WO2018220103A1 - Compositions - Google Patents

Compositions Download PDF

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Publication number
WO2018220103A1
WO2018220103A1 PCT/EP2018/064324 EP2018064324W WO2018220103A1 WO 2018220103 A1 WO2018220103 A1 WO 2018220103A1 EP 2018064324 W EP2018064324 W EP 2018064324W WO 2018220103 A1 WO2018220103 A1 WO 2018220103A1
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WO
WIPO (PCT)
Prior art keywords
sweetness
composition
sweetener
mogroside
ppm
Prior art date
Application number
PCT/EP2018/064324
Other languages
French (fr)
Inventor
Feng Shi
Jenifer AUGELLI
Uwe KOHRS
Wei Yuan
Original Assignee
Givaudan Sa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to BR112019024859-5A priority Critical patent/BR112019024859B1/en
Priority to MX2019014119A priority patent/MX2019014119A/en
Priority to CN202310559963.4A priority patent/CN116941752A/en
Priority to CA3064334A priority patent/CA3064334A1/en
Priority to JP2019566193A priority patent/JP7306999B2/en
Priority to RU2019138376A priority patent/RU2762719C2/en
Application filed by Givaudan Sa filed Critical Givaudan Sa
Priority to EP18728620.8A priority patent/EP3629764A1/en
Priority to CN201880036461.0A priority patent/CN110719738B/en
Priority to US16/617,687 priority patent/US20220104524A1/en
Publication of WO2018220103A1 publication Critical patent/WO2018220103A1/en
Priority to CONC2019/0013524A priority patent/CO2019013524A2/en
Priority to US16/853,562 priority patent/US20200345049A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/36—Terpene glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/86—Addition of bitterness inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/88—Taste or flavour enhancing agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates generally to the use of one or more low-potency sweetener(s) to improve one or more sweetness characteristics of one or more high- intensity sweetener(s).
  • the present invention thus also relates to compositions comprising a mixture of at least one high-intensity sweetener and at least one low- potency sweetener.
  • the present invention further 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 wh+en used in combination with at least one other sweetener and/or as a sweetener.
  • the present invention further relates to the use of one or more mogroside(s) as a sweetness enhancer in sweetened compositions and said sweetened compositions.
  • the present invention further relates to methods of making the sweeteners and compositions disclosed herein.
  • Sweetness in comestible products that is products intended to be taken by mouth either for permanent ingestion or temporarily for expectoration, is often a desirable characteristic.
  • sweetness has been provided by the addition of one or more sweeteners, particularly low-potency, nutritive sweeteners such as sucrose (table sugar), fructose, glucose, xylose, arabinose, rhamnose, sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol as well as sugar syrups such as high fructose corn syrup and starch syrup. These deliver considerable sweetness without any undesirable aftertaste.
  • nutritive sweeteners such as sucrose (table sugar), fructose, glucose, xylose, arabinose, rhamnose, sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol
  • sugar syrups such as high fructose corn syrup and starch
  • High-intensity sweeteners have been used for this purpose.
  • High-intensity sweeteners may be natural or artificial and have a sweetness that can be several hundred times that of sucrose and thus can theoretically replace a much larger quantity of sugar in a composition.
  • high-intensity sweeteners include sucralose, saccharin, aspartame, acesulfame potassium (AceK), neotame, advantame, sterviol glycosides, including stevioside, rebaudioside A, rebaudioside D or steviol glycoside mixture preparations with rebaudioside A and/or stevioside as predominant components.
  • these substances generally have the drawback that they may impart undesirable off-tastes to comestible products, typically bitter, metallic or liquorice tastes, or an undesirable lingering sweetness.
  • a sweetness modifying composition comprising:
  • one or more high-intensity sweetener(s) selected from the group consisting of steviol glycosides and/or mogrosides;
  • one or more low-intensity sweetener(s) selected from the group consisting of cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V;
  • the sweetness modifying composition increases the sweetness of a sweetened composition by more than the sweetness of the sweetness modifying composition alone;
  • ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) ranges from about 2:1 to about 12:1 .
  • a sweetened composition comprising:
  • At least one sweetener present in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence;
  • a sweetness modifying composition according to any aspect or embodiment of the present invention.
  • 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 recognition threshold and/or having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence, and adding 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 1 1 -O-mogroside V, wherein the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 2:1 to about 12:1 ; and/or wherein the one or more high- intensity sweetener(s) are added in a total amount equal to or greater than about 15
  • a method of making a sweetness modifying composition comprising combining one or more high-intensity sweetener(s) and one or more low-potency sweetener(s).
  • a method of making a sweetened composition comprising combining the base composition, one or more high-intensity sweetener(s), one or more low-intensity sweetener(s) and at least one other sweetener.
  • a sweetened composition comprising at least one sweetener present in an amount having a sweetness equal to or greater than 1 .5 % (w/v) sucrose equivalence; and one or more sweetness enhancer(s) selected from mogroside IV, siamenoside and neomogroside.
  • a use of one or more of mogroside IV, siamenoside and neomogroside to enhance 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.
  • a method of making a sweetened composition comprising combining the base composition, one or more sweetness enhancer(s) selected from mogroside IV, siamenoside and neomogroside and at least one other sweetener.
  • a sweetened composition comprising one or more mogroside(s).
  • the one or more mogroside(s) may, for example, be present as a sweetness enhancer and thus be present in an amount having a sweetness of less than 1 .5 % (w/v) sucrose equivalence.
  • the sweetened composition will then further comprise at least one sweetener present in an amount having a sweetness equal to or greater than 1 .5 % (w/v) sucrose equivalence.
  • a use of one or more mogroside(s) to enhance the sweetness of a sweetened composition comprising providing a base composition and adding at least one sweetener and one or more mogroside(s).
  • a method of making a sweetened composition comprising combining the base composition, one or more mogroside(s) and at least one other sweetener.
  • the one or more high-intensity sweetener may include or be mogroside V and/or the one or more low-potency sweetener may include or be 1 1 -O-mogroside V.
  • the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 2:1 to about 12:1 .
  • the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 5:1 to about 12:1 . In certain embodiments of any aspect of the present invention the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may be from about 6:1 to about 10:1 .
  • the one or more high-intensity sweetener(s) may be present in a total amount ranging from about 15 ppm to about 30 ppm and/or the one or more low-potency sweetener(s) may be present in a total amount ranging from about 2 ppm to about 10 ppm. In certain embodiments of any aspect of the present invention the one or more high-intensity sweetener(s) may be present in a total amount ranging from about 22 ppm to about 28 ppm and/or the one or more low-potency sweetener(s) may be present in a total amount ranging from about 2 ppm to about 5 ppm.
  • the one or more mogroside(s) or one or more sweetness enhancer(s) may be present in an amount ranging from about 15 ppm to about 50 ppm. In certain embodiments, the one or more mogroside(s) or one or more sweetness enhancer(s) may be present in an amount ranging from about 15 ppm to about 35 ppm.
  • the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) alone may have a sweetness less than about 1 .5 % (w/v) sucrose equivalence.
  • the concentration of the one or more high-intensity sweetener(s) and the one or more low-intensity sweetener(s) in a sweetened composition may have a sweetness less than about 1 .5 % (w/v) sucrose equivalence.
  • the one or more mogroside(s) or one or more sweetness enhancer(s) may have a total sweetness less than about 1 .5 % (w/v) sucrose equivalence. In certain embodiments, the one or more sweetness enhancer(s) increase the sweetness of a sweetened composition by more than the total sweetness of the one or more sweetness enhancer(s) alone.
  • the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) may increase the sweetness of a sweetened composition by more than the sweetness of the combination alone. In certain embodiments of any aspect of the present invention the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) may increase the sweetness of a composition by equal to or greater than about 1 .25 % (w/v) sucrose equivalence.
  • the one or more low-potency sweetener(s) weaken the lingering sweet taste of a sweetened composition comprising the one or more high-intensity sweetener(s) compared to the lingering sweet taste of the sweetened composition in the complete absence of the one or more low-potency sweetener(s). In certain embodiments of any aspect of the present invention the one or more low-potency sweetener(s) weakens the bitter and/or astringent taste of a sweetened composition comprising the one or more high-intensity sweetener(s) compared to the bitter and/or astringent taste of the sweetened composition in the complete absence of the one or more low-potency sweetener(s).
  • One or more (e.g. all) of the sweeteners used may be natural or synthetic (artificial).
  • One or more of the sweeteners may, for example, be made by a biological process or by an enzymatic process or by a synthetic process.
  • Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of Table 1 below).
  • Figure 2 shows the chemical structures of mogrosides 1 -6;
  • Figure 3 shows the LC-MS analysis of commercial Luo Han Guo extracts;
  • FIG. 4 shows the Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy (HSQC-TOCSY) (hsqcgpmlph) of iso-mogroside VI with different mixing time (d9).
  • A 10 ms mixing time.
  • B 30 ms mixing time.
  • C 60 ms mixing time.
  • D 100 ms mixing time. Because of the overlap of H-1 of Glc II and H-6a of Glc III, HSQC-COSY correlation intensity of Glc II was not analyzed here;
  • Figure 5 shows HSQC-TOCSY (hsqcgpmlph) peak intensity quantification of iso- mogroside VI glucopyranosyls with different mixing time.
  • C-3 and C-5 signals on HSQC-TOCSY appeared overlap for 100 ms mixing time. The total integration of C-3 and C-5 was therefore used in the bar chart;
  • Figure 6 shows the strategy to elucidate mogroside sugar chain ( * Numbers of C-2 to C- 6 appeared under certain mixing time might slightly change if adjusting peak intensity of HSQC-TOCSY. By observing the increasing intensity of C-2 to C-6 in different mixing time experiments connection sequence can be still determined. ** There is no natural glycosylation on C-3 of mogroside glucopyranosyl so far.
  • Step 1 Heteronuclear multiple bond correlation spectroscopy (HMBC) was used to determine anormeric C-1 and H-1 of the sugar. Start from the sugar link to aglycone.
  • HMBC Heteronuclear multiple bond correlation spectroscopy
  • Step 3 if a C-2 downshift from -575 to -581 , C-4 downshift from -571 to -581 or C-6 downshift from -562 to -69 is observed, check HMBC for glycosylation at these positions. **
  • Figure 7 shows the chemical structure for iso-mogroside VI which has the chemical formula C 6 6H 1 12 0 3 4 and an Exact Mass of 1448.70. This chemical structure is designated Formula I; and Figure 8 shows the chemical structure for 1 1 -epi-mogrosideV which has the chemical formula C 60 H 1 0 2O29 and an Exact Mass of 1286.65. This chemical structure is designated Formula II. DETAILED DESCRIPTION
  • 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. 1 1 -O-mogroside V) can act synergistically with at least one other sweetener (e.g. sucrose) to obtain a composition having a sweetness that is greater than the sum of the sweetness of the individual sweeteners.
  • the present invention is further based on the surprising finding that one or more low-potency sweetener(s) may offset one or more negative sweetness characteristics of one or more high-potency sweetener(s).
  • the combination of one or more high-intensity sweetener(s) e.g.
  • mogroside V and one or more low-potency sweetener(s) (e.g. 1 1 -O-mogroside V) may provide improved sweetness characteristics in a sweetened composition (i.e. a composition comprising at least one other sweetener such as sucrose in an amount above its sweetness recognition threshold and/or an amount equal to or greater than about 1 .5 % (w/v) sucrose equivalence) compared to using the one or more high-intensity sweetener(s) alone.
  • the sweetness characteristics may thus, for example, be closer to the sweetness characteristics of sucrose.
  • compositions comprising one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) as disclosed herein, particularly sweetened compositions comprising at least one sweetener in an amount above its sweetness recognition threshold and/or an amount equal to or greater than about 1 .5 % (w/v) sucrose equivalence and one or more high-intensity sweetener and one or more low-potency sweetener(s).
  • the sweetened compositions may also be referred to as comestible compositions.
  • compositions in particular sweetened compositions, comprising one or more of mogroside IV, siamenoside and neomogroside.
  • compositions comprising at least one high-intensity sweetener and at least one low-potency sweetener.
  • compositions comprising one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside.
  • the compositions are comestible compositions.
  • 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 1 1 -O-mogroside V.
  • the sweetness modifying composition comprises, consists essentially of or consists of one high-intensity sweetener and one low-potency sweetener.
  • the sweetness modifying composition may, for example, be a concentrate which may, for example, be diluted in a sweetened (e.g.
  • sweetened composition refers to a composition comprising at least one sweetener present in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • a sweetened composition comprising one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside.
  • a sweetened composition e.g. comestible composition
  • a sweetened composition comprising at least one high-intensity sweetener and at least one low-potency sweetener.
  • the combination of the high-intensity sweetener(s) and low-potency sweetener(s) may be referred to as a sweetness modifying composition.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may also be referred to herein as a sweetness modifying composition.
  • a sweetened composition comprising at least one sweetener present in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence 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 may, for example, be a comestible composition.
  • the term "enhancing" 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 a sweetened composition by more than the sweetness of the sweetness modifying composition alone. In other words, the sweetness of a composition comprising at least one sweetener and at least one sweetness modifying composition is greater than the sum of the sweetness of all the sweeteners in the composition.
  • the sweetness modifying compositions described herein are used in sweetened (e.g. comestible) compositions in amounts that have no detectable sweetness or no taste recognised as sweet (below its sweetness recognition threshold).
  • a sweetness modifying composition with a sweetness below 1 .5 % (w/v) sucrose equivalence is accepted as being "not intrinsically sweet” by FEMA (Flavor & Extract Manufacturers Association).
  • Sweetness modifiers may also be referred to as sweetness enhancers.
  • the sweetened composition comprising the sweetness modifying composition as disclosed herein and at least one sweetener in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may have a sweetness that is equal to or more than about 1 .0 % (w/v) sucrose equivalence greater than the sweetness of the sweetened composition in the absence of the sweetness modifying composition.
  • the sweetened composition may have a sweetness that is equal to or more than about 1 .1 % (w/v) or equal to or more than about 1 .15 % (w/v) or equal to or more than about 1 .2 % (w/v) or equal to or more than about 1 .25 % (w/v) sucrose equivalence greater than the sweetness of the sweetened composition in the absence of the sweetness modifying composition.
  • the sweetness modifying composition may increase the sweetness of a sweetened composition by equal to or more than about 1 % (w/v) or equal to or more than about 1 .1 % (w/v) or equal to or more than about 1 .15 % (w/v) or equal to or more than about 1 .2 % (w/v) or equal to or more than about 1 .25 % (w/v) sucrose equivalence.
  • the comparative composition is identical to the said composition except that it does not include the said sweetness modifying composition.
  • sucrose equivalence refers to the equivalence in sweetness of a composition containing at least one non-sucrose sweetener to a reference sucrose solution.
  • taste panellists are trained to detect sweetness of reference sucrose solutions containing between 1 % and 15 % sucrose (w/v).
  • Other non-sucrose sweeteners may then be tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet (i.e. isosweet) to a given sucrose reference.
  • isosweet refers to compositions that have equivalent sweetness. Typically, the sweetness of a given composition is measured with reference to a solution of sucrose.
  • the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) may, for example, have a sweetness less than about 1 .5 % (w/v) sucrose equivalence.
  • the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) e.g.
  • sweetness modifying composition may have a sweetness equal to or less than about 1 .45 % (w/v) sucrose equivalence or equal to or less than about 1 .4 % (w/v) sucrose equivalence or equal to or less than about 1 .35 % (w/v) sucrose equivalence or equal to or less than about 1 .3 % (w/v) sucrose equivalence.
  • the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) e.g.
  • sweetness modifying composition may have a sweetness equal to or greater than about 1 % (w/v) sucrose equivalence or equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence or equal to or greater than about 1 .3 % (w/v) sucrose equivalence.
  • the 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 about 1 .5 % (w/v) sucrose equivalence.
  • the 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 1 .45 % (w/v) sucrose equivalence or equal to or less than about 1 .4 % (w/v) sucrose equivalence or equal to or less than about 1 .35 % (w/v) sucrose equivalence or equal to or less than about 1 .3 % (w/v) sucrose equivalence.
  • the 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 about 1 % (w/v) sucrose equivalence or equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence or equal to or greater than about 1 .3 % (w/v) sucrose equivalence.
  • sweeteners and sweetness enhancers used in the compositions disclosed herein may be a natural or synthetic (artificial) sweetener.
  • non-naturally occurring (i.e. synthetic) mogrosides are disclosed in WO 2017/075257, the contents of which are incorporated herein by reference.
  • natural sweetener refers to sweeteners that are obtained from nature, including mixtures that may have been enzymatically treated (e.g. glycosylated) to form compounds not found in nature (this does not include purified compounds that have been enzymatically treated).
  • a modified extract having a mogrol glycoside distribution that is different (e.g. enhanced) from the naturally occurring mogrol glycoside distribution may be classed as natural.
  • a mixture of glucosylated steviol glycosides and/or glucosylated mogrosides may be classed as natural.
  • Each of the sweeteners used in the compositions disclosed herein may be food-derived.
  • a "food-derived" product refers to a product which is prepared under typical cooking conditions such as, for example, using temperatures similar to those used in cooking methods.
  • the high-intensity sweetener and the low-potency sweetener used in the compositions disclosed herein are both natural sweeteners.
  • all of the sweeteners used in the compositions disclosed herein are natural.
  • the sweeteners disclosed herein may be used in pure or purified form and may be chemically synthesised, produced by biotechnological processes (e.g. fermentation) or isolated from a natural source (e.g. a botanical source including, without limitation, fruits, sugar cane, sugar beet).
  • a natural source e.g. a botanical source including, without limitation, fruits, sugar cane, sugar beet.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be at least 80 wt% pure.
  • the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may be at least about 85 wt% or at least about 90 wt% or at least about 95 wt% or at least about 98 wt% or at least about 99 wt% pure.
  • the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may be up to 100 wt% or up to 99 wt% pure.
  • the term "high-intensity sweetener" refers to compounds having a sweetness that is at least 100 times the sweetness of sucrose.
  • 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 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 may, for example, have a sweetness that is up to 1000 times the sweetness of sucrose.
  • the high-intensity sweetener has a sweetness that is at least 100 times the sweetness of sucrose, in the context of its use in a sweetness modifying composition as described herein, they will be used in a sweetened composition in an amount that does not have any detectable sweetness or be recognised as sweet (amounts providing a sweetness less than 1 .5 % (w/v) sucrose equivalence, which is accepted as being "not intrinsically sweet” by FEMA.
  • the one or more high-intensity sweetener(s) may, for example, be one or more steviol glycosides and/or one or more mogrosides.
  • the one or more high-intensity sweetener may be a mixture of steviol glycosides and mogrosides.
  • the one or more high-intensity sweeteners may be one or more steviol glycosides.
  • the one or more high-intensity sweetener(s) may be one or more mogrosides.
  • mogrosides may perform better than steviol glycosides in terms of sweetness enhancement and off-note reduction (e.g. weakening of lingering sweet after taste).
  • the high-intensity sweetener may, for example, be one or more steviol glycoside(s).
  • 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: 12206
  • the high-intensity sweetener may, for example, be one or more mogroside(s). In certain embodiments, the high-intensity sweetener may be one or more of the mogrosides listed herein. In certain embodiments, the high-intensity sweetener may be one or more of mogroside IV, siamenoside, neomogroside and mogroside V (including all isomers thereof). For example, the high-intensity sweetener may be a mixture of mogroside IV, siamenoside and mogroside V (including all isomers thereof). The one or more mogroside(s) may, for example, be obtained or obtainable from Luo Han Guo fruit extracts.
  • low-potency sweetener refers to compounds having a sweetness that is less than 100 times the sweetness of sucrose. In certain 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 of cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V (CAS: 126105-1 1 -1 ).
  • the one or more low-intensity sweetener(s) may be one or more of cellobiose, psicose and 1 1 -O-mogroside V.
  • the one or more high-intensity sweetener(s) includes or is a high-intensity mogroside. In certain embodiments, the one or more low-potency sweetener(s) includes or is a low-potency mogroside. In certain embodiments, the one or more high-intensity sweetener includes or is a high-intensity mogroside and the one or more low-potency sweetener(s) includes or is a low-potency mogroside. In certain embodiments, the one or more high-intensity sweetener includes or is mogroside V. In certain embodiments, the one or more low-potency sweetener includes or is 1 1 -O-mogroside V.
  • the one or more high-intensity sweetener includes or is mogroside V and the one or more low-potency sweetener includes or is 1 1 -O-mogroside V.
  • Mogrosides are a group of triterpene glycosides and may be obtained from the fruit Luo Han Guo (Siraitia grosvenorii), also known as arhat fruit or longevity fruit or swingle fruit. Mogrosides make up approximately 1 % of the flesh of the fresh fruit. Through extraction, an extract in the form of a powder containing up to 80% mogrosides can be obtained.
  • Mogroside extract contains grosvenorine II, grosvenorine I, 1 1 -O-mogroside II (I), 1 1 -O-mogroside II (II), 1 1 -O-mogroside II (III), mogroside II (I), mogroside II (II), mogroside II (III), 1 1 -dehydroxy-mogroside III, 1 1 -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), 1 1 -O-mogroside V (I), mogroside V isomer, mogroside V, iso-mogroside V, 7-O-mogroside V, 1 1 -O-mogroside VI, mogroside VI (I), mogroside VI (I), mogroside VI (III) (neomogros
  • Mogroside(s) include both mogroside(s) that occur in nature and mogrosides that do not occur in nature.
