H-1 → COZY → H-2 to H-6 → HSQC / HMQC → C-2 to C-6, then promoted and validated by analysis of interaction constants and NOESY experiment.

Experiment ¹ H- ¹ H TOCSY (also known as NONANA (Hartmann-Hahn homonuclear spectroscopy) could be of great help in separating sugar proton signals into groups Magnetization transfer during TOCSY spin blocking from anomeric H-1 to the end of the furanose or pyranose ring will depend on the magnitude of the interference ³ J (H, H) of the scalar coupling constants. Adjacent axial-axial protons with a large coupling constant (> 7 Hz) provide a fast transfer of magnetization, while axial-equatorial or equatorial-equatorial protons with a small coupling constant (< 4 Hz) will significantly reduce the efficiency of transfer.Therefore, the TOCSY experiment can not only be used to group proton signals into spin systems, but also provide information about the stereochemistry of saccharide.For example, we should be able to see the transfer of glucose magnetization by H-1-H -6 with correct mixing time There are no gears for galactose and H-4 magnetization even with a mixing time of 200 ms.

However, in the case of mogrosides with five or six sets of glucopyranosyl signals, the use of COZY and TOCSY to bind H-1 to H-6 can be quite difficult. The proton signals of the glucopyranosyls of mogrosides have very similar chemical shifts and appear clustered in a small range from δ _H 3.8 to 4.5. It is difficult to obtain clear COZY bonds through such poorly resolved proton signals. The signals of the glucopyranosyl carbons are also very close, and the HSQC cross peaks overlap strongly with each other, making identification and assignment even more difficult.

Previously, HSQC-TOCSY was used to identify and assign the structure of saponins by grouping carbon signals together in a spin system [17, 18]. In their study, the present inventors demonstrated for the first time that the signal sequence in the range of the glucopyranosyl carbon group can be identified by applying different mixing times in HSQC-TOCSY experiments.

FIG. 6 summarizes a novel HSQC-TOCSY based strategy for identifying the structure of the glucopyranosyl oligosaccharide chain of mogrosides as follows: In Stage 1, heteronuclear multilink correlation spectroscopy (HMBC) was used to determine the anomeric C-1 and H-1 sugars. They start with the connection between sugar and aglycone. Stage 2 used HSQC-TOCSY with a mixing time of 100 ms to determine the entire group from C-2 to C-6. HSQC-COZY or HSQC-TOCSY (d9 = 10 ms) was used for C-2 assignment. HSQC-TOCSY (d9 = 30 ms) was used for C-3 assignment. HSQC-TOCSY (d9 = 60 ms) was used to assign C-4. HSQC-TOCSY (d9 = 100 ms) was used to assign C-5 and C-6. At Stage 3, if there is a shift in the weak field С-2 from ~ δ75 to ~ δ81, a shift in the weak field С-4 from ~ δ71 to ~ δ81, or a shift in the weak field С-6 from ~ δ62 to ~ 69 , is tested by HMBC for glycosylation at these positions. If there is a shift in the weak field C-2 from -δ75 to ~ δ81, a shift in the weak field C-4 from ~ δ71 to ~ δ81, or a shift in the weak field C-6 from ~ δ62 to ~ 69, the NMSC is checked in to glycosylation at these positions. * 1-D NMR data such as ¹ H coupling constants and C carbon signaling pattern and 2-D NMR experiments such as NOESY, HMBC, TOCSY, COZY and HSQC can facilitate the process and confirm the results ... A new strategy based on HSQC-TOCSY can provide a simple, fast and unambiguous method for identifying and assigning glucopyranosyl chains of any new or known mogrosides. This strategy can also be adapted to identify and assign other monosaccharides and oligosaccharides.

Structures of neomogroside and mogroside VI

Compound 3 was identified as a neomogroside as a result of large-scale 1-D and 2-D NMR analysis, as well as comparison with literature data [19]. To identify the oligosaccharide chain of compound 3, the signals were assigned by performing the HSQC-TOCSY and TOCSY experiments with different mixing times of 10, 30, 60, 100 ms. The connection of six saccharides was obtained from their NOESY and HMBC correlations. The oligosaccharide chain at C-3 of the aglycone can be clearly defined as β-D-glucopyranosyl- (1 → 2) -β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl. Glucopyranosil at the C-24 aglycone was branched β-D-glucopyranosyl- (1 → 2) ohm and β-D-glucopyranosyl- (1 → 6) ohm.