  • mogrosides include, for example, grosvenorine II, grosvenorine I, 1 1 -O-mogroside II (I), 1 1 -O-mogroside II (II), 1 1 -O-mogroside II (III), mogroside II (I), mogroside II (II), mogroside II (III), 1 1 -dehydroxy-mogroside III, 1 1 -O-mogroside III, mogroside III (I), mogroside III (II), mogroside llle, mogroside Mix, mogroside IV (I) (siamenoside), mogroside IV (II), mogroside IV (III), mogroside IV (IV), deoxymogroside V (I), deoxymogroside V (II), 1 1 -O-mogroside V (I), mogroside V isomer, mogroside V, iso-mogroside V
  • Mogroside V (CAS: 88901 -36-4) is a glycoside of a cucurbitane derivative and has the chemical formula C-60H102O29 and the chemical structure shown below. Mogroside V can be found in certain plant extracts such as extracts from the fruit Luo Han Guo (Siraitia grosvenorii). Pure mogroside V has been found to have a sweetness of at least 400 times the sweetness of sucrose.
  • Siamenoside (CAS: 126105-12-2) is a cucurbitane found in the fruit of Siraitia grosvenorii and has the following chemical structure.
  • Neomogroside (CAS: 189307-15-1 ) is a cucurbitane glycoside also found in the fruit of Siraitia grosvenorii and has the following chemical structure.
  • 1 1 -O-Mogroside V (CAS: 126105-1 1 -1 ) is derived from mogroside V and has the following chemical structure. It is also found in plant extracts such as extracts from the fruit Luo Han Guo (Siraitia grosvenorii). 1 1 -O-mogroside V has been found to have a sweetness that is about 84 times the sweetness of sucrose.
  • the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is equal to or greater than about 2:1 .
  • the ratio of the one or more high-intensity sweetener(s) to the 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 to or greater than about 5:1 or equal to or greater than about 5.5:1 or equal to 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 to or greater than about 8:1 .
  • the ratio of the high-intensity sweetener to the low-potency sweetener is equal to or less than about 12:1 .
  • the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may be equal to or less than about 1 1 .5:1 or equal to or less than about 1 1 :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 .
  • the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may range from about 5:1 to about 1 1 :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 .
  • the ratio of the one or more high-intensity sweetener(s) to the 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 may be weight or volume ratio.
  • the ratio only applies to the high-intensity sweetener(s) and low-potency sweeteners in the sweetness modifying composition (high-intensity and low-potency sweeteners that are used in a sweetened composition in an amount below the sweetness recognition threshold or having less than 1 .5 % (w/v) sucrose equivalence).
  • the one or more high-intensity sweetener(s) may be present in a composition in a total amount equal to or greater than about 15 ppm.
  • the one or more high-intensity sweetener(s) may be present in a 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.
  • the one or more high-intensity sweetener(s) may be present in a 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.
  • the one or more high-intensity sweetener(s) may be present in a composition in a total amount ranging from about 15 ppm to about 50 ppm or from about 15 ppm to about 45 ppm or from about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm or from about 15 ppm to about 30 ppm.
  • the one or more high-intensity sweetener(s) may be present in a composition in a total amount ranging from about 15 ppm to about 30 ppm or from about 20 ppm 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 to about 26 ppm.
  • the one or more high-intensity sweetener(s) may be present in a composition in a total amount of about 20 ppm or about 25 ppm.
  • the composition may, for example, be a sweetened composition comprising at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • the one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or greater than about 2 ppm.
  • the one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or greater than about 3 ppm.
  • the one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or less than about 12 ppm or equal to or less than about 1 1 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.
  • the one or more low-potency sweetener(s) may be present in a composition in a total amount ranging from about 2 ppm to about 12 ppm or from about 2 ppm to about 10 ppm or from about 2 ppm to about 5 ppm, for example in a total amount of about 3 ppm.
  • the composition may, for example, comprise at least one sweetener other than the combination of the high-intensity sweetener and the low-potency sweetener (e.g. sweetness modifying composition) as disclosed herein.
  • the concentration ranges may, for example, be particularly suitable for liquid compositions such as beverages or compositions that do not comprise any proteins or fats.
  • compositions having a base such as milk and yogurt or other compositions that do comprise proteins and fats higher concentrations of the one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) may be used. For example, concentrations that are about 1 .5 times higher than the concentrations used for liquid compositions or compositions that do not comprise any proteins or fats may be used. For example, concentrations that are from about 1 .5 times to about 3 times higher than the concentrations used for liquid compositions or compositions that do not comprise any proteins or fats may be used. Therefore, for example, the one or more high-intensity sweetener(s) may be present in a composition (e.g.
  • compositions having a base such as milk and yoghurt or other compositions that comprise 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 to about 66 ppm or from about 30 ppm to about 65 ppm or from about 30 ppm to about 60 ppm or from about 30 ppm to about 55 ppm or from about 30 ppm to about 50 ppm or from about 30 ppm to about 45 ppm.
  • a base such as milk and yoghurt or other compositions that comprise proteins and/or fats
  • the one or more low-potency sweetener(s) may be present in a composition (e.g. a composition having a base such as milk and yoghurt or other compositions that comprise proteins and/or fats) in a total amount ranging from about 3 ppm 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 to about 15 ppm or from about 6 ppm to about 15 ppm.
  • a composition e.g. a composition having a base such as milk and yoghurt or other compositions that comprise proteins and/or fats
  • a sweetened composition comprises at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or equal to or greater than about 1 .5 % (w/v) sucrose equivalence and a sweetness modifying composition consisting of 15 ppm to about 50 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 12 ppm of one or more low-potency sweetener(s) as described herein.
  • a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 15 ppm to about 30 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 10 ppm of one or more low-potency sweetener(s) as described herein.
  • a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 20 ppm to about 30 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 10 ppm of one or more low-potency sweetener(s) as described herein.
  • a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 22 ppm to about 28 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 5 ppm of one or more low-potency sweetener(s) as described herein.
  • the high- intensity sweetener is mogroside V.
  • the low-potency sweetener is 1 1 -O-mogroside V.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be present in a sweetened composition in a total amount equal to or greater than about 15 ppm.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siameonside and neomogroside may, for example, be present in a 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.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be present in a sweetened composition in a total amount equal to or less than about 50 ppm, for example equal to or less than about 45 ppm, for example equal to or less than about 40 ppm, for example equal to or less than about 35 ppm.
  • the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may be present in a sweetened composition in a total amount ranging from about 15 ppm to about 50 ppm or from about 15 ppm to about 45 ppm or from about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm or from about 20 ppm to about 35 ppm or from about 20 ppm to about 30 ppm.
  • ppm refers to part(s) per million by weight, for example the weight of a compound, such as Mogroside V (in milligrams) per kilogram of the product containing such compound (i.e.
  • a compound e.g. orally consumable/comestible product of the present disclosure
  • a compound e.g. orally consumable/comestible product of the present disclosure
  • Mogroside V in milligrams
  • a compound such as Mogroside V (in milligrams) per litre of the product containing such compound (i.e. mg/L) or by volume, for example, the volume of a compound, such as Mogroside V (in millilitres) per litre of the product containing such compound (i.e. ml/L).
  • the sweetness modifying compositions described herein may, for example, comprise higher concentrations of the high-intensity and low-intensity sweeteners and are then diluted in a sweetened composition to obtain the concentrations recited herein.
  • a sweetened composition comprises at least one sweetener in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • sweetness recognition threshold refers to the lowest known concentration of a compound that is perceivable as sweet by the human sense of taste.
  • a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence is accepted as being "intrinsically sweet” by FEMA.
  • the at least one sweetener may be nutritive or non-nutritive. Nutritive sweeteners add caloric value to the foods that contain them while non-nutritive sweeteners are very low in calories or contain no calories at all. Aspartame, the only approved nutritive high- intensity sweetener contains more than 2 % of the calories in an equivalent amount of sugar as opposed to non-nutritive sweeteners that contain less than 2 % of the calories in an equivalent amount of sugar.
  • 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 (AceK), aspartame, steviol glycoside(s), sucralose, high-fructose corn syrup, starch syrup, saccharin, sucralose, neotame, advantame, Luo Han Guo extract, neohespiridin, dihydrochalcone, naringin dihydrochalcone, neohesperidin dihydrochalcone, rubusoside, rebaudioside A, stevioside, stevia, trilobtain and sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol.
  • sweeteners
  • 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.
  • the amount of sweetener can be reduced by addition of a sweetness modifying composition as disclosed herein. For example, a reduction of about 1 0 to about 4° Brix or more may be achieved.
  • the at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may, for example, be used in a sweetened composition in an amount equal to or greater than about 0.01 % (w/v).
  • the at least one other sweetener may be used in a 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).
  • the at least one other sweetener may be used in a comestible 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 than 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).
  • the at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may be used in the sweetened compositions disclosed herein (e.g. comestible composition) in amounts isosweet to about 2 % (w/v) to about 15 % (w/v) sucrose.
  • a sweetness modifying composition consisting of mogroside V and 1 1 -O-mogroside V in a ratio ranging from about 2:1 to about 12:1 , for example from about 6:1 to about 10:1.
  • This sweetness modifying composition may, for example, be used as sweetness enhancer or modifier in a comestible composition.
  • the comestible composition may, for example, comprise at least one other sweetener such as sucrose.
  • the mogroside V may, for example, be used in the comestible composition in an amount ranging from about 15 ppm to about 30 ppm or from about 20 ppm to about 30 ppm (e.g. about 20 ppm or about 25 ppm).
  • the 1 1 -O-mogroside V may be used in the comestible composition in an amount ranging from about 2 ppm to about 12 ppm or from about 2 ppm to about 10 ppm (e.g. about 8.5 ppm or about 3 ppm).
  • the at least one other sweetener may, for example, be present in the comestible composition in an amount isosweet to about 2 % (w/v) to about 15 % (w/v) sucrose.
  • compositions may be in any suitable form, for example solid (e.g. powder, granules, tablets) or in solution (e.g. aqueous solution) or in an emulsion or in a suspension.
  • the compositions may further comprise a diluent or bulking agent such as dietary fibre.
  • compositions as disclosed herein include, for example, the following. - Wet/liquid soups regardless of concentration or container, including frozen soups.
  • soup(s) means a food prepared from meat, poultry, fish, vegetables, grains, fruit and other ingredients, cooked in a liquid which may include visible pieces of some or all of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth, pureed or chunky, ready-to-serve, semi-condensed or condensed and may be served hot or cold, as a first course or as the main course of a meal or as a between meal snack (sipped like a beverage). Soup may be used as an ingredient for preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups).
  • - Dehydrated and culinary foods including cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology).
  • cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology).
  • - Meal solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes.
  • - Meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salad, sold as a finished product or as an ingredient within a product, whether dehydrated, liquid or frozen.
  • - Beverages including beverage mixes and concentrates, including but not limited to, alcoholic and non-alcoholic ready to drink and dry powdered beverages, carbonated and non-carbonated beverages, e.g., sodas, fruit or vegetable juices, alcoholic and non-alcoholic beverages.
  • Confectionery products e.g., cakes, cookies, pies, candies, chewing gums, gelatins, ice creams, sorbets, puddings, jams, jellies, salad dressings, and other condiments, cereal, and other breakfast foods, canned fruits and fruit sauces and the like.
  • compositions which may, for example, be in the form of a syrup, an emulsion, a suspension, a solution or other liquid form.
  • compositions including, for example, mouth freshening agents, gargling agents, mouth rinsing agents, toothpaste, tooth polish, dentifrices, mouth sprays and dental floss.
  • compositions disclosed herein may further comprise a base composition.
  • the comestible compositions disclosed herein may further comprise a comestible base composition. This refers to all the ingredients necessary for the composition except the combination of the high-intensity sweetener and low-potency sweetener (e.g. sweetness modifying composition).
  • the base composition may, for example, be a sweetened base composition comprising at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • the ingredients in a base composition may include, but are not limited to, anti-caking agents, anti-foaming agents, anti-oxidants, binders, colourants, diluents, disintegrants, emulsifiers, encapsulating agents or formulations, enzymes, fats, flavour-enhancers, flavouring agents, gums, lubricants, polysaccharides, preservatives, proteins, solubilisers, solvents, stabilisers, sugar-derivatives, surfactants, sweetening agents, vitamins, waxes, and the like.
  • Solvents which may be used are known to those skilled in the art and include e.g. ethanol, ethylene glycol, propylene glycol, glycerine and triacetin.
  • Encapsulants and gums include maltodextrin, gum arabic, alginates, gelatine, modified starch, and polysaccharides.
  • the proportion of the combination of the one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) will depend on the nature of the composition and the degree and characteristics of the sweetness desired. The skilled person can readily ascertain the appropriate proportion in every case with only simple, non-inventive experimentation.
  • the amounts and proportions disclosed herein are exemplary only and the flavourist may seek particular effects by working outside this range, and it should be regarded as an indication only.
  • the pH of the composition disclosed herein may be any pH that does not adversely affect the taste of the sweetener blend.
  • the pH may range from about 1 .8 to about 8 or from about 2 to about 5.
  • a person skilled in the art would be able to identify a suitable concentration of each sweetener to use depending on the pH of the composition.
  • the use of the one or more low-potency sweetener(s) with the one or more high- intensity sweetener(s) may, for example, improve one or more sweetness characteristics in a sweetened composition in comparison to the use of the one or more high-intensity sweetener(s) alone.
  • the sweetened compositions disclosed herein may, for example, have one or more improved sweetness characteristics compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s).
  • sweetness enhancer(s) selected from mogroside IV, siamenoside and neomogroside may, for example, improve one or more sweetness characteristics of a sweetened composition in comparison to the use of a different sweetness enhancer such as Luo Han Guo extract in place of the one or more of mogroside IV, siamenoside and neomogroside.
  • the sweetened compositions disclosed herein may, for example, have one or more sweetness characteristics that are more similar to sucrose compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
  • the sweetened compositions disclosed herein may, for example, have a weakened lingering sweet taste in compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
  • the sweetened compositions disclosed herein may, for example, have a weakened bitter taste and/or astringent taste and/or metallic taste and/or liquorice taste compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
  • the sweetened compositions disclosed herein may, for example, have a strengthened sweetness impact compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
  • the comparative sweetened composition is identical except that it does not include any of the one or more low-potency sweetener(s) or identical except that it comprises a different sweetness enhancer in place of the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside.
  • a combination of one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) to enhance the sweetness of a composition comprising at least one other sweetener present an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • the combination of the one or more high-intensity sweetener(s) and one or more low-intensity sweetener(s) has a sweetness less than 1 .5 % (w/v) sucrose equivalence.
  • the 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.
  • mogroside(s) for example one or more of mogroside IV, siamenoside and neomogroside to enhance the sweetness of a composition comprising at least one other sweetener present an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
  • a method for enhancing the sweetness of a sweetened composition comprising providing a base composition comprising at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence, and adding at least one low-potency sweetener, at least one high-intensity sweetener; or adding one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside.
  • Each component of the final composition may be added in any order to obtain the desired final composition.
  • the method may, for example, comprise mixing the components.
  • the one or more high-intensity sweetener and/or the combination of the one or more high-intensity sweetener and the one or more low-potency sweetener (e.g. the sweetness modifying composition) and/or the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may, for example, increase the sweetness of a sweetened composition by equal to or more than about 1 .0 % (w/v) sucrose equivalence.
  • the high-intensity sweetener(s) and/or the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) and/or the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may increase the sweetness of a sweetened composition by equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence.
  • the composition may, for example, be a composition comprising at least one other sweetener.
  • one or more low-potency sweetener(s) to improve one or more sweetness characteristics of a sweetened composition comprising 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 of less than about 1 .5 % (w/v) sucrose equivalence.
  • a method for improving one or more sweetness characteristics of a sweetened composition comprising one or more high-intensity sweetener(s) in an amount having a sweetness less than 1 .5 % (w/v) sucrose equivalence, the method comprising providing a composition comprising one or more high-intensity sweetener(s) and adding one or more low-potency sweetener(s).
  • Each component of the final composition may be added in any order to obtain the desired final composition.
  • the method may, for example, comprise mixing the components.
  • the improvement of one or more sweetness characteristics of a sweetened composition comprising a high-intensity sweetener may, for example, provide sweetness characteristics that are more similar to the sweetness characteristics of sucrose.
  • the sweetness characteristics may refer to the flavour profile (taste profile), which refers to the intensity of the flavour and perceptual attributes of a given compound.
  • flavour profile refers to the intensity of the flavour and perceptual attributes of a given compound.
  • Exemplary flavour attributes of sweetness are sweetness intensity, bitterness, black liquorice etc.
  • the sweetness characteristics may refer to the temporal profile, which refers to the changes in perception of sweetness over time. Every sweetener exhibits a characteristic appearance time (AT) and extinction time (ET). Most high-potency sweeteners, in contrast to carbohydrate sweeteners, display prolonged ET (lingering). Generally, the detected sucrose equivalence spikes to a maximal response level, then tapers off over time. The longer the taper, the greater the detected sweetness linger of a compound.
  • the one or more low-potency sweetener(s) may be used to weaken the lingering sweet taste of the sweetened composition comprising one or more high-intensity sweetener(s).
  • the low-potency sweetener may be used to decrease the extinction time (ET) of the sweetened composition comprising one or more high-intensity sweetener(s).
  • ET extinction time
  • the lingering sweet taste may, for example, refer to the length of time that the sweetness taste remains after it is initially detected, how rapidly the intensity of the sweetness taste decreases or fades after it is initially detected and the intensity of the sweetness taste after it is initially detected.
  • the one or more low-potency sweetener(s) may, for example, decrease the length of time that the sweetness taste remains after it is initially detected and/or increase the speed at which the sweetness taste decreases after it is initially detected and/or decrease the intensity of the sweetness taste after it is initially detected.
  • the one or more low-potency sweetener(s) may be used to weaken the bitter taste and/or astringent taste and/or metallic taste and/or liquorice taste of the sweetened composition comprising the one or more high-intensity sweetener(s).
  • the term "liquorice” refers to a sweet taste of a compound.
  • the one or more low-potency sweetener(s) may be used to strengthen the sweetness impact of the sweetened composition comprising the one or more high-intensity sweetener(s).
  • the sweetness impact relates to the length of time it takes before the sweetness is initially detected and the intensity at which the sweetness is initially detected.
  • the one or more low-potency sweetener(s) may, for example, decrease the amount of time before the sweetness is initially detected and/or increase the intensity at which the sweetness is initially detected.
  • the degree of sweetness and other sweetness characteristics described herein may be evaluated by a tasting panel of trained experts, for example as described in the examples below. Manufacturing Methods
  • compositions disclosed herein may be in accordance with any embodiment disclosed herein.
  • the methods may comprise combining each component of a desired composition in the desired proportions and optionally mixing the components together.
  • the components may be combined and mixed in any suitable order.
  • the methods may, for example, comprise providing a desired base composition and adding the sweeteners thereto.
  • Each of the sweeteners disclosed herein may be made by a synthetic process or by a biological (e.g. enzymatic) process or a fermentation process or may be isolated from a natural source such as a plant or fruit.
  • the process may, for example, comprise 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 (e.g.
  • the enzyme may, for example, be a UGT enzyme (UDP-glucuronosyltransferase).
  • UGT enzyme UGT-glucuronosyltransferase
  • a mogroside compound may be made by the biosynthetic pathway disclosed in WO 2013/076577 or WO 2014/086842, the contents of which are incorporated herein by reference.
  • mogroside V may be made by the biosynthetic pathway disclosed in Itkin et at., "The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii', PNAS, 7 November 2016, E7619 - E7628 and WO 2016/038617, the contents of which are incorporated herein by reference.
  • a mogroside compound may be made by modifying (e.g. redistributing glycoside content) another mogroside compound.
  • a mogroside compound may be made by redistributing glycoside content of another mogroside compound using acid or enzymes as disclosed in WO 2014/150127, the contents of which are incorporated herein by reference.
  • the process may, for example, comprise extracting one or more sweetener compounds from a natural source such as a plant or fruit. This may, for example, be followed by a purification step to yield a high-intensity sweetener, low-intensity sweetener or mixture of sweeteners (e.g. 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 1 1 -O-mogroside V (e.g. at least about 30 wt% or at least about 40 wt%). This may, for example, involve fractioning, for example by flash chromatography.
  • One or more mogroside compounds (e.g. mogroside V) may be obtained from Luo Han Guo (Siraitia grosvenorii) fruit.
  • the target product e.g. target mogroside product
  • the target can be extracted from the aqueous fermentation reaction medium using an appropriate solvent (e.g., heptane) followed by fractional distillation.
  • the chemical composition of each fraction can be measured quantitatively by GC/MS (gas chromatography mass spectrometry).
  • Fractions can be blended to generate the desired mogroside compounds (e.g. mogroside V and 1 1 -O-mogroside V) for use in flavour or other applications. Verification of acceptability of the final blended product can be carried out by direct comparison to a reference mogroside flavouring product (for example, an existing natural flavouring commercial product obtained from a commercial supplier).
  • a reference mogroside flavouring product for example, an existing natural flavouring commercial product obtained from a commercial supplier.