Neomogroside was first isolated from S. grosvenorii and described by Si et al. [nineteen]. A search for neomogrozid in SciFinder databases returned CAS number 189307-15-1. However, SciFinder gave an imprecise structure for the neomogroside, although the literature referenced by the SciFinder database was from Si et al. 1996 year. The incorrect structure of 189307-15-1 was reported in SciFinder as 3-O-β-D-glucopyranosyl- (1 → 2) - [β-D-glucopyranosyl- (1 → 6)] - β-D-glucopyranosyl-mogrol- 24-O-β-D-glucopyranosyl- (1 → 6) - [β-D-glucopyranosyl- (1 → 2)] - β-D-glucopyranosyl (structure of compound 6 in Fig. 2). Si et al's article on neomogroside was written in China and published in the Chinese Journal in 1996. Comprehension and misunderstanding of this article could lead to incorrect structure in SciFinder.

In SciFinder, neomogroside and mogroside VI had the same CAS number 189307-15-1 and the same structure. Takemoto et al were the first to report on mogroside VI from S. grosvenorii [2]. But only pure mogroside with the molecular formula C ₆₆ H ₁₁₂ O ₃₄ was mentioned, and the structure was not determined [2]. Prakash et al. described the structure and NMR data of mogroside VI as a known compound in their article published in 2011 [6]. In their paper, the structure of mogroside VI was defined as the structure of compound 6 in FIG. 2. In the Prakash publication it is mentioned that the identification of the structure of mogroside VI was carried out according to the data of NMR analysis, as well as comparison with the literature values. However, no references have been given for these literary meanings.

For known compounds, comparison of NMR data with literature data can be useful in determining the structure. However, the complexity of the NMR data of mogrosides makes it difficult to determine the structure mainly by comparing the NMR data with the literature data. The ¹ H NMR data of known mogrosides in different reports differ due to the use of different solvents for NMR (the ratio of pyridine to D ₂ O can cause signal shifts) or simply incorrect assignments.

Even if the ¹³ C NMR data are in good agreement and have better resolution than the ¹ H NMR data, when determining the structure of the oligosaccharide chain, one cannot

rely on direct comparison of ¹³ C NMR data with literature data. Considering the case of neomogroside, if Glu-VI glucopyranosyl- (1 → 2) is branched to Glu-I, Glu-II, Glu-III, Glu-IV, or Glu-V, the five isomers may have very similar ¹³ C NMR data. Rather than comparing ¹³ C NMR data with literature data, a thorough 2-D NMR analysis must be performed carefully before the oligosaccharide chain of the mogroside can be determined unambiguously.

Example 11

Intensity of sweet taste of isomogroside VI and 11-epimogroside V

The ways

10 mg of isomogroside VI (Fig. 7) was dissolved in 31 ml of water to obtain a solution of 100 ppm. isomogroside VI. The working solution of 11-epimogroside V (Fig. 8) had a concentration of 374 ppm. (9.34 mg 11-epimogroside V in 25 ml of water). A series of standard sucrose solutions (0.50; 0.75; 1.00; 1.25; 1.50%) were prepared as sweetness standards.

results

Four members of the tasting panel rated 100 ppm. isomogroside VI and 374 ppm 11-epimogroside V and sucrose standards, and were asked to indicate concentrations giving a sweetness equivalent to that of sucrose. Average sweet taste equivalence concentrations of each compound were used to calculate sweet taste strength. The strengths of the isomogroside VI and 11-epimogroside V were determined to be 91 and 35 times the sweetness of sucrose, respectively (sweet taste 100 ppm isomogroside VI, equivalent to 0.91% sucrose; sweet taste 374 ppm 11- epimogroside V, equivalent to 1.31% sucrose).

Example 12

The ways

Isomogroside VI and 11-epimogroside V were prepared as described in Example 10. Isomogroside VI and 11-epimogroside V were each added to a solution containing 5% sucrose and 0.03% citric acid at a concentration of 25 ppm. These test solutions were tested by a panel of seven experts. To determine the various aspects of the sweet taste (sweet from start to finish, initially sweet, long-lasting sweet, astringent, volatile foreign note), each panel member scored the test solutions versus the stock solution (solution containing 5% sucrose and 0.03 % citric acid). A score of 0 meant the palatability was the same, 1 means slightly higher, 2 means higher, 3 means much higher, -1 means slightly lower, -2 means lower, and -3 means much lower.

results

The average score for each test solution was calculated for each taste aspect. The results are shown in Table 20 below.

Two new minor cucurbitan glycosides, in parallel with the known 11-oxo-mogroside and neomogroside, were purified from industrial extracts from Luo Han Guo (Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang). As a result of comprehensive NMR and LC / MS analyzes and chemical synthesis, the structures of two new compounds, isomogroside VI (1) and 11-epimogroside V (2), were identified as 3-O-β-D-glucopyranosyl (1 → 6) -β -D-glucopyranosyl-mogrol-24-O- (β-D-glucopyranosyl- (1 → 6) - [β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranosyl- (1 → 2)] - β-D-glucopyranosyl and 3-O-β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosyl-11β-OH-mogrol-24-O-β-D-glucopyranosyl- (1 → 6) - [ β-D-glucopyranosyl- (1 → 2)] - β-D-glucopyranosyl, respectively The strengths of the sweet taste of isomogroside VI and 11-epimogroside V were estimated to be 91 and 35 times the sweetness of sucrose, respectively (sweet taste 100 ppm). isomogroside VI, equivalent to 0.91% sucrose; sweet taste 374 ppm of 11-epimogroside V, equivalent to 1.31% sucrose) Thanks to the course used by the inventors to identify new and known mogrosides with five or six glucopyranosyls, a new strategy has been developed identification and assignment of the glucopyranosyl sugar chain. i on HSQC-TOCSY experiments with different mixing times, is a hallmark of the rapid and unambiguous implementation of the identification and assignment of glucopyranosyl oligosaccharide chains. The pre-existing confusion regarding the structures of neomogroside and mogroside VI was reviewed and clarified after confirming the structure of neomogroside through comprehensive NMR spectral analysis.

The foregoing broadly describes some embodiments of the present invention without limitation. Variants and modifications that will be apparent to those skilled in the art are intended to be within the range of the scope of the present invention as defined in the appended claims and by the appended claims.

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Claims (39)