  • Luo Han Guo fruit extracts obtained from Blue California (Tomas, Collinso Santa Margarita, California) (extract 4), Azile LCC (Rolling Hills Est, California) (extracts 1 and 2) and Chr. Olesen Group (Gentofte, Denmark) (extract 3) were analysed to identify the compounds present in the extract.
  • Sample solutions of the extracts were prepared by dissolving 16.52 ⁇ g of the sample in 25.0 ml_ solvent (acetonitrile/water 20/80 v/v). From this solution 100 ⁇ _ were transferred into a HPLC vial and 900 ⁇ _ solvent was added (66.1 ppm solution). From the sample solution 10 ⁇ _ was transferred to a HPLC vial and 990 ⁇ - solvent was added (6.61 ppm solution). Both the 66.1 and 6.61 ppm solutions were injected twice on the LC-MS.
  • Eluted compounds were detected using liquid chromatography mass spectrometry (LC/MS).
  • the mass spectrometer was operating in ESI negative mode measuring 150 to 2000 Amu with a resolution of 70000.
  • Gas flow rates were sheath 60, aux 20 and sweep 3.
  • Capillary temperature and aux gas heater temperature were set on 380°C and 400°C respectively.
  • V sample solvent volume in litres
  • Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of 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 (about 45 wt% in extract 1 ).
  • Fraction 1 -10 and 19-22 have an off-taste, which is the character of Luo Han Guo fruit, no sweet enhancement impact.
  • the better sweetness enhancement effect was observed within fractions 12-17, which contains mainly mogrosides.
  • the lingering, dirty fermented note is more noticeable, such as fractions 14 and 15.
  • pure mogroside V has inherent lingering off-taste.
  • Fraction 12 was the cleanest sweet, but less upfront due to small percentage mogroside V.
  • Fraction 13 has better sweet quality, but slightly higher astringency.
  • 1 1 - O-mogroside V and mogroside V are the two major mogrosides in those two fractions, but with different ratio (F12 mogroside V:1 1 -O-mogroside V is 4:9 and F13 mogroside V:1 1 -O-mogroside V is 13:3).
  • Mogroside V was very sweet, judged to be 425 times sweeter than sucrose, while 1 1 - O-mogroside V is rated as 84 times sweeter than sucrose.
  • a Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) containing about 68 wt% mogrosides was fractionated and the composition of each fraction determined by the chromatography method described above in relation to Example 1 .
  • Table 3 shows the chemical composition of fractions 1 1 to 20 of the extract.
  • Table 4 shows the tasting results for fractions 12 to 15. The results are similar to the results obtained in Example 2.
  • numbing/irritating effects are stronger than "wet dog” fermented effects, typical in Luo Han Guo extract
  • Luo Fractions 14 and 15 have a sharp, Han Guo extract numbing, burn, which negatively effects potency and quality of sweet
  • Mogroside V and 1 1 -O-mogroside V were isolated from extracts of Luo Han Guo using Agilent 1 100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 ⁇ , 210 X 21 .4 mm), and combined to form solutions of various concentrations.
  • a sweet sensitive taste panel ranked the sweetness of solutions of a mixture of sweeteners ("Mixture 1 ”) in relation to sucrose solutions at a range of concentrations to determine sucrose equivalence.
  • Mixture 1 was a combination of fractions 1 1 to 18 of Example 2 and contained 8.16 wt% 1 1 -O-mogroside V and 61 .6 wt% mogroside V. The results are shown in Table 6 below.
  • mixture 1 has a sweetness below 1 % sucrose equivalence (as determined by seven panellists), which is accepted as "not intrinsically sweet” by FEMA. Therefore, mixture 1 is suitable for use as sweetness modifiers or blends at the indicated concentrations because it does not have any detectable sweetness at these levels.
  • a concentration of mixture 1 having an iso-sweet threshold close to 1 % (35 ppm) was selected and added to 5 % (w/v) sucrose solutions. This solution was then ranked against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. This was repeated for 45 ppm Luo Han Guo extract. The average score of each solution was determined. The results are shown in Table 7 below.
  • mixture 1 and Luo Han Guo extract act as sweetness enhancers since 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 various concentrations of mixture 1 was tested by an expert panel of three people (trained flavourists) in solutions containing 5 % sucrose and 0.03 % citric acid.
  • the taste was compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) and containing about 68 wt% mogrosides) (combined with the same 5 % sucrose and 0.03 % citric acid solution). The results are shown in Table 8.
  • mixture 1 provides a better sweet quality (less baggage, sweeter) than the Luo Han Guo extract.
  • Example 6
  • a sweet sensitive taste panel ranked the sweetness of solutions of various sweeteners (mogroside V, mogroside IV, siamenoside, neomogroside, 1 1 -O-mogroside V) in relation to sucrose solutions at a range of concentrations to determine sucrose equivalence.
  • the sweeteners were obtained using an Agilent 1 100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 ⁇ , 210 X 21 .4 mm). The results are shown in Tables 9 to 13. Table 9.
  • Concentrations of the tested sweeteners were selected with an iso-sweet threshold close to 1 % and added to 5 % (w/v) sucrose solutions. These solutions were then ranked against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. The average score of each solution was determined. The results are shown in Table14 below.
  • mogroside V acts as sweetness enhancers since 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.
  • Siamenoside was described as having "more sweet body, sweeter, rounder with a little more upfront and more round lasting sweet”.
  • Mogroside IV was described as having "good and similar sweetness as mogroside V.
  • Neomogroside was described as having "sweetness, but slightly bitter aftertaste”. The results for mogroside V are shown in Table 15.
  • the mogroside V, siamenoside, mogroside IV and neomogroside were each added to a solution containing 5 % sucrose and 0.03 % citric acid in a concentration of 25 ppm (mogroside V), 25 ppm (siamenoside), 30 ppm (mogroside IV) and 30 ppm (neomogroside) respectively.
  • test solutions were tasted by an expert panel of seven people. For various aspects of sweet taste (upfront sweet, overall sweet, lingering sweet, astringent, volatile off-taste), each panellist scored the test solutions in comparison to the base solution (solution of 5 % sucrose and 0.03 % citric acid).
  • the average score for each test solution for each taste aspect was calculated. The results are shown in Table 16 below.
  • the taste of mogroside V with and without 1 1 -O-mogroside V in various milk or yoghurt bases is evaluated as shown in Table 18.
  • the iso-sweet threshold for mogroside V in milk and yoghurt is also evaluated.
  • a milk base (2% fat) includes 2% fat milk and 5 % sucrose.
  • a non-fat yoghurt base includes plain non-fat yoghurt and 5 % sucrose.
  • a full fat yoghurt base includes plain full fat yoghurt and 5 % sucrose.
  • a higher dose level is used for milk and yoghurt compositions due to the fat, protein and other ingredients. Luo Han Guo extract is used at 75 ppm for these applications.
  • Siraitia grosvenorii Swingle C. Jeffrey ex Lu et Z. Y. Zhang is a herbaceous perennial vine of Cucurbitaceae family endemic to southern China and northern Thailand.
  • the fruit of S. grosvenorii commonly known as 'luo han guo' has been used for traditional medicine in China for centuries as a treatment of respiratory infection, bronchitis, gastritis, constipation etc.
  • Modern pharmacological research have confirmed that S. grosvenorii extracts or its components possess variety of bioactivities, such as antibacterial, anti-inflammation, anti-diabetic, anti-cancer, and immunostimulatory [1 ].
  • Luo Han Guo has been used as a sweetener in food and beverages in China.
  • Optical rotations were measure with a Rudolph Autopol IV polarimeter.
  • the NMR spectra were recorded on Bruker DRX Avance 300 or 500 spectrometers. Chemical shifts are given in ⁇ (ppm) referring to the residual solvent peak.
  • Low pressure chromatography was performed on Biotage Flash System SP1 .
  • Preparative HPLC was performed on an Agilent 1 100 preparative HPLC system with a Phenomenex Lunar C18(2) column (5 ⁇ , 210 x 21 .4 mm) or a TSKgel Amide-80 (5 ⁇ , 300 x 21 .5 mm) (Tosoh Bioscience LLC).
  • Analytical HPLC was performed on an Agilent 1 100 analytical HPLC system equipped with ESA Corona CAD detector.
  • LC-MS was performed using Waters Q-Tof micro mass spectrometer coupled with Waters 2795 separation module.
  • the Luo Han Guo extract (commercial name Swingle, -60% mogrosides) was purchased from Blue California Co., Ltd.
  • Sample was eluted from the LC column using the following linear gradient (curve number 6): 0-40min: 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 re-equilibration.
  • the U-HPLC 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 was operated in the positive electrospray ionization mode (EST).
  • the sample cone voltage 40, capillary voltage 0.7 kv, source temperature 40 5 C, desolvation temperature 450 5 C, desolvation gas flow 800 L/h, and cone gas flow 50 L/h were optimized.
  • Leucine enkephalin was used as the lock mass [M+H] " at m/ z 556.2771 .
  • Sodium formate solution was used for external instrument calibration.
  • Fractions 36-38 with iso-mogroside VI and 1 1 -epi-mogroside V were combined to evaporate solvents. Further preparative HPLC purification of fractions 36- 38 afforded iso-mogroside VI (1 , 22 mg) and 1 1 -epi-mogroside V (2, 17 mg) (24% acetonitrile in water, 10 mL/min, retention time 13.1 min and 14.3 min, respectively).
  • 1 1 -oxo-mogroside V (4) and neomogroside (3) were mainly in flash fractions 39-40 with 1 1 -oxo-mogroside V as the major component.
  • neomogroside appeared as a tail shoulder of 1 1 -oxo-mogroside (24% acetonitrile in water, 10 mL/min, retention time 17.0 min and 18.0 min, respectively). Collection of the peak front gave 105 mg of the compound 1 1 -oxo-mogroside V (4). Further purification of the shoulder neomogroside (3, 15 mg) was achieved by preparative HPLC on TSKgel Amide-80 (65% acetonitrile in water, 20 mL/min, rt 15.5 min).
  • the n-hexane extract was analyzed by GC-MS under the following conditions: capillary column HP-5MS (30 m ⁇ 0.25 mm ⁇ 0.25 ⁇ , Agilent); column temperature, 180 to 230 ⁇ at a ramp of 5 ⁇ C/min ; injection tern perature, 250 ⁇ ; carrier, He gas; split ratio, 20:1 .
  • the O-silylated derivatives of D-glucose and L-glucose showed retention time at 16.02 and 16.39 min, respectively. By comparing the retention time and co-chromatography, the sugar residues after acid hydrolysis of 1 and 2 were determined to be D-glucose.
  • the NMR spectral data of 1 suggested the structure of a hexasaccharide triterpenoid saponin: 30 of the 66 carbons were assigned to the triterpenoid aglycone, and 36 of 66 to six hexose moieties.
  • the 13 C and 1 H NMR spectra of 1 showed the signals of seven singlet tertiary methyls, a doublet secondary methyl, and an olefinic methine (Table 19), which suggested a typical (24R)-cucurbit-5-ene-33,1 1 a,24,25-tetraol mogrol aglycone.
  • the mogrol aglycone of 1 was further confirmed by extensive analysis of its 1 H, 13 C, and 2D (COSY, TOCSY, HSQC and NOESY) NMR data, as well as comparison with NMR data of mogroside V standard.
  • the 13 C signals of Glc-I (5 C 75.1 , 77.8, 71 .4, 77.2, 70.1 ) as determined by HSQC-TOCSY missed a typical C-6 carbon signal at around 5 C 62.
  • the downfield shift of Glc-I C-6 (5 C 70.1 ) indicated glycosylation at this position.
  • the 13 C pattern of Glc-lll (5c 81 .6, 78.3, 71 .4, 76.4, 70.0) suggested C-2 and C-6 glycosylation shifts.
  • Analysis of HSQC-TOCSY with 10, 30, 60, and 100 ms mixing time resulted in the sequential assignment and confirmation of C-2 and C-6 downfield shifts.
  • Glc-IV was a regular terminal glucopyranosyl without any substitution (5 C 74.5, 77.7, 71 .2, 78.2, 62.4).
  • Glc-VI was a terminal glucopyranosyl without further sugar branch.
  • iso-mogroside (1 ) was assigned as 3- ⁇ - ⁇ - D-glucopyranosyl(1 ⁇ 6)-3-D-glucopyranosyl-mogrol-24-0-3-D-glucopyranosyl-(1 ⁇ 6- [3-D-glucopyranosyl-(1 ⁇ 4)-B-D-glucopyranosyl-(1 ⁇ 2)]-B-D-glucopyranosyl.
  • Compound 2 was assigned a molecular formula of C6oH 102 029 from its HR-ESI-MS data ([M-H] " m/z, 1285.6429).
  • the NMR data of the oligosaccharide portion of 2 were superimposable with those of mogroside V.
  • Detailed 2-D NMR experiments including HSQC, HMBC, NOESY, COSY and HSQC-TOCSY confirmed that 2 had the same sugar moieties as mogroside V. Attentions were then turned onto the aglycone NMR data.
  • the HMBC correlation between C-1 1 and H 3 -19 revealed significant upfield shift of C-1 1 (5 C 72.5) as compared with mogroside V (5 C 77.8).
  • the 13 C NMR data of compound 2 aglycone had a good match with the data of the semi-synthetic 1 1 - ⁇ - ⁇ cucurbitane glycoside, which was recorded in pyridine-d 6 [14].
  • the 13 C NMR data of natural 1 1 - ⁇ - ⁇ cucurbitane by Matsuda et al was obtained in methanol-c/4 and were quite different in terms of chemical shifts at C-1 1 , C-8, C-10 and C-12 [15].
  • semi-synthesis of 2 was carried out by chemical reduction of 1 1 -oxo-mogroside V (4) to the 1 1 - ⁇ - ⁇ and 1 1 -a-OH isomers of mogroside V.
  • the semisynthetic 1 1 -epi-mogroside V was determined to be identical to the isolated 1 1 -epi- mogroside V. Therefore, the structure of 1 1 -epi-mogroside V (2) was elucidated as 3- ⁇ - ⁇ -D-glucopyranosy I ⁇ 6) ⁇ -D-glucopyranosyl-1 1 ⁇ - ⁇ - ⁇ -24-0- ⁇ - ⁇ - glucopyranosyl-(1 ⁇ 2)-[B-D-glucopyranosyl-(1 ⁇ 6)]-3-D-glucopyranosyl. To our best of knowledge, this is the first report of natural mogroside with a 1 1 - ⁇ hydroxyl group.
  • the chemical shifts of the carbon signals as determined by HSQC/HMQC are very important information to confirm the monosaccharide type since the pattern of C-1 to C-6 chemical shifts for different type of monosaccharides is characteristic and consistent.
  • the glycosylation position on the sugar chain can be identified and further confirmed by HMBC correlation.
  • traditional way to elucidate saponin sugar is: HMBC ⁇ C-1 , H-1 ⁇ COSY ⁇ H-2 to H-6 ⁇ HSQC/HMQC ⁇ C-2 to C-6, then assisted and confirmed by coupling constant analysis and NOESY experiment.
  • TOCSY experiment not only can be used to group proton signals into spin systems, but also provide the stereochemistry information of the saccharide. For example, we should be able to see the magnetization relay of glucose through H-1 to H-6 with the right mixing time. For galactose, there is no magnetization relay over H-4 even with 200 ms mixing time.
  • HSQC-TOCSY have been applied in the structure elucidation and assignment of saponins by grouping carbon signals in each spin system together [17, 18].
  • Figure 6 summarized the new HSQC-TOCSY based strategy to elucidate the glucopyranosyl oligosaccharide chain of mogrosides as follows:
  • HMBC Heteronuclear multiple bond correlation spectroscopy
  • Step 2 HSQC-TOCSY was used with 100ms mixing time to determine the whole group of C-2 to C-6.
  • Step 3 if a C-2 downshift from -575 to -581 , C-4 downshift from -571 to -581 or C-6 downshift from -562 to -69 is observed, check HMBC for glycosylation at these positions.
  • HMBC glycosylation at these positions.
  • 1 -D NMR data such as 1 H coupling constants and 13 C carbon signal pattern
  • 2-D NMR experiments such as NOESY, HMBC, TOCSY, COSY and HSQC could assist the process and confirm the results.
  • the new HSQC- TOCSY based strategy may provide a simple, fast and unambiguous way for elucidation and assignment of glucopyranosyl chains of any new or known mogrosides. The strategy can also be adapted for elucidation and assignment of other monosaccharides and oligosaccharides.
  • Compound 3 was determined to be neomogroside by extensive 1 -D and 2-D NMR analysis, as well as comparison with literature data [19].
  • signals were assigned by HSQC-TOCSY and TOCSY experiments with different mixing time at 10, 30, 60, 100 ms.
  • the linkage of the six saccharides were made by their NOESY and HMBC correlations.
  • the oligosaccharide chain on C-3 of aglycone can be clearly assigned as 3-D-glucopyranosyl-(1 ⁇ 2)-B-D-glucopyranosyl- (1 ⁇ 6)-3-D-glucopyranosyl.
  • the glucopyranosyl on C-24 of aglycone was branched with a 3-D-glucopyranosyl-(1 ⁇ 2) and a 3-D-glucopyranosyl-(1 ⁇ 6).
  • Neomogroside was firstly discovered from S. grosvenorii and described by Si et al.
  • neomogroside and mogroside VI had the same CAS number 189307-15-1 and same structure.
  • mogroside VI Takemoto et al firstly reported mogroside VI from S. grosvenorii [2]. But it only referred a pure mogroside with a molecular formula of C 6 6H 1 12 0 34 and no structure was determined [2].
  • Prakash et al. reported the structure and NMR data of mogroside VI as a known compound in their article published in 201 1 [6]. In their article, the structure of mogroside VI was assigned as the structure of 6 in Figure 2. Prakash mentioned that the structure elucidation of mogroside VI was made by NMR analysis and also by comparing with the literature values. However, no citation was given for the literature values.
  • Example 12 Four sweet sensitive panelists evaluated 100 ppm iso-mogroside VI and 374 ppm 1 1 - epi-mogroside V and the sucrose standards, and were asked to give sweet equivalence concentrations to sucrose. The mean sweet equivalence concentrations of each compound were used to calculate the iso-sweet potency. The iso-sweet potency values of iso-mogroside VI and 1 1 -epi-mogroside V were determined as 91 and 35 times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet equivalent to 0.91 % sucrose; 374 ppm 1 1 -epi-mogroside V sweet equivalent to 1 .31 % sucrose). Example 12
  • Iso-Mogroside VI and 1 1 -epi-mogroside V were obtained as described in Example 10.
  • the iso-Mogroside VI and 1 1 -epi-mogroside V were each added to a solution containing 5 % sucrose and 0.03 % citric acid in a concentration of 25 ppm.
  • These test solutions were tasted by an expert panel of seven people.
  • sweet taste overall sweet, upfront sweet, lingering sweet, astringent, volatile off-note
  • each panellist scored the test solutions in comparison to the base solution (solution of 5 % sucrose and 0.03 % citric acid). A score of 0 indicated that the taste aspect was the same, 1 indicates slightly higher, 2 indicates higher, 3 indicates much higher, -1 indicates slightly lower, -2 indicates lower and -3 indicates much lower.
  • the sweet potency of iso-mogroside VI and 1 1 -epi-mogroside V were evaluated as 91 and 35 times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet equivalent to 0.91 % sucrose; 374 ppm 1 1 -epi-mogroside V sweet equivalent to 1 .31 % sucrose).
  • the new strategy based on HSQC-TOCSY experiments with different mixing times featured a quick and unambiguous elucidation and assignment of glucopyranosyl oligosaccharide chains.
  • the previous confusion regarding the structures of neomogroside and mogroside VI was reviewed and clarified after the confirmation of neomogroside structure by our extensive NMR spectral analysis.

Abstract

A sweetness modifying composition comprising at least one high-intensity sweetener chosen from steviol glycosides and/or mogrosides and at least one low-potency sweetener chosen from cellobiose, psicose, cyclamate and/or 11-O-mogroside V, and a sweetened composition comprising said sweetness modifying composition and at least one other sweetener; the uses of said sweetened compositions and sweetness modifying compositions; methods of making said sweetened compositions and sweetness modifying compositions.

Description

COMPOSITIONS
TECHNICAL FIELD The present invention relates generally to the use of one or more low-potency sweetener(s) to improve one or more sweetness characteristics of one or more high- intensity sweetener(s). The present invention thus also relates to compositions comprising a mixture of at least one high-intensity sweetener and at least one low- potency sweetener. The present invention further 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 wh+en used in combination with at least one other sweetener and/or as a sweetener. The present invention further relates to the use of one or more mogroside(s) as a sweetness enhancer in sweetened compositions and said sweetened compositions. The present invention further relates to methods of making the sweeteners and compositions disclosed herein.
BACKGROUND
Sweetness in comestible products, that is products intended to be taken by mouth either for permanent ingestion or temporarily for expectoration, is often a desirable characteristic. Traditionally, sweetness has been provided by the addition of one or more sweeteners, particularly low-potency, nutritive sweeteners such as sucrose (table sugar), fructose, glucose, xylose, arabinose, rhamnose, sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol as well as sugar syrups such as high fructose corn syrup and starch syrup. These deliver considerable sweetness without any undesirable aftertaste. However, it is desirable to use a reduced amounts of these sweeteners to reduce the caloric value of the comestible product. It is therefore desirable to provide alternative sweeteners that can reduce the caloric value of the comestible product whilst maintaining the same or a similar sweetness taste.