1. A sweetness-modifying composition comprising: one or more high-intensity sweetener (s) selected from steviol glycosides such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside I, N , rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubuzoside and naringenin dihydrochalcone; and / or mogroside such as mogroside III, mogroside IIIx, mogroside IV, siamenoside, neomogroside and mogroside V; and a low-potency sweetener selected as 11-O-mogroside V; wherein said sweetness modifying composition increases the sweetness of the sweetened composition by more than the sweetness of the sweetness modifying composition itself; and / or where the ratio of one or more high intensity sweetener (s) to low potency sweetener is in the range from 2: 1 to 12: 1; and wherein said sweetness modifying composition is for use in a sweetened composition in an amount having a sweetness less than sweetness equivalent to 1.5% (w / v) sucrose, or below the sweetness detection threshold. 2. The sweetness-modifying composition of claim 1, which increases the sweetness of the sweetened composition by an amount equal to or greater than the sweetness equivalent to about 1.25% sucrose. 3. A sweetness-modifying composition according to claim 1 or 2, wherein the ratio of high intensity sweetener (s) to low potency sweetener is in the range of 5: 1 to 12: 1, or 6: 1 to 10: 1. 4. Composition, modifying sweetness, according to any one of paragraphs. 1-3, where one or more high intensity sweetener (s) is selected from mogroside such as mogroside III, mogroside IIIx, mogroside IV, siamenoside, neomogroside, and mogroside V. 5. Composition, modifying sweetness, according to any one of paragraphs. 1-4, wherein one or both of the high intensity sweetener (s) and the low potency sweetener are / are natural (s). 6. Composition, modifying sweetness, according to any one of paragraphs. 1-5, which improves one or more sweetness characteristics of the sweetened composition over the corresponding sweetness characteristics (s) of the sweetened composition in the absence of the low potency sweetener in the sweetness-modifying composition. 7. Composition, modifying sweetness, according to any one of paragraphs. 1-6, which attenuates the long-term sweet taste of the sweetened composition as compared to the long-term sweet taste of the sweetened composition in the absence of the low potency sweetener in the sweetness-modifying composition. 8. Composition, modifying sweetness, according to any one of paragraphs. 1-7, which reduces the bitter and / or astringent taste of the sweetened composition relative to the corresponding bitter and / or astringent taste of the sweetened composition in the absence of the low potency sweetener in the sweetness-modifying composition. 9. Composition, modifying sweetness, according to any one of paragraphs. 1-8, which is intended for use in a sweetened composition in an amount having less sweetness than the equivalent of 1.5% (w / v) sucrose. 10. A sweetened composition comprising: at least one sweetener present in an amount having a sweetness equal to or greater than a sweetness equivalent to 1.5% (w / v) sucrose; and a sweetness modifying composition according to any one of claims. 1-9. 11. The sweetened composition of claim 10, wherein the sweetness-modifying composition is present in the composition in an amount having a sweetness less than the sweetness equivalent to 1.5% (w / v) sucrose. 12. The sweetened composition of claim 10 or 11, wherein one or more high intensity sweetener (s) of the sweetness-modifying composition is present in the sweetened composition in a total amount equal to or greater than about 15 ppm. (ppm) and optionally equal to or less than about 50 ppm. 13. The sweetened composition according to any one of paragraphs. 10-12, wherein one or more high intensity sweetener (s) of the sweetness-modifying composition is present in the sweetened composition in a total amount ranging from 15 ppm. up to 30 ppm 14. The sweetened composition according to any one of paragraphs. 10-13, wherein the low potency sweetener of the sweetness-modifying composition is present in the sweetened composition in a total amount equal to or greater than about 2 ppm. and possibly equal to or less than about 12 ppm. 15. The sweetened composition according to any one of paragraphs. 10-14, wherein the low potency sweetener of the sweetness-modifying composition is present in the sweetened composition in a total amount ranging from 2 ppm. up to 10 ppm 16. The sweetened composition according to any one of paragraphs. 10-15, wherein the at least one sweetener is a nutritive or non-nutritive sweetener. 17. The sweetened composition according to any one of paragraphs. 10-16, wherein at least one sweetener is selected from the group consisting of sucrose, high fructose corn syrup, acesulfame potassium (AceK), aspartame, steviol glycosides such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcosidoside and nabaudioside 18. The sweetened composition according to any one of paragraphs. 10-17, wherein one or more of the sweetness characteristics of the sweetened composition are improved over the corresponding one or more sweetness characteristics of the sweetened composition in the absence of a low potency sweetener in the sweetness-modifying composition. 19. The sweetened composition according to any one of paragraphs. 10-18, wherein the long-term sweet taste of the sweetened composition is less than the long-term sweet taste of the sweetened composition in the absence of the low potency sweetener in the sweetness-modifying composition. 20. The sweetened composition according to any one of paragraphs. 10-19, wherein the bitter and / or astringent taste of the sweetened composition is less than the corresponding bitter and / or astringent taste of the sweetened composition in the absence of the low potency sweetener in the sweetness-modifying composition. 21. The sweetened composition according to any one of paragraphs. 10-20, which is a wet / runny soup, a dehydrated or culinary food, a meal solution product, a food supplement, a drink, or a dairy product such as milk, cheese, and yogurt. 22. The use of 11-O-mogroside V as a low potency sweetener to improve one or more sweetness enhancing characteristics selected from flavor profile and a time profile, which refers to changes in sweetness perception over time, of a sweetened composition containing one or more high intensity ( s) sweetener (s) selected from steviol glycosides such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside I, rebaudioside H L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubuzoside and naringenin dihydrochalcone; and a mogroside such as mogroside III, mogroside IIIx, mogroside IV, siamenoside, neomogroside, and mogroside V, where the combined amount of low potency sweetener and high intensity sweetener (s) has a sweetness less than that equivalent to 1.5% (w / v .) sucrose. 23. The use of claim 22, wherein the low potency sweetener attenuates the long-term sweet taste of the sweetened composition. 24. Use according to claim 22 or 23, wherein the low potency sweetener reduces the bitter and / or astringent taste of the sweetened composition. 25. The use according to any one of paragraphs. 22-24, wherein the ratio of high intensity sweetener (s) to low potency sweetener is in the range of 5: 1 to 12: 1, or 6: 1 to 10: 1. 26. Application according to any one of paragraphs. 22-25, wherein one or more high intensity sweetener (s) is mogroside V. 27. Application according to any one of paragraphs. 22-26, wherein the combination of low potency sweetener and one or more high intensity sweetener (s) are used to enhance the sweetness of the sweetened composition by an amount greater than the sweetness of the combination of the low potency sweetener and one or more high intensity sweetener (s) by themselves. ... 28. The use according to any one of paragraphs. 22-27, wherein the sweetened composition is a wet / runny soup, a dehydrated or culinary food product, a meal solution product, a food addition product, a drink or dairy product such as milk, cheese and yogurt. 29. Application according to any one of paragraphs. 22-28, wherein one or more high intensity sweetener (s) are present in the sweetened composition in a total amount equal to or greater than about 15 ppm. and possibly equal to or greater than about 50 ppm. 30. The use according to any one of paragraphs. 22-29, wherein one or more high intensity sweetener (s) are present in the sweetened composition in a total amount ranging from 15 ppm. up to 30 ppm 31. Application according to any one of paragraphs. 22-30, wherein the low potency sweetener is present in the sweetened composition in a total amount equal to or greater than about 2 ppm. and possibly equal to or greater than about 12 ppm. 32. Application according to any one of paragraphs. 22-31, wherein the low potency sweetener is present in the sweetened composition in a total amount ranging from 2 ppm. up to 10 ppm 33. The use according to any one of paragraphs. 22-32, wherein the low potency sweetener and one or more high intensity sweetener (s) increase the sweetness of the sweetened composition by more than a sweetness equivalent to about 1.25% (w / v) sucrose.

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