High-intensity sweeteners (HIS) have been used for this purpose. High-intensity sweeteners may be natural or artificial and have a sweetness that can be several hundred times that of sucrose and thus can theoretically replace a much larger quantity of sugar in a composition. Examples of high-intensity sweeteners include sucralose, saccharin, aspartame, acesulfame potassium (AceK), neotame, advantame, sterviol glycosides, including stevioside, rebaudioside A, rebaudioside D or steviol glycoside mixture preparations with rebaudioside A and/or stevioside as predominant components. However, these substances generally have the drawback that they may impart undesirable off-tastes to comestible products, typically bitter, metallic or liquorice tastes, or an undesirable lingering sweetness.
It is therefore desirable to provide alternative and/or improved sweetness modifying composition and sweetened compositions to address one or more of these issues.
SUMMARY
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 1 1 -O-mogroside V;
wherein the sweetness modifying composition increases the sweetness of a sweetened composition by more than the sweetness of the sweetness modifying composition alone; and/or
wherein the ratio of the one or more high-intensity sweetener(s) to the 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 about 1 .5 % (w/v) sucrose equivalence; and
a sweetness modifying composition according to any aspect or embodiment of the present invention. In accordance with a third aspect of the present invention there is provided a use of one or more low-potency sweetener(s) selected from the group consisting of cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V to improve one or more sweetness characteristic(s) of a sweetened composition comprising one or more high- intensity sweetener(s) selected from the group consisting of steviol glycosides and/or mogrosides, wherein the total concentration of the one or more low-potency sweetener(s) and the one or more high-potency sweetener(s) that is used has a sweetness of less than 1 .5 % (w/v) sucrose equivalence.
In accordance with a fourth aspect of the present invention there is provided a method of enhancing the sweetness of a sweetened composition, the method comprising providing a base composition comprising at least one sweetener present in an amount at or above its sweetness recognition threshold and/or having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence, and adding 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 1 1 -O-mogroside V, wherein the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 2:1 to about 12:1 ; and/or wherein the 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 the one or more low- potency sweetener(s) are added in a total amount equal to or greater than about 2 ppm and optionally equal to or less than about 12 ppm; and/or wherein the total concentration of the one or more high-intensity sweetener(s) and the one or more low- potency sweetener(s) that is added has a sweetness less than 1 .5 % (w/v) sucrose equivalence.
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, the method 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, the method comprising combining the base composition, one or more high-intensity sweetener(s), one or more low-intensity sweetener(s) and at least one other sweetener.
In accordance with 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 1 .5 % (w/v) sucrose equivalence; 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 a use of one or more of mogroside IV, siamenoside and neomogroside to enhance the sweetness of a sweetened composition. Thus, in a further aspect there is provided a method for enhancing the sweetness of a sweetened composition, the method 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, the method comprising combining the 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). The one or more mogroside(s) may, for example, be present as a sweetness enhancer and thus be present in an amount having a sweetness of less than 1 .5 % (w/v) sucrose equivalence. The sweetened composition will then further comprise at least one sweetener present in an amount having a sweetness equal to or greater than 1 .5 % (w/v) sucrose equivalence.
In accordance with an eleventh aspect of the present invention there is provided a use of one or more mogroside(s) to enhance the sweetness of a sweetened composition. Thus, there is provided a method for enhancing the sweetness of a sweetened composition, the method comprising providing 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, the method comprising combining the base composition, one or more mogroside(s) and at least one other sweetener. In certain embodiments of any aspect of the present invention the one or more high-intensity sweetener may include or be mogroside V and/or the one or more low-potency sweetener may include or be 1 1 -O-mogroside V. In certain embodiments of any aspect of the present invention the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 2:1 to about 12:1 . In certain embodiments of any aspect of the present invention the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 5:1 to about 12:1 . In certain embodiments of any aspect of the present invention the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may be from about 6:1 to about 10:1 .
In certain embodiments of any aspect of the present invention the one or more high-intensity sweetener(s) may be present in a total amount ranging from about 15 ppm to about 30 ppm and/or the one or more low-potency sweetener(s) may be present in a total amount ranging from about 2 ppm to about 10 ppm. In certain embodiments of any aspect of the present invention the one or more high-intensity sweetener(s) may be present in a total amount ranging from about 22 ppm to about 28 ppm and/or the one or more low-potency sweetener(s) may be present in a total amount ranging from about 2 ppm to about 5 ppm.
In certain embodiments of the seventh to twelfth aspect of the present invention, the one or more mogroside(s) or one or more sweetness enhancer(s) may be present in an amount ranging from about 15 ppm to about 50 ppm. In certain embodiments, the one or more mogroside(s) or one or more sweetness enhancer(s) may be present in an amount ranging from about 15 ppm to about 35 ppm.
In certain embodiments of any aspect of the present invention the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) alone may have a sweetness less than about 1 .5 % (w/v) sucrose equivalence. In particular, the concentration of the one or more high-intensity sweetener(s) and the one or more low-intensity sweetener(s) in a sweetened composition may have a sweetness less than about 1 .5 % (w/v) sucrose equivalence. In certain embodiments of the seventh to twelfth aspect of the present invention, the one or more mogroside(s) or one or more sweetness enhancer(s) may have a total sweetness less than about 1 .5 % (w/v) sucrose equivalence. In certain embodiments, the one or more sweetness enhancer(s) increase the sweetness of a sweetened composition by more than the total sweetness of the one or more sweetness enhancer(s) alone.
In certain embodiments of any aspect of the present invention the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) may increase the sweetness of a sweetened composition by more than the sweetness of the combination alone. In certain embodiments of any aspect of the present invention the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) may increase the sweetness of a composition by equal to or greater than about 1 .25 % (w/v) sucrose equivalence.
In certain embodiments of any aspect of the present invention the one or more low-potency sweetener(s) weaken the lingering sweet taste of a sweetened composition comprising the one or more high-intensity sweetener(s) compared to the lingering sweet taste of the sweetened composition in the complete absence of the one or more low-potency sweetener(s). In certain embodiments of any aspect of the present invention the one or more low-potency sweetener(s) weakens the bitter and/or astringent taste of a sweetened composition comprising the one or more high-intensity sweetener(s) compared to the bitter and/or astringent taste of the sweetened composition in the complete absence of the one or more low-potency sweetener(s).
One or more (e.g. all) of the sweeteners used may be natural or synthetic (artificial). One or more of the sweeteners may, for example, be made by a biological process or by an enzymatic process or by a synthetic process. Certain embodiments of any aspect of the present invention may provide one or more of the following advantages:
• increased sweetness in a composition;
• enhanced sweetness in a composition including at least one sweetener;
· decrease in the amount of caloric sweetener required to obtain desired sweetness; • improvement of one or more sweetness characteristics to make sweet taste more similar to sugar (sucrose);
• weakening of lingering sweetness (e.g. decreasing the length of time the sweet taste remains and/or decreasing the intensity of the sweet taste more rapidly); · weakening of bitter taste and/or astringent taste and/or liquorice taste and/or metallic taste;
• improvement in sweetness impact (e.g. increasing the maximum intensity of the sweet taste and/or decreases the length of time for the sweet taste to be detected) (e.g. decreasing the lingering sweetness).
The details, examples and preferences provided in relation to any particulate one or more of the stated aspects of the present invention will be further described herein and apply equally to all aspects of the present invention. Any combination of the embodiments, examples and preferences described herein in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein, or otherwise clearly contradicted by context.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of Table 1 below). Figure 2 shows the chemical structures of mogrosides 1 -6; Figure 3 shows the LC-MS analysis of commercial Luo Han Guo extracts;
Figure 4 shows the Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy (HSQC-TOCSY) (hsqcgpmlph) of iso-mogroside VI with different mixing time (d9). A: 10 ms mixing time. B: 30 ms mixing time. C: 60 ms mixing time. D: 100 ms mixing time. Because of the overlap of H-1 of Glc II and H-6a of Glc III, HSQC-COSY correlation intensity of Glc II was not analyzed here;
Figure 5 shows HSQC-TOCSY (hsqcgpmlph) peak intensity quantification of iso- mogroside VI glucopyranosyls with different mixing time. (* C-3 and C-5 signals on HSQC-TOCSY appeared overlap for 100 ms mixing time. The total integration of C-3 and C-5 was therefore used in the bar chart); Figure 6 shows the strategy to elucidate mogroside sugar chain (*Numbers of C-2 to C- 6 appeared under certain mixing time might slightly change if adjusting peak intensity of HSQC-TOCSY. By observing the increasing intensity of C-2 to C-6 in different mixing time experiments connection sequence can be still determined. **There is no natural glycosylation on C-3 of mogroside glucopyranosyl so far. C-3 glycosylation on glucopyranosyl would cause the downshift from 576 to 581 and can be easily determined by HSQC-TOCSY experiments). The sequence of steps in Figure 6 can be outlined as follows: In Step 1 , Heteronuclear multiple bond correlation spectroscopy (HMBC) was used to determine anormeric C-1 and H-1 of the sugar. Start from the sugar link to aglycone. In Step 2, HSQC-TOCSY was used with 100ms mixing time to determine the whole group of C-2 to C-6. HSQC-COSY or HSQC-TOCSY (d9=10 ms) to assign C-2. HSQC- TOCSY (d9=30 ms) to assign C-3. HSQC-TOCSY (d9=60 ms) to assign C-4. HSQC- TOCSY (d9=100 ms) to assign C-5 and C-6. In Step 3, if a C-2 downshift from -575 to -581 , C-4 downshift from -571 to -581 or C-6 downshift from -562 to -69 is observed, check HMBC for glycosylation at these positions.**
If a C-2 downshift from -575 to -581 , C-4 downshift from -571 to -581 or C-6 downshift from -562 to -69, check HMBC for glycosylation at these positions.**
Figure 7 shows the chemical structure for iso-mogroside VI which has the chemical formula C66H1 12034 and an Exact Mass of 1448.70. This chemical structure is designated Formula I; and Figure 8 shows the chemical structure for 1 1 -epi-mogrosideV which has the chemical formula C60H1 02O29 and an Exact Mass of 1286.65. This chemical structure is designated Formula II. DETAILED DESCRIPTION
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. 1 1 -O-mogroside V) can act synergistically with at least one other sweetener (e.g. sucrose) to obtain a composition having a sweetness that is greater than the sum of the sweetness of the individual sweeteners. The present invention is further based on the surprising finding that one or more low-potency sweetener(s) may offset one or more negative sweetness characteristics of one or more high-potency sweetener(s). For example, the combination of one or more high-intensity sweetener(s) (e.g. mogroside V) and one or more low-potency sweetener(s) (e.g. 1 1 -O-mogroside V) may provide improved sweetness characteristics in a sweetened composition (i.e. a composition comprising at least one other sweetener such as sucrose in an amount above its sweetness recognition threshold and/or an amount equal to or greater than about 1 .5 % (w/v) sucrose equivalence) compared to using the one or more high-intensity sweetener(s) alone. The sweetness characteristics may thus, for example, be closer to the sweetness characteristics of sucrose.
Thus, there is provided herein various compositions comprising one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) as disclosed herein, particularly sweetened compositions comprising at least one sweetener in an amount above its sweetness recognition threshold and/or an amount equal to or greater than about 1 .5 % (w/v) sucrose equivalence and one or more high-intensity sweetener and one or more low-potency sweetener(s). The sweetened compositions may also be referred to as comestible compositions. There is also provided herein various uses of one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) as disclosed herein and methods of making the various compositions disclosed herein. The present invention is further based on the surprising finding that mogrosides 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 alone). Thus, there is provided herein various compositions, in particular sweetened compositions, comprising one or more of mogroside IV, siamenoside and neomogroside. Compositions
There is provided herein various compositions comprising at least one high-intensity sweetener and at least one low-potency sweetener. There is also provided herein compositions comprising one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside. In certain embodiments, the compositions are comestible compositions.
In certain 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 1 1 -O-mogroside V. In certain 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 may, for example, be a concentrate which may, for example, be diluted in a sweetened (e.g. comestible) composition to give the comestible composition a desired sweetness. The term "sweetened composition" refers to a composition comprising at least one sweetener present in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
In certain embodiments, there is provided a sweetened composition (e.g. comestible composition) comprising one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside. In certain embodiments, there is provided a sweetened composition (e.g. comestible composition) comprising at least one high-intensity sweetener and at least one low-potency sweetener. The combination of the high-intensity sweetener(s) and low-potency sweetener(s) may be referred to as a sweetness modifying composition. The one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may also be referred to herein as a sweetness modifying composition. Thus, in certain embodiments, there is provided a sweetened composition comprising at least one sweetener present in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence 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 may, for example, be a comestible composition.
The term "enhancing" 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 a sweetened composition by more than the sweetness of the sweetness modifying composition alone. In other words, the sweetness of a composition comprising at least one sweetener and at least one sweetness modifying composition is greater than the sum of the sweetness of all the sweeteners in the composition. The sweetness modifying compositions described herein are used in sweetened (e.g. comestible) compositions in amounts that have no detectable sweetness or no taste recognised as sweet (below its sweetness recognition threshold). Typically, a sweetness modifying composition with a sweetness below 1 .5 % (w/v) sucrose equivalence is accepted as being "not intrinsically sweet" by FEMA (Flavor & Extract Manufacturers Association). Sweetness modifiers may also be referred to as sweetness enhancers.
The sweetened composition comprising the sweetness modifying composition as disclosed herein and at least one sweetener in an amount above its sweetness recognition threshold and/or in an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may have a sweetness that is equal to or more than about 1 .0 % (w/v) sucrose equivalence greater than the sweetness of the sweetened composition in the absence of the sweetness modifying composition. For example, the sweetened composition may have a sweetness that is equal to or more than about 1 .1 % (w/v) or equal to or more than about 1 .15 % (w/v) or equal to or more than about 1 .2 % (w/v) or equal to or more than about 1 .25 % (w/v) sucrose equivalence greater than the sweetness of the sweetened composition in the absence of the sweetness modifying composition. In other words, the sweetness modifying composition may increase the sweetness of a sweetened composition by equal to or more than about 1 % (w/v) or equal to or more than about 1 .1 % (w/v) or equal to or more than about 1 .15 % (w/v) or equal to or more than about 1 .2 % (w/v) or equal to or more than about 1 .25 % (w/v) sucrose equivalence. The comparative composition is identical to the said composition except that it does not include the said sweetness modifying composition.
The term "sucrose equivalence" refers to the equivalence in sweetness of a composition containing at least one non-sucrose sweetener to a reference sucrose solution. Typically, taste panellists are trained to detect sweetness of reference sucrose solutions containing between 1 % and 15 % sucrose (w/v). Other non-sucrose sweeteners may then be tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet (i.e. isosweet) to a given sucrose reference. The term "isosweet" refers to compositions that have equivalent sweetness. Typically, the sweetness of a given composition is measured with reference to a solution of sucrose. See "A Systematic Study of Concentration-Response Relationships of Sweeteners," G.E. DuBois, D.E. Walters, S.S. Schiffman, Z.S. Warwick, B.J. Booth, S.D. Pecore, K. Gibes, B.T. Carr, and L.M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, D.E. Walters, FT. Orthoefer, and G.E. DuBois, Eds., American Chemical Society, Washington, DC (1991 ), pp 261 -276.
The combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) (e.g. the sweetness modifying composition) may, for example, have a sweetness less than about 1 .5 % (w/v) sucrose equivalence. For example, the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) (e.g. sweetness modifying composition) may have a sweetness equal to or less than about 1 .45 % (w/v) sucrose equivalence or equal to or less than about 1 .4 % (w/v) sucrose equivalence or equal to or less than about 1 .35 % (w/v) sucrose equivalence or equal to or less than about 1 .3 % (w/v) sucrose equivalence. For example, the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) (e.g. sweetness modifying composition) may have a sweetness equal to or greater than about 1 % (w/v) sucrose equivalence or equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence or equal to or greater than about 1 .3 % (w/v) sucrose equivalence.
The 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 about 1 .5 % (w/v) sucrose equivalence. For example, the 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 1 .45 % (w/v) sucrose equivalence or equal to or less than about 1 .4 % (w/v) sucrose equivalence or equal to or less than about 1 .35 % (w/v) sucrose equivalence or equal to or less than about 1 .3 % (w/v) sucrose equivalence. For example, the 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 about 1 % (w/v) sucrose equivalence or equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence or equal to or greater than about 1 .3 % (w/v) sucrose equivalence.
Each of the sweeteners and sweetness enhancers used in the compositions disclosed herein may be a natural or synthetic (artificial) sweetener. Examples of non-naturally occurring (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 obtained from nature, including mixtures that may have been enzymatically treated (e.g. glycosylated) to form compounds not found in nature (this does not include purified compounds that have been enzymatically treated). For example, a modified extract having a mogrol glycoside distribution that is different (e.g. enhanced) from the naturally occurring mogrol glycoside distribution may be classed as natural. For example, a mixture of glucosylated steviol glycosides and/or glucosylated mogrosides may be classed as natural. Each of the sweeteners used in the compositions disclosed herein may be food-derived. A "food-derived" product refers to a product which is prepared under typical cooking conditions such as, for example, using temperatures similar to those used in cooking methods. In certain embodiments, the high-intensity sweetener and the low-potency sweetener used in the compositions disclosed herein (e.g. in the sweetness modifying composition disclosed herein) are both natural sweeteners. In certain embodiments, all of the sweeteners used in the compositions disclosed herein are natural.
The sweeteners disclosed herein may be used in pure or purified form and may be chemically synthesised, produced by biotechnological processes (e.g. fermentation) or isolated from a natural source (e.g. a botanical source including, without limitation, fruits, sugar cane, sugar beet). The one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be at least 80 wt% pure. For example, the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may be at least about 85 wt% or at least about 90 wt% or at least about 95 wt% or at least about 98 wt% or at least about 99 wt% pure. For example, the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may be up to 100 wt% or up to 99 wt% pure. The term "high-intensity sweetener" refers to compounds having a sweetness that is at least 100 times the sweetness of sucrose. In certain 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 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 may, for example, have a sweetness that is up to 1000 times the sweetness of sucrose. Although the high-intensity sweetener has a sweetness that is at least 100 times the sweetness of sucrose, in the context of its use in a sweetness modifying composition as described herein, they will be used in a sweetened composition in an amount that does not have any detectable sweetness or be recognised as sweet (amounts providing a sweetness less than 1 .5 % (w/v) sucrose equivalence, which is accepted as being "not intrinsically sweet" by FEMA.
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, the one or more high-intensity sweetener may be a mixture of steviol glycosides and mogrosides. For example, the one or more high-intensity sweeteners may be one or more steviol glycosides. For example, the one or more high-intensity sweetener(s) may be one or more mogrosides. In certain embodiments, mogrosides may perform better than steviol glycosides in terms of sweetness enhancement and off-note reduction (e.g. weakening of lingering sweet after taste). 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 may, for example, be one or more mogroside(s). In certain embodiments, the high-intensity sweetener may be one or more of the mogrosides listed herein. In certain embodiments, the high-intensity sweetener may be one or more of mogroside IV, siamenoside, neomogroside and mogroside V (including all isomers thereof). For example, the high-intensity sweetener may be a mixture of mogroside IV, siamenoside and mogroside V (including all isomers thereof). The one or more mogroside(s) may, for example, be obtained or obtainable from Luo Han Guo fruit extracts.
The term "low-potency sweetener" refers to compounds having a sweetness that is less than 100 times the sweetness of sucrose. In certain 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 of cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V (CAS: 126105-1 1 -1 ). For example, the one or more low-intensity sweetener(s) may be one or more of cellobiose, psicose and 1 1 -O-mogroside V.
In certain embodiments, the one or more high-intensity sweetener(s) includes or is a high-intensity mogroside. In certain embodiments, the one or more low-potency sweetener(s) includes or is a low-potency mogroside. In certain embodiments, the one or more high-intensity sweetener includes or is a high-intensity mogroside and the one or more low-potency sweetener(s) includes or is a low-potency mogroside. In certain embodiments, the one or more high-intensity sweetener includes or is mogroside V. In certain embodiments, the one or more low-potency sweetener includes or is 1 1 -O-mogroside V. In certain embodiments, the one or more high-intensity sweetener includes or is mogroside V and the one or more low-potency sweetener includes or is 1 1 -O-mogroside V. Mogrosides are a group of triterpene glycosides and may be obtained from the fruit Luo Han Guo (Siraitia grosvenorii), also known as arhat fruit or longevity fruit or swingle fruit. Mogrosides make up approximately 1 % of the flesh of the fresh fruit. Through extraction, an extract in the form of a powder containing up to 80% mogrosides can be obtained. Mogroside extract contains grosvenorine II, grosvenorine I, 1 1 -O-mogroside II (I), 1 1 -O-mogroside II (II), 1 1 -O-mogroside II (III), mogroside II (I), mogroside II (II), mogroside II (III), 1 1 -dehydroxy-mogroside III, 1 1 -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), 1 1 -O-mogroside V (I), mogroside V isomer, mogroside V, iso-mogroside V, 7-O-mogroside V, 1 1 -O-mogroside VI, mogroside VI (I), mogroside VI (II), mogroside VI (III) (neomogroside) and mogroside VI (IV). The precise amount of mogroside V may vary depending on the ripeness of the fruit and/or extraction process used.
Mogroside(s) include both mogroside(s) that occur in nature and mogrosides that do not occur in nature. Examples of mogrosides include, for example, grosvenorine II, grosvenorine I, 1 1 -O-mogroside II (I), 1 1 -O-mogroside II (II), 1 1 -O-mogroside II (III), mogroside II (I), mogroside II (II), mogroside II (III), 1 1 -dehydroxy-mogroside III, 1 1 -O-mogroside III, mogroside III (I), mogroside III (II), mogroside llle, mogroside Mix, mogroside IV (I) (siamenoside), mogroside IV (II), mogroside IV (III), mogroside IV (IV), deoxymogroside V (I), deoxymogroside V (II), 1 1 -O-mogroside V (I), mogroside V isomer, mogroside V, iso-mogroside V, 7-O-mogroside V, 1 1 -O-mogroside VI, mogroside VI (I), mogroside VI (II), mogroside VI (III) (neomogroside) and mogroside VI (IV). The mogroside(s) may, for example, be obtained or obtainable from Luo Han Guo extracts.
Mogroside V (CAS: 88901 -36-4) is a glycoside of a cucurbitane derivative and has the chemical formula C-60H102O29 and the chemical structure shown below. Mogroside V can be found in certain plant extracts such as extracts from the fruit Luo Han Guo (Siraitia grosvenorii). Pure mogroside V has been found to have a sweetness of at least 400 times the sweetness of sucrose.
Figure imgf000018_0001
Siamenoside (CAS: 126105-12-2) is a cucurbitane found in the fruit of Siraitia grosvenorii and has the following chemical structure.
Figure imgf000018_0002
Neomogroside (CAS: 189307-15-1 ) is a cucurbitane glycoside also found in the fruit of Siraitia grosvenorii and has the following chemical structure.
Figure imgf000019_0001
1 1 -O-Mogroside V (CAS: 126105-1 1 -1 ) is derived from mogroside V and has the following chemical structure. It is also found in plant extracts such as extracts from the fruit Luo Han Guo (Siraitia grosvenorii). 1 1 -O-mogroside V has been found to have a sweetness that is about 84 times the sweetness of sucrose.
Figure imgf000019_0002
The ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is equal to or greater than about 2:1 . For example, the ratio of the one or more high-intensity sweetener(s) to the 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 to or greater than about 5:1 or equal to or greater than about 5.5:1 or equal to 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 to or greater than about 8:1 . The ratio of the high-intensity sweetener to the low-potency sweetener is equal to or less than about 12:1 . For example, the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may be equal to or less than about 1 1 .5:1 or equal to or less than about 1 1 :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 the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) may range from about 5:1 to about 1 1 :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 certain embodiments, the ratio of the one or more high-intensity sweetener(s) to the 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 may be weight or volume ratio. The ratio only applies to the high-intensity sweetener(s) and low-potency sweeteners in the sweetness modifying composition (high-intensity and low-potency sweeteners that are used in a sweetened composition in an amount below the sweetness recognition threshold or having less than 1 .5 % (w/v) sucrose equivalence).
The one or more high-intensity sweetener(s) may be present in a composition in a total amount equal to or greater than about 15 ppm. For example, the one or more high-intensity sweetener(s) may be present in a 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, the one or more high-intensity sweetener(s) may be present in a 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, the one or more high-intensity sweetener(s) may be present in a composition in a total amount ranging from about 15 ppm to about 50 ppm or from about 15 ppm to about 45 ppm or from about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm or from about 15 ppm to about 30 ppm. For example, the one or more high-intensity sweetener(s) may be present in a composition in a total amount ranging from about 15 ppm to about 30 ppm or from about 20 ppm 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 to about 26 ppm. For example, the one or more high-intensity sweetener(s) may be present in a composition in a total amount of about 20 ppm or about 25 ppm. The composition may, for example, be a sweetened composition comprising at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or equal to or greater than about 1 .5 % (w/v) sucrose equivalence. The one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or greater than about 2 ppm. For example, the one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or greater than about 3 ppm. For example, the one or more low-potency sweetener(s) may be present in a composition in a total amount equal to or less than about 12 ppm or equal to or less than about 1 1 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, the one or more low-potency sweetener(s) may be present in a composition in a total amount ranging from about 2 ppm to about 12 ppm or from about 2 ppm to about 10 ppm or from about 2 ppm to about 5 ppm, for example in a total amount of about 3 ppm. The composition may, for example, comprise at least one sweetener other than the combination of the high-intensity sweetener and the low-potency sweetener (e.g. sweetness modifying composition) as disclosed herein. The concentration ranges may, for example, be particularly suitable for liquid compositions such as beverages or compositions that do not comprise any proteins or fats. In compositions having a base such as milk and yogurt or other compositions that do comprise proteins and fats, higher concentrations of the one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) may be used. For example, concentrations that are about 1 .5 times higher than the concentrations used for liquid compositions or compositions that do not comprise any proteins or fats may be used. For example, concentrations that are from about 1 .5 times to about 3 times higher than the concentrations used for liquid compositions or compositions that do not comprise any proteins or fats may be used. Therefore, for example, the one or more high-intensity sweetener(s) may be present in a composition (e.g. a composition having a base such as milk and yoghurt or other compositions that comprise 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 to about 66 ppm or from about 30 ppm to about 65 ppm or from about 30 ppm to about 60 ppm or from about 30 ppm to about 55 ppm or from about 30 ppm to about 50 ppm or from about 30 ppm to about 45 ppm.
Therefore, for example, the one or more low-potency sweetener(s) may be present in a composition (e.g. a composition having a base such as milk and yoghurt or other compositions that comprise proteins and/or fats) in a total amount ranging from about 3 ppm 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 to about 15 ppm or from about 6 ppm to about 15 ppm. In certain embodiments, a sweetened composition comprises at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or equal to or greater than about 1 .5 % (w/v) sucrose equivalence and a sweetness modifying composition consisting of 15 ppm to about 50 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 12 ppm of one or more low-potency sweetener(s) as described herein. In certain embodiments, a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 15 ppm to about 30 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 10 ppm of one or more low-potency sweetener(s) as described herein. In certain embodiments, a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 20 ppm to about 30 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 10 ppm of one or more low-potency sweetener(s) as described herein. In certain embodiments, a comestible composition comprises at least one sweetener and a sweetness modifying composition consisting of 22 ppm to about 28 ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm to 5 ppm of one or more low-potency sweetener(s) as described herein. In certain embodiments, the high- intensity sweetener is mogroside V. In certain embodiments, the low-potency sweetener is 1 1 -O-mogroside V. The one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be present in a sweetened composition in a total amount equal to or greater than about 15 ppm. For example, the one or more mogroside(s), for example the one or more of mogroside IV, siameonside and neomogroside may, for example, be present in a 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, the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may, for example, be present in a sweetened composition in a total amount equal to or less than about 50 ppm, for example equal to or less than about 45 ppm, for example equal to or less than about 40 ppm, for example equal to or less than about 35 ppm. For example, the one or more mogroside(s), for example the one or more of mogroside IV, siamenoside and neomogroside may be present in a sweetened composition in a total amount ranging from about 15 ppm to about 50 ppm or from about 15 ppm to about 45 ppm or from about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm or from about 20 ppm to about 35 ppm or from about 20 ppm to about 30 ppm. The term "ppm" refers to part(s) per million by weight, for example the weight of a compound, such as Mogroside V (in milligrams) per kilogram of the product containing such compound (i.e. mg/Kg) or the weight of a compound (e.g. orally consumable/comestible product of the present disclosure), such as Mogroside V (in milligrams) per litre of the product containing such compound (i.e. mg/L) or by volume, for example, the volume of a compound, such as Mogroside V (in millilitres) per litre of the product containing such compound (i.e. ml/L).
The sweetness modifying compositions described herein may, for example, comprise higher concentrations of the high-intensity and low-intensity sweeteners and are then diluted in a sweetened composition to obtain the concentrations recited herein.
A sweetened composition comprises at least one sweetener in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence. The term "sweetness recognition threshold" refers to the lowest known concentration of a compound that is perceivable as sweet by the human sense of taste. A sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence is accepted as being "intrinsically sweet" by FEMA.
The at least one sweetener may be nutritive or non-nutritive. Nutritive sweeteners add caloric value to the foods that contain them while non-nutritive sweeteners are very low in calories or contain no calories at all. Aspartame, the only approved nutritive high- intensity sweetener contains more than 2 % of the calories in an equivalent amount of sugar as opposed to non-nutritive sweeteners that contain less than 2 % of the calories in an equivalent amount of sugar.
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 (AceK), aspartame, steviol glycoside(s), sucralose, high-fructose corn syrup, starch syrup, saccharin, sucralose, neotame, advantame, Luo Han Guo extract, neohespiridin, dihydrochalcone, naringin dihydrochalcone, neohesperidin dihydrochalcone, rubusoside, rebaudioside A, stevioside, stevia, trilobtain and sugar alcohols such as erythritol, xylitol, mannitol, sorbitol and inositol. Examples of sweeteners that may be used in the 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 consumables using sweeteners in a sufficient amount is well-known in the art. Depending on the consumable, the amount of sweetener can be reduced by addition of a sweetness modifying composition as disclosed herein. For example, a reduction of about 10 to about 4° Brix or more may be achieved.
The at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may, for example, be used in a sweetened composition in an amount equal to or greater than about 0.01 % (w/v). For example, the at least one other sweetener may be used in a 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, the at least one other sweetener may be used in a comestible 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 than 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).
The at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence may be used in the sweetened compositions disclosed herein (e.g. comestible composition) in amounts isosweet to about 2 % (w/v) to about 15 % (w/v) sucrose.
In certain embodiments, there is provided herein a sweetness modifying composition consisting of mogroside V and 1 1 -O-mogroside V in a ratio ranging from about 2:1 to about 12:1 , for example from about 6:1 to about 10:1. This sweetness modifying composition may, for example, be used as sweetness enhancer or modifier in a comestible composition. The comestible composition may, for example, comprise at least one other sweetener such as sucrose. The mogroside V may, for example, be used in the comestible composition in an amount ranging from about 15 ppm to about 30 ppm or from about 20 ppm to about 30 ppm (e.g. about 20 ppm or about 25 ppm). The 1 1 -O-mogroside V may be used in the comestible composition in an amount ranging from about 2 ppm to about 12 ppm or from about 2 ppm to about 10 ppm (e.g. about 8.5 ppm or about 3 ppm). The at least one other sweetener may, for example, be present in the comestible composition in an amount isosweet to about 2 % (w/v) to about 15 % (w/v) sucrose.
The compositions may be in any suitable form, for example solid (e.g. powder, granules, tablets) or in solution (e.g. aqueous solution) or in an emulsion or in a suspension. For example, the compositions may further comprise a diluent or bulking agent such as dietary fibre.
Comestible compositions as disclosed herein include, for example, the following. - Wet/liquid soups regardless of concentration or container, including frozen soups. For the purpose of this definition soup(s) means a food prepared from meat, poultry, fish, vegetables, grains, fruit and other ingredients, cooked in a liquid which may include visible pieces of some or all of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth, pureed or chunky, ready-to-serve, semi-condensed or condensed and may be served hot or cold, as a first course or as the main course of a meal or as a between meal snack (sipped like a beverage). Soup may be used as an ingredient for preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups).
- Dehydrated and culinary foods, including cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology). - Meal solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes.
- Meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salad, sold as a finished product or as an ingredient within a product, whether dehydrated, liquid or frozen. - Beverages, including beverage mixes and concentrates, including but not limited to, alcoholic and non-alcoholic ready to drink and dry powdered beverages, carbonated and non-carbonated beverages, e.g., sodas, fruit or vegetable juices, alcoholic and non-alcoholic beverages. - Confectionery products, e.g., cakes, cookies, pies, candies, chewing gums, gelatins, ice creams, sorbets, puddings, jams, jellies, salad dressings, and other condiments, cereal, and other breakfast foods, canned fruits and fruit sauces and the like.
- Dairy products such as milk, cheese, yoghurt. - Pharmaceutical compositions which may, for example, be in the form of a syrup, an emulsion, a suspension, a solution or other liquid form.
- Dental compositions including, for example, mouth freshening agents, gargling agents, mouth rinsing agents, toothpaste, tooth polish, dentifrices, mouth sprays and dental floss.
- Edible gel compositions The compositions disclosed herein may further comprise a base composition. For example, the comestible compositions disclosed herein may further comprise a comestible base composition. This refers to all the ingredients necessary for the composition except the combination of the high-intensity sweetener and low-potency sweetener (e.g. sweetness modifying composition). The base composition may, for example, be a sweetened base composition comprising at least one other sweetener present in an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence. These will naturally vary in both nature and proportion, depending on the nature and use of the composition, but they are well known in the art and may be used in art-recognised proportions. The formulation of such a base composition for every conceivable purpose is therefore within the ordinary skill in the art.
The ingredients in a base composition may include, but are not limited to, anti-caking agents, anti-foaming agents, anti-oxidants, binders, colourants, diluents, disintegrants, emulsifiers, encapsulating agents or formulations, enzymes, fats, flavour-enhancers, flavouring agents, gums, lubricants, polysaccharides, preservatives, proteins, solubilisers, solvents, stabilisers, sugar-derivatives, surfactants, sweetening agents, vitamins, waxes, and the like. Solvents which may be used are known to those skilled in the art and include e.g. ethanol, ethylene glycol, propylene glycol, glycerine and triacetin. Encapsulants and gums include maltodextrin, gum arabic, alginates, gelatine, modified starch, and polysaccharides.
Examples of additives, excipients, carriers, diluents or solvents for flavour or fragrance compounds may be found e.g. in "Perfume and Flavour Materials of Natural Origin", S. Arctander, Ed., Elizabeth, N.J., 1960; in "Perfume and Flavour 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 "CTFA Cosmetic Ingredient Handbook", J.M. Nikitakis (ed.), 1 st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988.
The proportion of the combination of the one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) (e.g. sweetness modifying composition) or the 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 the sweetness desired. The skilled person can readily ascertain the appropriate proportion in every case with only simple, non-inventive experimentation. The amounts and proportions disclosed herein are exemplary only and the flavourist may seek particular effects by working outside this range, and it should be regarded as an indication only.
The pH of the composition disclosed herein may be any pH that does not adversely affect the taste of the sweetener blend. For example, the pH may range from about 1 .8 to about 8 or from about 2 to about 5. A person skilled in the art would be able to identify a suitable concentration of each sweetener to use depending on the pH of the composition.
The use of the one or more low-potency sweetener(s) with the one or more high- intensity sweetener(s) may, for example, improve one or more sweetness characteristics in a sweetened composition in comparison to the use of the one or more high-intensity sweetener(s) alone. Thus, the sweetened compositions disclosed herein may, for example, have one or more improved sweetness characteristics compared to the sweetened compositions in the absence of the 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 a sweetened composition in comparison to the use of a different sweetness enhancer such as Luo Han Guo extract in place of the one or more of mogroside IV, siamenoside and neomogroside.
The sweetened compositions disclosed herein may, for example, have one or more sweetness characteristics that are more similar to sucrose compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer. The sweetened compositions disclosed herein may, for example, have a weakened lingering sweet taste in compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
The sweetened compositions disclosed herein may, for example, have a weakened bitter taste and/or astringent taste and/or metallic taste and/or liquorice taste compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
The sweetened compositions disclosed herein may, for example, have a strengthened sweetness impact compared to the sweetened compositions in the absence of the one or more low-potency sweetener(s) or compared to the sweetened compositions comprising a different sweetness enhancer.
The comparative sweetened composition is identical except that it does not include any of the one or more low-potency sweetener(s) or identical except that it comprises a different sweetness enhancer in place of the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside.
Uses
There is provided herein the use of a combination of one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) to enhance the sweetness of a composition comprising at least one other sweetener present an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence. The combination of the one or more high-intensity sweetener(s) and one or more low-intensity sweetener(s) has a sweetness less than 1 .5 % (w/v) sucrose equivalence. The 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 herein 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 comprising at least one other sweetener present an amount equal to or greater than its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence.
Thus, there is provided a method for enhancing the sweetness of a sweetened composition, the method comprising providing a base composition comprising at least one sweetener in an amount having a sweetness above its sweetness recognition threshold and/or an amount having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence, and adding at least one low-potency sweetener, at least one high-intensity sweetener; or adding one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside. Each component of the final composition may be added in any order to obtain the desired final composition. The method may, for example, comprise mixing the components.
The one or more high-intensity sweetener and/or the combination of the one or more high-intensity sweetener and the one or more low-potency sweetener (e.g. the sweetness modifying composition) and/or the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may, for example, increase the sweetness of a sweetened composition by equal to or more than about 1 .0 % (w/v) sucrose equivalence. For example, the high-intensity sweetener(s) and/or the combination of the high-intensity sweetener(s) and the low-potency sweetener(s) and/or the one or more mogroside(s), for example one or more of mogroside IV, siamenoside and neomogroside may increase the sweetness of a sweetened composition by equal to or greater than about 1 .1 % (w/v) sucrose equivalence or equal to or greater than about 1 .15 % (w/v) sucrose equivalence or equal to or greater than about 1 .2 % (w/v) sucrose equivalence or equal to or greater than about 1 .25 % (w/v) sucrose equivalence. The composition may, for example, be a composition comprising at least one other sweetener.
There is also provided herein the use of one or more low-potency sweetener(s) to improve one or more sweetness characteristics of a sweetened composition comprising 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 of less than about 1 .5 % (w/v) sucrose equivalence. Thus, there is provided a method for improving one or more sweetness characteristics of a sweetened composition comprising one or more high-intensity sweetener(s) in an amount having a sweetness less than 1 .5 % (w/v) sucrose equivalence, the method comprising providing a composition comprising one or more high-intensity sweetener(s) and adding one or more low-potency sweetener(s). Each component of the final composition may be added in any order to obtain the desired final composition. The method may, for example, comprise mixing the components.
The improvement of one or more sweetness characteristics of a sweetened composition comprising a high-intensity sweetener may, for example, provide sweetness characteristics that are more similar to the sweetness characteristics of sucrose.
The sweetness characteristics may refer to the flavour profile (taste profile), which refers to the intensity of the flavour and perceptual attributes of a given compound. Exemplary flavour attributes of sweetness are sweetness intensity, bitterness, black liquorice etc.
The sweetness characteristics may refer to the temporal profile, which refers to the changes in perception of sweetness over time. Every sweetener exhibits a characteristic appearance time (AT) and extinction time (ET). Most high-potency sweeteners, in contrast to carbohydrate sweeteners, display prolonged ET (lingering). Generally, the detected sucrose equivalence spikes to a maximal response level, then tapers off over time. The longer the taper, the greater the detected sweetness linger of a compound. In certain embodiments, the one or more low-potency sweetener(s) may be used to weaken the lingering sweet taste of the sweetened composition comprising one or more high-intensity sweetener(s). In other words, the low-potency sweetener may be used to decrease the extinction time (ET) of the sweetened composition comprising one or more high-intensity sweetener(s). This relates to the undesirable lingering of the sweetness taste in the mouth after the composition is initially ingested or expectorated. The lingering sweet taste may, for example, refer to the length of time that the sweetness taste remains after it is initially detected, how rapidly the intensity of the sweetness taste decreases or fades after it is initially detected and the intensity of the sweetness taste after it is initially detected. The one or more low-potency sweetener(s) may, for example, decrease the length of time that the sweetness taste remains after it is initially detected and/or increase the speed at which the sweetness taste decreases after it is initially detected and/or decrease the intensity of the sweetness taste after it is initially detected.
In certain embodiments, the one or more low-potency sweetener(s) may be used to weaken the bitter taste and/or astringent taste and/or metallic taste and/or liquorice taste of the sweetened composition comprising the one or more high-intensity sweetener(s). The term "liquorice" refers to a sweet taste of a compound.
In certain embodiments, the one or more low-potency sweetener(s) may be used to strengthen the sweetness impact of the sweetened composition comprising the one or more high-intensity sweetener(s). The sweetness impact relates to the length of time it takes before the sweetness is initially detected and the intensity at which the sweetness is initially detected. The one or more low-potency sweetener(s) may, for example, decrease the amount of time before the sweetness is initially detected and/or increase the intensity at which the sweetness is initially detected.
The degree of sweetness and other sweetness characteristics described herein may be evaluated by a tasting panel of trained experts, for example as described in the examples below. Manufacturing Methods
There is further provided herein methods of making the compositions disclosed herein. The compositions may be in accordance with any embodiment disclosed herein. The methods may comprise combining each component of a desired composition in the desired proportions and optionally mixing the components together. The components may be combined and mixed in any suitable order.
A person skilled in the art would identify a suitable method to make the composition (e.g. suitable order in which to combine or mix the components) depending on the nature of the composition and the degree and characteristics of the sweetness desired. The methods may, for example, comprise providing a desired base composition and adding the sweeteners thereto. Each of the sweeteners disclosed herein may be made by a synthetic process or by a biological (e.g. enzymatic) process or a fermentation process or may be isolated from a natural source such as a plant or fruit. The process may, for example, comprise 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 (e.g. a recombinant host cell). The enzyme may, for example, be a UGT enzyme (UDP-glucuronosyltransferase). For example, a mogroside compound may be made by the biosynthetic pathway disclosed in WO 2013/076577 or WO 2014/086842, the contents of which are incorporated herein by reference.
For example, mogroside V may be made by the biosynthetic pathway disclosed in Itkin et at., "The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii', PNAS, 7 November 2016, E7619 - E7628 and WO 2016/038617, the contents of which are incorporated herein by reference.
For example, a mogroside compound may be made by modifying (e.g. redistributing glycoside content) another mogroside compound. For example, a mogroside compound may be made by redistributing glycoside content of another mogroside compound using acid or enzymes as disclosed in WO 2014/150127, the contents of which are incorporated herein by reference.
The process may, for example, comprise extracting one or more sweetener compounds from a natural source such as a plant or fruit. This may, for example, be followed by a purification step to yield a high-intensity sweetener, low-intensity sweetener or mixture of sweeteners (e.g. 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 1 1 -O-mogroside V (e.g. at least about 30 wt% or at least about 40 wt%). This may, for example, involve fractioning, for example by flash chromatography. One or more mogroside compounds (e.g. mogroside V) may be obtained from Luo Han Guo (Siraitia grosvenorii) fruit.
When a fermentation process is used to make the target product (e.g. target mogroside product), the target can be extracted from the aqueous fermentation reaction medium using an appropriate solvent (e.g., heptane) followed by fractional distillation. The chemical composition of each fraction can be measured quantitatively by GC/MS (gas chromatography mass spectrometry). Fractions can be blended to generate the desired mogroside compounds (e.g. mogroside V and 1 1 -O-mogroside V) for use in flavour or other applications. Verification of acceptability of the final blended product can be carried out by direct comparison to a reference mogroside flavouring product (for example, an existing natural flavouring commercial product 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 analysed 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_ solvent (acetonitrile/water 20/80 v/v). From this solution 100 μΙ_ were transferred into a HPLC vial and 900 μΙ_ solvent was added (66.1 ppm solution). From the sample solution 10 μΙ_ was transferred to a HPLC vial and 990 μ\- solvent was added (6.61 ppm solution). Both the 66.1 and 6.61 ppm solutions were injected twice on the LC-MS.
Calibration (reference) solutions of mogroside V were made by dissolving 9.22 mg mogroside V (98.5 % mogroside V obtained from AAPIN chemicals Ltd., Oxfordshire, UK) in 10.0 mL solvent (acetonitrile/water 20/80 v/v). The stock solution was stored in the freezer and used to prepare solutions of mogroside V at various concentrations (0.1 1 ppm, 0.34 ppm, 1 .02 ppm, 3.07 ppm and 9.22 ppm). These solutions were also injected twice on the LC-MS.
2 μ\- of each solution was injected on an Acquity C18 BEH 1 .7 μιτι 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 going up to 50% acetonitrile in 14 minutes. The gradient was back on the starting values in 1 minute and stabilized for 5 minutes. The flow was set on 400 μ\- during the whole run.
Eluted compounds were detected using liquid chromatography mass spectrometry (LC/MS). The mass spectrometer was operating in ESI negative mode measuring 150 to 2000 Amu with a resolution of 70000. Gas flow rates were sheath 60, aux 20 and sweep 3. Capillary temperature and aux gas heater temperature were set on 380°C and 400°C respectively.
The % of each component in the extract was calculated using the following equation and calibrated against a curve of the various concentration calibration mogroside V (reference) solutions described above. area * V * d
slope * SW ratio = % component
area = component area in sample (average area from 2 injections)
V = sample solvent volume in litres
d = sample dilution (from sample solution to vial)
slope = slope from mogroside V calibration curve with b (intercept) = 0
SW = sample weight in mg
Results
Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of 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 (about 45 wt% in extract 1 ).
Table 1.
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Example 2
Methods A Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) containing about 68 wt% mogrosides 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 flushed with 80% MeOH. The solvents were introduced at the flow rate of 30ml/min throughout the separation procedure. Eluted compounds were visualized with a UV detector set at 210 nm and a coronal light scattering detector. The % of each component in the extract was calculated using the equation described in Example 1 above.
Collected fractions were pooled according to Table 2 below, and then freeze dried to powders. The powder corresponding to various pooled fractions as given in Table 2 below was dissolved in various concentrations on top of 5% sucrose. The taste of these samples was compared by three expert panellists (trained flavourists) to controls of 5% sucrose. Thus the sweetness enhancement effect of each fraction or pool of fractions exhibited in 5% sucrose was determined.
Results
The results are indicated in the table below. The whole extract was collected into 22 fractions. Fractions 1 -10 contain no mogroside V.
Table 2.
Fraction # Mogroside Dose level
(combined) V content of Fraction Taste evaluation (on top of 5%
(%) (ppm) sucrose)
1 ( tube 1 -7) 0 15
2(tube 8-12) 0 15 Some in this set have sugary notes, most
3(tube 13-16) 0 15 also have fermented off notes 4 (tube 17-20) 0 15
5 (tube 21 -24) 0 15 Some astringency in this part
6 (tube 25-28) 0 15
Sweetness suppressed
7 (tube 29-32) 0 15
Bitter, metallic, fermented off notes
8 (tube 33-34) 0 15
No enhancement
9 (tube 35-36) 0 15
10 (tube 37) 0 15 Fermented, typical white dog notes, astringent, cooked delayed sweet
1 1 (tube 38) 1 .15 45 Sweeter, some mouthfeel, some upfront
12 (tube 39) 15.9 45 Slightly higher in enhancement, less off notes
13 (tube 40) 64.5 30 Sweeter, higher licorice, less dirty fermented, si higher astringency
14 (tube 41 - 100 25 Very lingering, very licorice, dirty sweet, 42) numbing, sharp sweetness, mouth drying
15 (tube 43- 97 25 Strong fermented dirty note, higher 44) sweet, numbing delayed sharp, strong licorice, linger, metallic
16 (tube 45- 70.3 25
46)
17 (tube 47) 18.7 45 Astringent, some enhancement, mostly
18 (tube 48) 4.6 45 licorice lingering
19 (tube 49) 1 .95 45
20 (tube 50- 1 .56 5
51 ) Negative notes, typical fermented
21 (tube 52- 0 5 lingering
54)
22 (tube 55- 0
end)
Fraction 1 -10 and 19-22 have an off-taste, which is the character of Luo Han Guo fruit, no sweet enhancement impact. The better sweetness enhancement effect was observed within fractions 12-17, which contains mainly mogrosides. When the fractions almost have pure mogroside V, the lingering, dirty fermented note is more noticeable, such as fractions 14 and 15. Thus, pure mogroside V has inherent lingering off-taste.
Fraction 12 was the cleanest sweet, but less upfront due to small percentage mogroside V. Fraction 13 has better sweet quality, but slightly higher astringency. 1 1 - O-mogroside V and mogroside V are the two major mogrosides in those two fractions, but with different ratio (F12 mogroside V:1 1 -O-mogroside V is 4:9 and F13 mogroside V:1 1 -O-mogroside V is 13:3).
Mogroside V was very sweet, judged to be 425 times sweeter than sucrose, while 1 1 - O-mogroside V is rated as 84 times sweeter than sucrose. Example 3
Methods
A Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) containing about 68 wt% mogrosides was fractionated and the composition of each fraction determined by the chromatography method described above in relation to Example 1 .
Each fraction was combined with a solution of 5 % sucrose and the taste of these samples was compared by three expert panelists (trained flavourists) to controls of 5% sucrose.
Results Table 3 shows the chemical composition of fractions 1 1 to 20 of the extract. Table 3.
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Table 4 shows the tasting results for fractions 12 to 15. The results are similar to the results obtained in Example 2.
Table 4.
Iso-sweet Comments for
Fraction Level Iso-sweet Comments for 5% Sucrose Tasting
(ppm) tasting
Slightly higher in enhancement, less off- notes, this was the cleanest sweet of all the samples, less upfront sweet than Luo Han Guo extract, good quality of sweet except for lower upfront sweet impression.
Closest to Luo
Han Guo extract,
F12 45 The other fractions have different off- less overall off- notes compared to Luo Han Guo extract, notes
but are comparable in terms of quality of sweet.
Basically, numbing/irritating effects are stronger than "wet dog" fermented effects, typical in Luo Han Guo extract
Very close in
Sweeter, higher licorice, less dirty
F13 30 sweetness,
fermented, si higher astringency lingering
Very lingering, very licorice, dirty sweet,
Slightly sweeter, lower overall quality of sweet compared very likely would to Luo Han Guo extract, numbing, sharp match iso-sweet sweetness, mouth drying, irritating,
F14 25 of Luo Han Guo lingers
extract, less dirty
finish than Luo Fractions 14 and 15 have a sharp, Han Guo extract numbing, burn, which negatively effects potency and quality of sweet
F15 25 Very close in Strong fermented dirty note, higher profile, less off- sweet, numbing, delayed sharp, strong notes licorice, lingers, metallic
Example 4
Mogroside V and 1 1 -O-mogroside V were isolated from extracts of Luo Han Guo using Agilent 1 100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 μηι, 210 X 21 .4 mm), and combined to form solutions of various concentrations.
These solutions were combined with a solution containing 5 % (w/v) sucrose and 0.03 % (w/v) citric acid to give test samples and evaluated by a sweet sensitive taste panel of five experts (trained flavourists).
The results are shown in Table 5 below.
Table 5.
Figure imgf000048_0001
It was surprisingly found that blending 1 1 -O-mogroside V with mogroside V improves the sweet quality compared to mogroside V alone. 1 1 -O-mogroside V on top of mogroside V helped to reduce sweetness lingering (weaken later sweetness taste) and reduce astringent and bitter aftertastes compared to mogroside V alone. Thus, the 1 1 -O-mogroside V made the sweetness taste more similar to sugar than mogroside V alone (i.e. assists in providing a temporal profile that is closer to sugar). This enables the use of higher concentrations of mogroside V to obtain higher sweetness whilst eliminating the disadvantages associated with using higher concentrations of this sweetener (e.g. lingering, bitter and astringent aftertastes). This was surprising given that mogroside V is the most potent mogroside sweetener and 1 1 -O-mogroside V is of much lower potency.
Example 5
A sweet sensitive taste panel ranked the sweetness of solutions of a mixture of sweeteners ("Mixture 1 ") in relation to sucrose solutions at a range of concentrations to determine sucrose equivalence. Mixture 1 was a combination of fractions 1 1 to 18 of Example 2 and contained 8.16 wt% 1 1 -O-mogroside V and 61 .6 wt% mogroside V. The results are shown in Table 6 below.
Table 6.
Figure imgf000049_0001
The data demonstrates that mixture 1 has a sweetness below 1 % sucrose equivalence (as determined by seven panellists), which is accepted as "not intrinsically sweet" by FEMA. Therefore, mixture 1 is suitable for use as sweetness modifiers or blends at the indicated concentrations because it does not have any detectable sweetness at these levels. A concentration of mixture 1 having an iso-sweet threshold close to 1 % (35 ppm) was selected and added to 5 % (w/v) sucrose solutions. This solution was then ranked against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. This was repeated for 45 ppm Luo Han Guo extract. The average score of each solution was determined. The results are shown in Table 7 below.
Table 7.
Figure imgf000050_0001
It was surprisingly found that mixture 1 and Luo Han Guo extract act as sweetness enhancers since 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 various concentrations of mixture 1 was tested by an expert panel of three people (trained flavourists) in solutions containing 5 % sucrose and 0.03 % citric acid. The taste was compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) and containing about 68 wt% mogrosides) (combined with the same 5 % sucrose and 0.03 % citric acid solution). The results are shown in Table 8.
Table 8.
Figure imgf000050_0002
Overall, mixture 1 provides a better sweet quality (less baggage, sweeter) than the Luo Han Guo extract. Example 6
A sweet sensitive taste panel ranked the sweetness of solutions of various sweeteners (mogroside V, mogroside IV, siamenoside, neomogroside, 1 1 -O-mogroside V) in relation to sucrose solutions at a range of concentrations to determine sucrose equivalence. The sweeteners were obtained using an Agilent 1 100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 μηι, 210 X 21 .4 mm). The results are shown in Tables 9 to 13. Table 9.
Figure imgf000051_0001
Table 10.
25 ppm 30 ppm
Sucrose 0.5 Sucrose 1 Sucrose 1.5
Panelist Mogroside Mogroside
% % % IV IV
1 3 1 4 2 5
2 2 3 1 4 5
3 1 2 3 4 5
4 2 4 3 1 5
5 2 1 3 4 5
6 1 2 3 4 5
7 2 1 3 4 5
Total 13 14 20 23 35 Table 11.
Figure imgf000052_0001
Table 12.
Mogroside
V (25 ppm)
Sucrose 0.5 Sucrose 1 Sucrose 1.5
Panelist + 11-0-
% % %
Mogroside
V (3 ppm)
1 1 2 3 4
2 3 2 1 4
3 1 3 2 4
4 1 2 4 3
5 1 2 3 4
6 1 3 2 4
7 1 3 2 4
Total 9 17 17 27
Table 13.
Figure imgf000053_0001
The data demonstrates that mogroside V (25 ppm), mogroside IV (30 ppm), siamenoside (25 ppm), mogroside V (25 ppm) in combination with 1 1 -O-mogroside V (3 ppm) and neomogroside (30 ppm) all have a sweetness below 1 .5 % sucrose equivalence (as determined by seven panellists), which is accepted as "not intrinsically sweet" by FEMA. Therefore, these compounds and mixtures are suitable for use as sweetness modifiers at the indicated concentrations because they do not have any detectable sweetness at these levels.
Concentrations of the tested sweeteners were selected with an iso-sweet threshold close to 1 % and added to 5 % (w/v) sucrose solutions. These solutions were then ranked against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. The average score of each solution was determined. The results are shown in Table14 below.
Table 14.
Figure imgf000053_0002
It was surprisingly found that mogroside V, siamenoside, neomogroside and mogroside V act as sweetness enhancers since 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 an expert panel of three people (trained flavourists) in solutions containing 5 % sucrose and 0.03 % citric acid. The taste was compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) and containing about 68 wt% mogrosides). Mogroside IV, siamenoside and neomogroside are all better than Luo Han Guo extract in terms of sweet quality when added to 5 % sucrose and 0.03 % citric acid. These 3 compounds provide a sugar like taste with less lingering sweet taste. Siamenoside was described as having "more sweet body, sweeter, rounder with a little more upfront and more round lasting sweet". Mogroside IV was described as having "good and similar sweetness as mogroside V". Neomogroside was described as having "sweetness, but slightly bitter aftertaste". The results for mogroside V are shown in Table 15.
Table 15.
Figure imgf000054_0001
In general, the tasters agreed that 80% mogroside V does not have the same full round sweet profile as the Luo Han Guo extract. The 80% mogroside V is more acidic tasting when applied to a sugar/acid/water solution. Example 7
Methods Mogroside V, siamenoside, mogroside IV and neomogroside were obtained using an Agilent 1 100 preparative HPLC system with a Phenomenex Luna C18 (2) column (5 μπΊ, 210 X 21 .4 mm)..
The mogroside V, siamenoside, mogroside IV and neomogroside were each added to a solution containing 5 % sucrose and 0.03 % citric acid in a concentration of 25 ppm (mogroside V), 25 ppm (siamenoside), 30 ppm (mogroside IV) and 30 ppm (neomogroside) respectively.
These test solutions were tasted by an expert panel of seven people. For various aspects of sweet taste (upfront sweet, overall sweet, lingering sweet, astringent, volatile off-taste), each panellist scored the test solutions in comparison to the base solution (solution of 5 % sucrose and 0.03 % citric acid).
A score of 0 indicated that the taste aspect was the same, 1 indicates slightly higher, 2 indicates higher, 3 indicates much higher, -1 indicates slightly lower, -2 indicates lower and -3 indicates much lower. The average score for each test solution for each taste aspect was calculated. The results are shown in Table 16 below.
Table 16.
Mogroside V Siamenoside Mogroside IV Neomogroside
Overall Sweet 1 1 .8 0.4 1 .4
Upfront Sweet 0.8 1 .4 0.4 1
Lingering
0.4 0.6 0 0.4 Sweet
Astringent 0 0.4 0 0
Volatile Off-
0 0.2 0 0 Note Example 8
As shown above, siamenoside, neomogroside and mogroside IV all have similar or better sweet taste quality on top of 5 % sucrose and 0.03 % citric acid compared to mogroside V. Therefore, the taste of 1 1 -O-mogroside V with each of these mogrosides is evaluated as shown in Table 17.
Table 17.
Figure imgf000056_0001
Example 9
The taste of mogroside V with and without 1 1 -O-mogroside V in various milk or yoghurt bases is evaluated as shown in Table 18. The iso-sweet threshold for mogroside V in milk and yoghurt is also evaluated.
A milk base (2% fat) includes 2% fat milk and 5 % sucrose. A non-fat yoghurt base includes plain non-fat yoghurt and 5 % sucrose. A full fat yoghurt base includes plain full fat yoghurt and 5 % sucrose. A higher dose level is used for milk and yoghurt compositions due to the fat, protein and other ingredients. Luo Han Guo extract is used at 75 ppm for these applications.
Table 18.
Figure imgf000057_0001
mogros e
Example 10
The identification of new minor cucurbitane glycosides from Siraitia grosvenorii Introduction
Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang is a herbaceous perennial vine of Cucurbitaceae family endemic to southern China and northern Thailand. The fruit of S. grosvenorii, commonly known as 'luo han guo' has been used for traditional medicine in China for centuries as a treatment of respiratory infection, bronchitis, gastritis, constipation etc. Modern pharmacological research have confirmed that S. grosvenorii extracts or its components possess variety of bioactivities, such as antibacterial, anti-inflammation, anti-diabetic, anti-cancer, and immunostimulatory [1 ]. Luo Han Guo has been used as a sweetener in food and beverages in China. It is now one of the best known natural high intensity sweeteners throughout the world. Since cucurbitane glycoside mogroside V has been discovered as the sweet principle of S. grosvenorii, more than 40 cucurbitane triterpenoids have been reported from S. grosvenorii so far [1 -4]. Food and flavor industry researchers have been actively discovering and adding more new compounds into the mogroside pool in order to find new mogrosides with better sweet performance [5-7]. New molecules under known natural sweetener categories with better sweet performance have been a sought-after for food and flavor industries. The commercialization of rebaubioside M (also known as rebaubioside X) is a good example. Even it is a minor natural product from Stevia (less than 0.1 %) discovered in 2010, rebaubioside M quickly progressed into commercialization stage thanks to cost reduction by technology development in plant biology, molecular biology and enzyme engineering [8, 9]. Rebaudioside M received Letter of No Objection concerning its Generally Recognized as Safe (GRAS) status from US FDA in 2013, 2014 and 2017 (GRN No. 473, 512 and 667) [10-12].
We have been conducting investigations to seek the best performance mogrosides or their combinations by using commercial Luo Han Guo extracts [13]. Herein, we report two new minor cucurbitane glycosides from S. grosvenorii and emphasize our new oligosaccharide elucidation strategy based on HSQC-TOCSY experiments with different mixing times. Materials and Methods
General experimental procedures
Optical rotations were measure with a Rudolph Autopol IV polarimeter. The NMR spectra were recorded on Bruker DRX Avance 300 or 500 spectrometers. Chemical shifts are given in δ (ppm) referring to the residual solvent peak. Low pressure chromatography was performed on Biotage Flash System SP1 . Preparative HPLC was performed on an Agilent 1 100 preparative HPLC system with a Phenomenex Lunar C18(2) column (5 μιτι, 210 x 21 .4 mm) or a TSKgel Amide-80 (5 μιτι, 300 x 21 .5 mm) (Tosoh Bioscience LLC). Analytical HPLC was performed on an Agilent 1 100 analytical HPLC system equipped with ESA Corona CAD detector. LC-MS was performed using Waters Q-Tof micro mass spectrometer coupled with Waters 2795 separation module.
Plant material
The Luo Han Guo extract (commercial name Swingle, -60% mogrosides) was purchased from Blue California Co., Ltd.
Instrumentation
Chromatographic conditions: The chromatography was performed on a Waters Acquity H UPLC. Separation was carried out at 25 C using a 1.0 x100 mm, Acquity UPLC HSS T3 column (Waters), with a particle size of 1.8 mm, equipped with a 0.2 mm prefilter. Solvent A was water and solvent B was acetonitrile, both solvents contained 0.1 % formic acid. Injection volume was set to 10 μΙ. The chromatography flow rate was 200 μΙ/min. Sample was eluted from the LC column using the following linear gradient (curve number 6): 0-40min: 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 re-equilibration.
Mass spectrometry
The U-HPLC 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 was operated in the positive electrospray ionization mode (EST). The sample cone voltage 40, capillary voltage 0.7 kv, source temperature 40 5C, desolvation temperature 450 5C, desolvation gas flow 800 L/h, and cone gas flow 50 L/h were optimized. Leucine enkephalin was used as the lock mass [M+H]" at m/ z 556.2771 . Sodium formate solution was used for external instrument calibration.
Purification 3 g Luo Han Guo extract was dissolved in 15 mL water and loaded onto a pre- equilibrated C-18 Snap cartridge (KP-C18-HS, 120 g, 132 mL column volume). The gradient system (A: water; B: methanol) used was: 30% 2 CV, 30%~80% 10 CV, 80%~100% 2 CV, 100% 2 CV. The flow rate was 30 mL/min. Fractions were collected for 27 mL per tube. Four loading of total 12 g Luo Han Guo was fractionated. All the fractions were analyzed by analytical HPLC to locate the fractions with the target mogrosides (isocratic mobile phase: 24% acetonitrile in water. Column: Luna C18 5μηι 4.6x 150mm). Fractions 36-38 with iso-mogroside VI and 1 1 -epi-mogroside V were combined to evaporate solvents. Further preparative HPLC purification of fractions 36- 38 afforded iso-mogroside VI (1 , 22 mg) and 1 1 -epi-mogroside V (2, 17 mg) (24% acetonitrile in water, 10 mL/min, retention time 13.1 min and 14.3 min, respectively). 1 1 -oxo-mogroside V (4) and neomogroside (3) were mainly in flash fractions 39-40 with 1 1 -oxo-mogroside V as the major component. On reverse phase C-18 preparative HPLC, neomogroside appeared as a tail shoulder of 1 1 -oxo-mogroside (24% acetonitrile in water, 10 mL/min, retention time 17.0 min and 18.0 min, respectively). Collection of the peak front gave 105 mg of the compound 1 1 -oxo-mogroside V (4). Further purification of the shoulder neomogroside (3, 15 mg) was achieved by preparative HPLC on TSKgel Amide-80 (65% acetonitrile in water, 20 mL/min, rt 15.5 min).
iso-mogroside VI iJ.White amorphous powder; [a]20 D -8.2 (c 0.12, MeOH); For 1 H NMR and 13C spectroscopic data, see Tables 1 ;-HRESIMS: m/z 1449.7075 [M+H] " (calcd. for C66H 1 13034, 1447.71 13, Δ2.6 ppm). epi-mogroside V (2) .White amorphous powder; [a]20 D +4.5 (c 0.13, MeOH); For 1 H NMR and 13C spectroscopic data, see Tables 1 ; HRESIMS: m/z 1287.6558 [M+H] " (calcd. for C60HW3O29, 1287.6585, Δ2.1 ppm).
Acid hydrolysis and determination of absolute configuration of sugars
Compounds 1 (1 .2 mg) or 2 (1 .8 mg) were incubated in 1 mL 1 M HCI at 80 <C f or 3 hrs. After hydrolysis, the solution was extracted with EtOAc (1 mL X 3). The remaining aqueous solutions were evaporated by blowing nitrogen gas and freezing dried. The absolute configuration of the sugar in the residue was determined by GC-MS analysis of its O-silylated derivative and comparing with the derivatives of D-glucose and L- glucose standards. Briefly, the sugar residues, D-glucose (2 mg) or L-glucose (2 mg) were dissolved in pyridine (0.5 mL). 0.1 M L-cysteine methyl ester hydrochloride (Aldrich, Milwaukee, Wl) in pyridine (0.5 mL) was added into the solution. The mixture was kept at 60 Ό for 2 h and dried by blowing nitr ogen gas. The residue was added with 1 -trimethylsilylimidazole (Fluka, Buchs, Switzerland) (0.5 mL) and incubated under 60 Ό for 1 h. The mixture was partitioned by addin g n-hexane and water (1 .0 mL each). The n-hexane extract was analyzed by GC-MS under the following conditions: capillary column HP-5MS (30 m χ 0.25 mm χ 0.25 μηι, Agilent); column temperature, 180 to 230 Ό at a ramp of 5 <C/min ; injection tern perature, 250 Ό; carrier, He gas; split ratio, 20:1 . The O-silylated derivatives of D-glucose and L-glucose showed retention time at 16.02 and 16.39 min, respectively. By comparing the retention time and co-chromatography, the sugar residues after acid hydrolysis of 1 and 2 were determined to be D-glucose.
Reduction of 11-oxo-mogroside V with NaBH4
25 mg of 1 1 -oxo-mogroside V (4) was dissolved in 50% dioxane and added with 20 mg NaBH4 and heated at 50 C for 3 days. The reaction mixture was periodically analyzed by HPLC to monitor the reaction progress. After the reaction, the mixture was acidified by acetate acid and concentrated to dryness by blowing nitrogen gas. The residue was re-dissolved in water and passed through a pre-equilibrated C-18 SPE column. The methanol eluents from SPE column were concentrated. The residue was then separated by semi-preparative HPLC. The two reduced products had same retention time and molecular weight as the isolated mogroside V and 1 1 -epi-mogroside V by LC-MS analysis and co-chromatography on analytical HPLC. The 1 -D and 2-D NMR data also confirmed that the structures of the two reduced products were mogroside V and 1 1 -epi-mogroside V.
Results and Discussion
Isolation and elucidation of iso-mogroside VI (1 ) and 11 -epi-mogroside V (2)
During the course of investigating a commercial Luo Han Guo extracts with 60% mogrosides by LC-MS, several mogrosides with six or five sugar moieties in the extracts attracted our attention (Figure 2 and 3). Since there were little report on the sweet properties of mogroside V and VI isomers, we decided to purify and identify these isomers for our evaluation. The concentrations of 1 , 2, 3 and 4 in the extracts were estimated to be 0.8%, 0.5%, 0.6%, 4.9%, respectively, according to the universal Corona detector. After fractionated on flash chromatography system and followed by preparative HPLC purification, the four targeted mogrosides 1 -4 were purified and determined to be iso-mogroside VI (1 ), 1 1 -epi-mogroside V (2), neomogroside (3), 1 1 - oxo-mogroside V(4). The molecular formula of 1 was deduced as C66H112034 by its HR-ESI-MS spectral data ([M-H] " m/z, 1447.6957, calcd. for C66H111034, 1447.6957). The NMR spectral data of 1 suggested the structure of a hexasaccharide triterpenoid saponin: 30 of the 66 carbons were assigned to the triterpenoid aglycone, and 36 of 66 to six hexose moieties. The 13C and 1 H NMR spectra of 1 showed the signals of seven singlet tertiary methyls, a doublet secondary methyl, and an olefinic methine (Table 19), which suggested a typical (24R)-cucurbit-5-ene-33,1 1 a,24,25-tetraol mogrol aglycone. The mogrol aglycone of 1 was further confirmed by extensive analysis of its 1 H, 13C, and 2D (COSY, TOCSY, HSQC and NOESY) NMR data, as well as comparison with NMR data of mogroside V standard.
Table 19. 1 H NMR and 13C NMR spectroscopic data for iso-mogroside VI and 1 1 -ep/- mogroside V (1 H 300 MHz and 13C 75 MHz in pyridine-d5/D20 10:1 )
Figure imgf000062_0001
Figure imgf000063_0001
GC-MS analysis of water-soluble acid hydrolysis products of 1 showed that D-glucose was the only monosaccharide in the structure of 1. The HSQC spectra clearly displayed the anomeric cross-peaks of six glucosyls: Glc-I (5C 106.8 and 5H 4.73), Glc- II (5c 105.1 and δΗ 5.10), Glc-lll (5C 103.7 and δΗ 4.85), Glc-IV (5C 104.6 and δΗ 4.78), Glc-V (5c 104.7 and δΗ 5.43), Glc-VI (5C 104.8 and δΗ 5.03). The stereochemistry of all the six glucopyranosyls were determined to be β configuration from their anomeric proton coupling constants 3Jm , H2- From HSQC-TOCSY experiment (hsqcgpmlph) with 100 ms mixing time, the glucopranosyl carbon signals can be divided into six groups (Figure 4). The oligosaccharide elucidation was started from the glucopyranosyl connected at C-3 of the cucurbitane aglycone. Glc-I was determined to link with aglycone C-3 according to the HMBC correlation of its anomeric proton (δΗ 4.73, d, J = 7.9 Hz) with aglycone C-3 (5C 87.6) and the NOESY correlation of Glc-I H-1 and aglycone H-3. The 13C signals of Glc-I (5C 75.1 , 77.8, 71 .4, 77.2, 70.1 ) as determined by HSQC-TOCSY missed a typical C-6 carbon signal at around 5C 62. The downfield shift of Glc-I C-6 (5C 70.1 ) indicated glycosylation at this position. By comparing HSQC- TOCSY spectra (hsqcgpmlph) with increased mixing time from 10, 30, 60, and 100 ms, the magnetization transfer relay can be observed gradually extending from C-2 to C-6 (Figure 4). As shown in Figure 4, HSQC-TOCSY under 10 ms mixing time displayed the correlation of glucopyranosyl H-1 and C-2. Under 30 ms mixing time, the correlation of H-1 and C-3 appeared in addition to H-1 and C-2 correlation. Under 60 ms, the carbon chain as indicated by the HSQC-TOCSY correlation extend to C-4. The full HSQC-TOCSY correlation of H-1 with C-2 to C-6 can be observed under 100 ms. Therefore, signals of C-2 to C-6 can be unambiguously assigned. The linkage of Glc-ll to Glc-I was established by the HMBC correlation of anomeric Glc-ll H-1 (δΗ 5.10, d, J=7.8 Hz) to Glc-I C-6 (5C 70.1 ). The 13C signals of Glc-ll (5C 75.0, 77.8, 71 .5, 78.0, 62.5) suggested no glycosylation on Glc-ll. As a result, the sugar chain on aglycone C- 3 was furnished as 3-0-(3-D-glucopyranosyl(1→6)-3-D-glucopyranosyl.
HMBC correlation of anomeric proton (5H 4.85, d, J=7.5 Hz) to aglycone carbon signal (5c 92.3) indicated the connection of Glc-lll H-1 to aglycone C-24. The 13C pattern of Glc-lll (5c 81 .6, 78.3, 71 .4, 76.4, 70.0) suggested C-2 and C-6 glycosylation shifts. Analysis of HSQC-TOCSY with 10, 30, 60, and 100 ms mixing time resulted in the sequential assignment and confirmation of C-2 and C-6 downfield shifts. Glc-IV was determined to connect to C-6 of Glc-lll as from its H-1 (5H 4.78, d, J=7.5 Hz) HMBC correlation with C-6 of Glc-lll (5C 70.0). Glc-IV was a regular terminal glucopyranosyl without any substitution (5C 74.5, 77.7, 71 .2, 78.2, 62.4). The linkage of Glc-V to C-2 of Glc-lll was established by HMBC correlation of anomeric Glc-V H-1 (5H 5.43, d, J=7.8 Hz) to Glc-lll C-2 (5C 81 .6). The relatively down-field shift of Glc-V H-1 (δΗ 5.43) was consistent with previous reports with similar structure. The 13C chemical shift of C-4 normally at 5C 70-71 was missing in the Glc-V signal set (5C 104.7, 75.4, 76.4, 82.0, 76.5, 62.6), which suggested glycosylation at C-4. By observing the C-2 to C-6 relay from HSQC-TOCSY with 10, 30, 60 and 100 ms mixing time, 5C 82.0 was clearly assigned to C-4 of Glc-V (Figure 4). HMBC cross-peak between C-4 of Glc-V (5C 82.0) and H-1 of Glc-VI (5H 5.03, d, J=7.7 Hz) further confirmed that Glc-VI linked to Glc-V at this position. Glc-VI was a terminal glucopyranosyl without further sugar branch. Based upon the above evidences, the structure of iso-mogroside (1 ) was assigned as 3-Ο-β- D-glucopyranosyl(1→6)-3-D-glucopyranosyl-mogrol-24-0-3-D-glucopyranosyl-(1→6- [3-D-glucopyranosyl-(1→4)-B-D-glucopyranosyl-(1→2)]-B-D-glucopyranosyl.
Compound 2 was assigned a molecular formula of C6oH102029 from its HR-ESI-MS data ([M-H] " m/z, 1285.6429). The NMR data of the oligosaccharide portion of 2 were superimposable with those of mogroside V. Detailed 2-D NMR experiments including HSQC, HMBC, NOESY, COSY and HSQC-TOCSY confirmed that 2 had the same sugar moieties as mogroside V. Attentions were then turned onto the aglycone NMR data. The HMBC correlation between C-1 1 and H3-19 revealed significant upfield shift of C-1 1 (5C 72.5) as compared with mogroside V (5C 77.8). Further assignment of aglycone data by 2-D NMR experiments showed that major 13C chemical shift changes occurred on C-8, C-10 and C-12 when comparing with the data of mogroside V (Table 19). This suggested β-ΟΗ instead of a-OH at C-1 1 . The β-ΟΗ stereo structure of 2 was further established by NOE correlations between H-8 and H3-18, 19; H-10 and H3-28, H3-30; H-1 1 and H3-30; H-17 and H3-30. There were one natural 1 1 -β-ΟΗ cucurbitane and one semi-synthetic 1 1 -β-ΟΗ cucurbitane reported before [14, 15]. The 13C NMR data of compound 2 aglycone had a good match with the data of the semi-synthetic 1 1 - β-ΟΗ cucurbitane glycoside, which was recorded in pyridine-d6 [14]. The 13C NMR data of natural 1 1 -β-ΟΗ cucurbitane by Matsuda et al was obtained in methanol-c/4 and were quite different in terms of chemical shifts at C-1 1 , C-8, C-10 and C-12 [15]. To further confirm the 1 1 -β-ΟΗ structure of 2, semi-synthesis of 2 was carried out by chemical reduction of 1 1 -oxo-mogroside V (4) to the 1 1 -β-ΟΗ and 1 1 -a-OH isomers of mogroside V. By LC-MS, HPLC co-chromatography and NMR data analysis, the semisynthetic 1 1 -epi-mogroside V was determined to be identical to the isolated 1 1 -epi- mogroside V. Therefore, the structure of 1 1 -epi-mogroside V (2) was elucidated as 3- Ο-β-D-glucopyranosy I→6)^-D-glucopyranosyl-1 1 β-ΟΗ-ΓΤ^οΙ-24-0-β-ϋ- glucopyranosyl-(1→2)-[B-D-glucopyranosyl-(1→6)]-3-D-glucopyranosyl. To our best of knowledge, this is the first report of natural mogroside with a 1 1 -β hydroxyl group.
HSQC-TOCSY with different mixing time for oligosaccharide chain elucidation. Gheysen et al investigated TOCSY experiments with different mixing time and concluded 100 ms as the optimal spin lock time to discriminate D-glucose, D-galactose and D-mannose [16]. Through their results, we noticed that the spin lock time could significantly affect the magnetization transfer efficiency of H-1 of D-glucose. The correlation between H-1 and H-2 through H-6 gradually extended to H-6 as the spin lock time increased. Inspired from their investigation, we hypothesized that by increasing spin lock time of HSQC-TOCSY, we should be able to see that the correlations of glucose H-1 with C-2 to C-6 gradually extend from C-2 to C-6 as the chain of the magnetization transfer extends. HSQC-TOCSY with increasing spin lock time should tell the carbon sequence information, which would be very useful for oligosaccharide elucidation and assignment. Figure 4 showed HSQC-TOCSY (hsqcgpmlph) of iso-mogroside VI with 10, 30, 60 and 100 ms mixing time. The cross- peaks in Figure 4 were quantified by their integrals and compared in Figure 5. The peak intensity (as presented by the integrals) could be an indication of their distance from H-1 in some cases. For example, all the C-3 peaks were significantly weaker than C-2 peaks under 30 ms mixing time experiments. However, under 60 ms C-3 peaks become bigger than C-2 peaks. To ensure correct elucidation, the carbon sequence should be determined through the overview of all the HSQC-TOCSY spectra with different mixing time, not just by the peak intensity under one mixing time. Traditionally, NMR elucidation and assignment of sugar chain of saponins start from the sugar linked to aglycone. By HMBC or NOESY, the well-resolved anomeric H-1 and C-1 signals can be identified. Then through COSY correlations and matching 3J (H,H) coupling constants, the proton signals of the monosaccharide can be assigned. Since a large coupling constant (>7 Hz) typically indicate two neighboring axial C-H bonds and small coupling constant (<4 Hz) for an axial-equatorial or equatorial-equatorial C-H bond, the type of monosaccharide can be determined. NOE correlations are useful for confirmation of the stereochemistry of axial-axial, axial-equatorial or equatorial- equatorial relations. The carbon signals of the saccharide (C-2 to C-6) are assigned according to HSQC or HMQC. The chemical shifts of the carbon signals as determined by HSQC/HMQC are very important information to confirm the monosaccharide type since the pattern of C-1 to C-6 chemical shifts for different type of monosaccharides is characteristic and consistent. Through the observation of carbon chemical shifts changes, the glycosylation position on the sugar chain can be identified and further confirmed by HMBC correlation. In summary, traditional way to elucidate saponin sugar is: HMBC→ C-1 , H-1 → COSY→ H-2 to H-6→ HSQC/HMQC→ C-2 to C-6, then assisted and confirmed by coupling constant analysis and NOESY experiment.
1 H-1 H TOCSY (Total Correlated Spectroscopy also known as HOHAHA - Homonuclear Hartmann Hahn) experiment could be a big help to divide the complicated sugar proton signals into groups. The transfer of magnetization during the TOCSY spin lock from the anomeric H-1 to the end of the furanose or pyranose ring will depend on the magnitude of the intervening 3J (H,H) scalar coupling constants. Neighboring axial-axial protons with large coupling constant (>7 Hz) allow a fast transfer of magnetization, whereas axial-equatorial or equatorial-equatorial with small coupling constant (<4 Hz) will considerably reduce transfer efficiency. Therefore, TOCSY experiment not only can be used to group proton signals into spin systems, but also provide the stereochemistry information of the saccharide. For example, we should be able to see the magnetization relay of glucose through H-1 to H-6 with the right mixing time. For galactose, there is no magnetization relay over H-4 even with 200 ms mixing time.
However, for the case of mogrosides with five or six sets of glucopyranosyl signals, using COSY and TOCSY to connect H-1 to H-6 can be quite tricky. The proton signals of the mogroside glucopyranosyls have very similar chemical shifts and appear crowded in a small range from δΗ 3.8 to 4.5. It is hard to make clear COSY connections through such poorly-resolved proton signals. The glucopyranosyl carbon signals are also very close and the HSQC cross-peaks heavily overlap to each other , which make the elucidation and assignment even more difficult.
Previously, HSQC-TOCSY have been applied in the structure elucidation and assignment of saponins by grouping carbon signals in each spin system together [17, 18]. Through our investigation, we demonstrated for the first time that the signal sequence within the glucopyranosyl carbon group can be identified by applying different mixing time in HSQC-TOCSY experiments. Figure 6 summarized the new HSQC-TOCSY based strategy to elucidate the glucopyranosyl oligosaccharide chain of mogrosides as follows: In Step 1 , Heteronuclear multiple bond correlation spectroscopy (HMBC) was used to determine anormeric C-1 and H-1 of the sugar. Start from the sugar link to aglycone. In Step 2, HSQC-TOCSY was used with 100ms mixing time to determine the whole group of C-2 to C-6. HSQC-COSY or HSQC-TOCSY (d9=10 ms) to assign C-2. HSQC-TOCSY (d9=30 ms) to assign C-3. HSQC-TOCSY (d9=60 ms) to assign C-4. HSQC-TOCSY (d9=100 ms) to assign C-5 and C-6. In Step 3, if a C-2 downshift from -575 to -581 , C-4 downshift from -571 to -581 or C-6 downshift from -562 to -69 is observed, check HMBC for glycosylation at these positions.** If a C-2 downshift from -575 to -581 , C- 4 downshift from -571 to -581 or C-6 downshift from -562 to -69, check HMBC for glycosylation at these positions.**. 1 -D NMR data such as 1 H coupling constants and 13C carbon signal pattern and 2-D NMR experiments such as NOESY, HMBC, TOCSY, COSY and HSQC could assist the process and confirm the results. The new HSQC- TOCSY based strategy may provide a simple, fast and unambiguous way for elucidation and assignment of glucopyranosyl chains of any new or known mogrosides. The strategy can also be adapted for elucidation and assignment of other monosaccharides and oligosaccharides.
Structures of neomogroside and mogroside VI
Compound 3 was determined to be neomogroside by extensive 1 -D and 2-D NMR analysis, as well as comparison with literature data [19]. For the oligosaccharide chain elucidation of 3, signals were assigned by HSQC-TOCSY and TOCSY experiments with different mixing time at 10, 30, 60, 100 ms. The linkage of the six saccharides were made by their NOESY and HMBC correlations. The oligosaccharide chain on C-3 of aglycone can be clearly assigned as 3-D-glucopyranosyl-(1→2)-B-D-glucopyranosyl- (1→6)-3-D-glucopyranosyl. The glucopyranosyl on C-24 of aglycone was branched with a 3-D-glucopyranosyl-(1→2) and a 3-D-glucopyranosyl-(1→6).
Neomogroside was firstly discovered from S. grosvenorii and described by Si et al.
[19]. Searching neomogroside in Scifinder returned the CAS number 189307-15-1 . However, the incorrect structure of neomogroside was given in Scifinder even though the literature referred by Scifinder was the 1996 article by Si et al. The incorrect structure of 189307-15-1 was given as 3-0-3-D-glucopyranosyl-(1→2) -[β-D- glucopyranosyl-(1→6)]-3-D-glucopyranosyl-mogrol-24-0-3-D-glucopyranosyl-(1→6)- [3-D-glucopyranosyl-(1→2)]-B-D-glucopyranosyl (structure of 6 in Figure 2) in Scifinder. The report of neomogroside by Si et al was written in Chinese and published in a Chinese journal in 1996. The accessibility and misunderstanding of this article might lead to the incorrect structure in Scifinder.
In Scifinder, neomogroside and mogroside VI had the same CAS number 189307-15-1 and same structure. Takemoto et al firstly reported mogroside VI from S. grosvenorii [2]. But it only referred a pure mogroside with a molecular formula of C66H1 12034 and no structure was determined [2]. Prakash et al. reported the structure and NMR data of mogroside VI as a known compound in their article published in 201 1 [6]. In their article, the structure of mogroside VI was assigned as the structure of 6 in Figure 2. Prakash mentioned that the structure elucidation of mogroside VI was made by NMR analysis and also by comparing with the literature values. However, no citation was given for the literature values.
For known compounds, comparison of NMR data with literature data could be useful for structure determination. However, the complexity of mogroside NMR data makes it difficult to determine the structure mainly by comparison of NMR data with literature data. 1 H NMR data of known mogrosides in different reports showed variations due to different NMR solvents used (the ratio of pyridine and D20 could cause signal shifts) or simply incorrect assignments.
Even though 13C NMR data are quite consistent and have better resolution than 1 H NMR data, structure determination of oligosaccharide chain of known mogrosides cannot be relied on directly comparing 13C NMR data with literature data. Considering the case of neomogroside, if the Glu-VI glucopyranosyl-(1→2) branched on Glu-I, Glu- II, Glu-lll, Glu-IV, or GluV, the five isomers may have very similar 13C NMR data. Rather than comparing 13C NMR data with literature data, extensive 2-D NMR analysis should be carefully done before the oligosaccharide chain of mogrosides are unambiguously determined. Example 1 1
Sweet intensity of iso-mogroside VI and 1 1 -epi-mogroside V Methods
10 mg iso-mogroside VI (Figure 7) was dissolved in 31 mL water to make 100 ppm iso- mogroside VI solution. The working solution of 1 1 -epi-mogroside V (Figure 8) was 374 ppm (9.34 mg 1 1 -epi-mogroside V in 25 mL water). A series of standard sucrose solutions were prepared (0.50, 0.75, 1 .00, 1 .25, 1 .50%) as sweetness references. Results
Four sweet sensitive panelists evaluated 100 ppm iso-mogroside VI and 374 ppm 1 1 - epi-mogroside V and the sucrose standards, and were asked to give sweet equivalence concentrations to sucrose. The mean sweet equivalence concentrations of each compound were used to calculate the iso-sweet potency. The iso-sweet potency values of iso-mogroside VI and 1 1 -epi-mogroside V were determined as 91 and 35 times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet equivalent to 0.91 % sucrose; 374 ppm 1 1 -epi-mogroside V sweet equivalent to 1 .31 % sucrose). Example 12
Methods
Iso-Mogroside VI and 1 1 -epi-mogroside V were obtained as described in Example 10. The iso-Mogroside VI and 1 1 -epi-mogroside V were each added to a solution containing 5 % sucrose and 0.03 % citric acid in a concentration of 25 ppm. These test solutions were tasted by an expert panel of seven people. For various aspects of sweet taste (overall sweet, upfront sweet, lingering sweet, astringent, volatile off-note), each panellist scored the test solutions in comparison to the base solution (solution of 5 % sucrose and 0.03 % citric acid). A score of 0 indicated that the taste aspect was the same, 1 indicates slightly higher, 2 indicates higher, 3 indicates much higher, -1 indicates slightly lower, -2 indicates lower and -3 indicates much lower.
Results
The average score for each test solution for each taste aspect was calculated. The results are shown in Table 20 below.
Table 20 Iso-Mogroside Epi-Mogroside
VI V
Overall Sweet 0.2 0.17
Upfront Sweet 0.2 0.33
Lingering
0.2 0
Sweet
Astringent 0 0
Volatile Off- n U n U
Note
Higher upfront
sweetness
offsets slightly
Comments Slightly acidic lower linger to
give overall
higher
sweetness
Two new minor cucurbitane glycosides along with known 1 1 -oxo-mogroside and neomogroside were purified from the commercial extracts of Luo Han Guo (Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang). By extensive NMR and LC-MS analyses and chemical synthesis, the structures of the two new compounds iso- mogroside VI (1 ) and 1 1 -epi-mogroside V (2) were elucidated as 3-0-3-D- glucopyranosyl(1→6)-3-D-glucopyranosyl-mogrol-24-0-(3-D-glucopyranosyl-(1→6)-[3- D-glucopyranosyl-(1→4)-B-D-glucopyranosyl-(1→2)]-B-D-glucopyranosyl and 3-0-3-D- glucopyranosyl(1→6)-3-D-glucopyranosyl-1 1 B-OH-mogrol-24-0-3-D-glucopyranosyl- (1→6)-[3-D-glucopyranosyl-(1→2)]-B-D-glucopyranosyl, respectively. The sweet potency of iso-mogroside VI and 1 1 -epi-mogroside V were evaluated as 91 and 35 times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet equivalent to 0.91 % sucrose; 374 ppm 1 1 -epi-mogroside V sweet equivalent to 1 .31 % sucrose). Through our course of identifying the new and known mogrosides with five or six glucopyranosyls, a new strategy for glucopyranosyl sugar chain elucidation and assignment was developed. The new strategy based on HSQC-TOCSY experiments with different mixing times featured a quick and unambiguous elucidation and assignment of glucopyranosyl oligosaccharide chains. The previous confusion regarding the structures of neomogroside and mogroside VI was reviewed and clarified after the confirmation of neomogroside structure by our extensive NMR spectral analysis.
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Claims

A sweetness modifying composition comprising:
one or more high-intensity sweetener(s) selected from steviol glycosides and/or mogrosides; and
one or more low-potency sweetener(s) selected from cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V;
wherein the sweetness modifying composition increases the sweetness of a sweetened composition by more than the sweetness of the sweetness modifying composition alone; and/or
wherein the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) ranges from about 2:1 to about 12:1 .
The sweetness modifying composition of claim 1 , wherein the sweetness modifying composition increases the sweetness of a sweetened composition by equal to or more than about 1 .25 % sucrose equivalence.
The sweetness modifying composition of claim 1 or 2, wherein the ratio of the high-intensity sweetener(s) to the low-potency sweetener(s) ranges from about 5:1 to about 12:1 or from about 6:1 to about 10:1 .
The sweetness modifying composition of any preceding claim, wherein the one or more high-intensity sweetener(s) includes a steviol glycoside 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, rubusoside and Naringin Dihydrochalcone.
The sweetness modifying composition of any one of claims 1 to 4, wherein the one or more high-intensity sweetener(s) includes a mogroside such as mogroside III, mogroside Mix, mogroside IV, siamenoside, neomogroside and mogroside V.
The sweetness modifying composition of any preceding claim, wherein the one or more high-intensity sweetener is mogroside V.
7. The sweetness modifying composition of any preceding claim, wherein the one or more low-potency sweetener is selected from one or more of cellobiose, psicose, and 1 1 -O-mogroside V.
8. The sweetness modifying composition of any preceding claim, wherein the one or more low-potency sweetener is 1 1 -O-mogroside V.
9. The sweetness modifying composition of any preceding claim, wherein one or both of the high-intensity sweetener(s) and the low-potency sweetener(s) is/are natural.
10. The sweetness modifying composition of any preceding claim, wherein the sweetness modifying composition improves one or more sweetness characteristic(s) of a sweetened composition compared to the respective sweetness characteristic(s) of the sweetened composition in the absence of the one or more low-potency sweetener(s) in the sweetness modifying composition.
1 1 . The sweetness modifying composition of any preceding claim, wherein the sweetness modifying composition weakens the lingering sweet taste of a sweetened composition compared to the lingering sweet taste of the sweetened composition in the absence of the one or more low-potency sweetener(s) in the sweetness modifying composition.
12. The sweetness modifying composition of any preceding claim, wherein the sweetness modifying composition weakens the bitter and/or astringent taste of a sweetened composition compared to the respective bitter and/or astringent taste of the sweetened composition in the absence of the one or more low-potency sweetener(s) in the sweetness modifying composition.
13. The sweetness modifying composition of any preceding claim, wherein the sweetness modifying composition is to be used in a sweetened composition in an amount having a sweetness less than 1 .5 % (w/v) sucrose equivalence.
14. A sweetened composition comprising:
at least one sweetener present in an amount having a sweetness equal to or greater than 1 .5 % (w/v) sucrose equivalence; and
the sweetness modifying composition of any preceding claim.
15. The sweetened composition of claim 14, wherein the sweetness modifying composition is present in the composition in an amount having a sweetness less than 1 .5 % (w/v) sucrose equivalence.
16. The sweetened composition of claim 14 or 15, wherein the one or more high- intensity sweetener(s) of the sweetness modifying composition are present in the sweetened composition in a total amount equal to or greater than about 15 ppm and optionally equal to or less than about 50 ppm.
17. The sweetened composition of any of claims 14 to 16, wherein the one or more high-intensity sweetener(s) of the sweetness modifying composition are present in the sweetened composition in a total amount ranging from about 15 ppm to about 30 ppm.
18. The sweetened composition of any of claims 14 to 17, wherein the one or more low-potency sweetener(s) of the sweetness modifying composition are present in the sweetened composition in a total amount equal to or greater than about 2 ppm and optionally equal to or less than about 12 ppm.
19. The sweetened composition of any of claims 14 to 18, wherein the one or more low-potency sweetener(s) of the sweetness modifying composition are present in the sweetened composition in a total amount ranging from about 2 ppm to about 10 ppm.
20. The sweetened composition of any of claims 14 to 19, wherein the at least one sweetener is a nutritive or non-nutritive sweetener.
21 . The sweetened composition of any of claims 14 to 20, wherein the at least one sweetener is selected from the group consisting of sucrose, high fructose corn syrup, acesulfame potassium (AceK), aspartame, steviol glycosides and sucralose.
22. The sweetened composition of any of claims 14 to 21 , wherein one or more sweetness characteristic(s) of the sweetened composition are improved compared to the respective one or more sweetness characteristic(s) of the sweetened composition in the absence of the one or more low-potency sweetener in the sweetness modifying composition.
23. The sweetened composition of any of claims 14 to 22, wherein the lingering sweet taste of the sweetened composition is less than the lingering sweet taste of the sweetened composition in the absence of the one or more low-potency sweetener in the sweetness modifying composition.
24. The sweetened composition of any of claims 14 to 23, wherein the bitter and/or astringent taste of the sweetened composition is less than the respective bitter and/or astringent taste of the sweetened composition in the absence of the one or more low-potency sweetener in the sweetness modifying composition.
25. The sweetened composition of any of claims 14 to 24, wherein the sweetened composition is a wet/liquid soup, a dehydrated and culinary food, a meal solution product, a meal embellishment product, a beverage or a dairy product such as milk, cheese and yoghurt.
26. Use of one or more low-potency sweetener(s) selected from cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V to improve one or more sweetness characteristic(s) of a sweetened composition comprising one or more high-intensity sweetener(s) selected from steviol glycosides and/or mogrosides, wherein the total amount of the low-potency sweetener(s) and the high-intensity sweetener(s) has a sweetness of less than 1 .5 % (w/v) sucrose equivalence.
27. The use of claim 26, wherein the one or more low-potency sweetener(s) weaken the lingering sweet taste of the sweetened composition.
28. The use of claim 26 or 27, wherein the one or more low-potency sweetener(s) weaken the bitter and/or astringent taste of the sweetened composition.
29. The use of any of claims 26 to 28, wherein the ratio of the high-intensity sweetener(s) to the low-potency sweetener(s) ranges from about 2:1 to about 12:1 .
30. The use of any of claims 26 to 29, wherein the ratio of the high-intensity sweetener(s) to the low-potency sweetener(s) ranges from about 5:1 to about
12:1 or from about 6:1 to about 10:1 .
31 . The use of any of claims 26 to 30, wherein the one or more high-intensity sweetener(s) includes a steviol glycoside 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, rubusoside and Naringin Dihydrochalcone.
32. The use of any of claims 26 to 31 , wherein the one or more high-intensity sweetener(s) includes a mogroside such as mogroside III, mogroside Mix, mogroside IV, siamenoside, neomogroside and mogroside V.
33. The use of any of claims 26 to 32, wherein the one or more high-intensity sweetener is mogroside V.
34. The use of any of claims 26 to 33, wherein the one or more low-potency sweetener(s) is selected from one or more of cellobiose, psicose and 1 1 -0- mogroside V.
35. The use of any of claims 26 to 34, wherein the one or more low-potency sweetener is 1 1 -O-mogroside V.
36. The use of any of claims 26 to 35, wherein the combination of the one or more low-potency sweetener(s) and one or more high-intensity sweetener(s) is used to increase the sweetness of the sweetened composition by an amount more than the sweetness of the combination of the low-potency sweetener(s) and the high-intensity sweetener(s) alone.
37. The use of any of claims 26 to 36, wherein the sweetened composition is a wet/liquid soup, a dehydrated and culinary food, a meal solution product, a meal embellishment product, a beverage or a dairy product such as milk, cheese and yoghurt.
38. The use of any of claims 26 to 37, wherein the 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 optionally equal to or less than about 50 ppm.
39. The use of any of claims 26 to 38, wherein the one or more high-intensity sweetener(s) are present in the sweetened composition in a total amount ranging from about 15 ppm to about 30 ppm.
40. The use of any of claims 26 to 39, wherein the one or more low-potency sweetener(s) are present in the sweetened composition in a total amount equal to or greater than about 2 ppm and optionally equal to or less than about 12 ppm.
41 . The use of any of claims 26 to 40, wherein the one or more low-potency sweetener(s) are present in the sweetened composition in a total amount ranging from about 2 ppm to about 10 ppm.
42. The use of any of claims 26 to 41 , wherein the one or more low-potency sweetener(s) and one or more high-intensity sweetener(s) increase the sweetness of the sweetened composition by more than about 1 .25 % (w/v) sucrose equivalence.
43. A method of modifying the sweetness of a sweetened composition, the method comprising providing a base composition comprising at least one sweetener in an amount above its sweetness recognition threshold and/or having a sweetness equal to or greater than about 1 .5 % (w/v) sucrose equivalence, and adding one or more high-intensity sweetener(s) selected from steviol glycosides and/or mogrosides and one or more low-potency sweetener(s) selected from cellobiose, psicose, cyclamate and/or 1 1 -O-mogroside V,
wherein the ratio of the one or more high-intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 2:1 to about 12:1 ; and/or
wherein the 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 the one or more low-potency sweetener(s) are added in a total amount equal to or greater than about 2 ppm and optionally equal to or less than about 12 ppm; and/or
wherein the total concentration of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) that are added has a sweetness less than 1 .5 % (w/v) sucrose equivalence.
44. The method of claim 43, wherein one or more sweetness characteristic(s) of the composition is/are improved compared to the respective one or more sweetness characteristic(s) of the composition in the absence of the one or more low-potency sweetener(s).
45. The method of claim 43 or 44, wherein the sweetness of the composition is enhanced compared to the sweetness of the composition in the absence of the one or more low-potency sweetener(s).
46. The method of any one of claims 43 to 45, wherein the lingering sweet taste of the composition is weakened compared to the lingering sweet taste of the composition in the absence of the one or more low-potency sweetener(s).
47. The method of any one of claims 43 to 46, wherein the bitter and/or astringent taste of the composition is weakened compared to the bitter and/or astringent taste of the composition in the absence of the one or more low-potency sweetener(s).
48. The method of any of claims 43 to 47, wherein the ratio of the one or more high- intensity sweetener(s) to the one or more low-potency sweetener(s) is from about 5:1 to about 12:1 or from about 6:1 to about 12:1 .
49. The method of any of claims 43 to 48, wherein the combination of the one or more high-intensity sweetener(s) and the one or more low-potency sweetener(s) increases the sweetness of the sweetened composition by equal to or more than about 1 .25 % (w/v) sucrose equivalence.
50. The method of any of claims 43 to 49, wherein the one or more high-intensity sweetener(s) are added to the base composition in a total amount ranging from about 15 ppm to about 30 ppm or from about 20 ppm to about 30 ppm.
51 . The method of any of claims 43 to 50, wherein the one or more low-potency sweetener(s) are added to the base composition in a total amount ranging from about 2 ppm to about 10 ppm.
52. The method of any of claims 43 to 51 , wherein the one or more high-intensity sweetener(s) include a steviol glycoside 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, rubusoside and Naringin Dihydrochalcone.
53. The method of any of claims 43 to 52, wherein the one or more high-intensity sweetener(s) include a mogroside such as mogroside III, mogroside Mix, mogroside IV, siamenoside, neomogroside and mogroside V.
54. The method of any of claims 43 to 53, wherein the one or more high-intensity sweetener is mogroside V.
55. The method of any of claims 43 to 54, wherein the one or more low-potency sweetener are selected from one or more of cellobiose, psicose and 1 1 -0- mogroside V.
56. The method of any of claims 43 to 55, wherein the one or more low-potency sweetener(s) is 1 1 -O-mogroside V.
57. The method of any of claims 43 to 56, wherein the at least one other sweetener is selected from the group consisting of sucrose, high fructose corn syrup, acesulfame potassium (AceK), aspartame, steviol glycosides and sucralose.
58. The method of any of claims 43 to 57, wherein the base composition is a wet/liquid soup, a dehydrated and culinary food, a meal solution product, a meal embellishment product, a beverage or a dairy product such as milk, cheese and yoghurt.
59. A method for preparing the sweetened composition of any of claims 14 to 25 comprising combining a base composition, one or more high-intensity sweetener(s) and one or more low-potency sweetener(s) and at least one other sweetener.
60. A method for preparing the sweetness modifying composition of any of claims 1 to 13 comprising combining one or more high-intensity sweetener(s) and one or more low-potency sweetener(s).
61 . The method of claim 59 or 60, wherein one or more of the sweeteners are made by a biological process.
62. The method of any of claims 59 to 61 , wherein one or more of the sweeteners are made by an enzymatic process.
63. The method of claim 59 or 60, wherein one or more of the sweeteners are made by a synthetic process.
64. A sweetened composition comprising:
at least one sweetener present in an amount having a sweetness equal to or greater than 1 .5 % (w/v) sucrose equivalence; and
one or more sweetness enhancer(s) selected from mogroside IV, siamenoside and neomogroside.
65. The sweetened composition of claim 64, wherein the one or more sweetness enhancer(s) are present in the composition in a total amount having a sweetness less than 1 .5 % (w/v) sucrose equivalence.
66. The sweetened composition of claim 64 or 65, wherein the one or more sweetness enhancer(s) are present in the sweetened composition in a total amount equal to or greater than about 15 ppm and optionally equal to or less than about 50 ppm.
67. The sweetened composition of any of claims 64 to 66, wherein the one or more sweetness enhancer(s) are present in the sweetened composition in a total amount ranging from about 15 ppm to about 35 ppm.
68. The sweetened composition of any of claims 64 to 67, wherein the at least one sweetener is a nutritive or non-nutritive sweetener.
69. The sweetened composition of any of claims 64 to 68, wherein the at least one sweetener is selected from the group consisting of sucrose, high fructose corn syrup, acesulfame potassium (AceK), aspartame, steviol glycosides and sucralose.
70. The sweetened composition of any of claims 64 to 69, wherein the sweetened composition is a wet/liquid soup, a dehydrated and culinary food, a meal solution product, a meal embellishment product, a beverage or a dairy product such as milk, cheese and yoghurt.
71 . The sweetened composition of any of claims 64 to 70, wherein the one or more sweetness enhancers are at least 80 wt% pure.
72. Use of one or more of mogroside IV, siamenoside and neomogroside as a sweetness enhancer in a sweetened composition.
73. The use of claim 72, wherein the one or more sweetness enhancer(s) are used in the sweetened composition in a total amount having a sweetness less than
1 .5 % (w/v) sucrose equivalence.
74. The use of claim 72 or 73, wherein the one or more sweetness enhancer(s) are used in the sweetened composition in a total amount equal to or greater than about 15 ppm and optionally equal to or less than about 50 ppm.
75. The use of any of claims 72 to 74, wherein the one or more sweetness enhancer(s) are used in the sweetened composition in a total amount ranging from about 15 ppm to about 35 ppm.
76. The use of any of claims 72 to 75, wherein the sweetened composition is a wet/liquid soup, a dehydrated and culinary food, a meal solution product, a meal embellishment product, a beverage or a dairy product such as milk, cheese and yoghurt.
77. The use of any of claims 72 to 76, wherein the one or more sweetness enhancers are at least 80 wt% pure.
78. A compound having the structure of Formula I or a salt thereof wherein the compound is in an isolated and/or purified form.
79. A compound having the structure of Formula II or a salt thereof wherein the compound is in an isolated and/or purified form.
80. Use of the compound of claim 78 or claim 79 as a sweetener.
81 . Use of the compound of claim 78 or claim 79 as a sweetener modifier and/or sweetener enhancer in a sweetened composition.
82. A composition comprising the compound of claim 78 and the compound of claim 79 and optionally one or more additional compounds.
83. The composition of claim 82 wherein the one or more additional compounds are sweetener compounds, preferably mogroside sweetener compounds.
84. Use of the composition of claim 82 or claim 83 as a sweetener modifier and/or sweetener enhancer in a sweetened composition.
PCT/EP2018/064324 2017-06-02 2018-05-31 Compositions WO2018220103A1 (en)

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